Physiol Rev 94: 1143–1218, 2014
doi:10.1152/physrev.00014.2014
BONE DEVELOPMENT AND MINERAL
HOMEOSTASIS IN THE FETUS AND NEONATE:
ROLES OF THE CALCIOTROPIC AND
PHOSPHOTROPIC HORMONES
Christopher S. Kovacs
Faculty of Medicine-Endocrinology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
Kovacs CS. Bone Development and Mineral Homeostasis in the Fetus and Neonate:
Roles of the Calciotropic and Phosphotropic Hormones. Physiol Rev 94: 1143–1218,
2014; doi:10.1152/physrev.00014.2014.—Mineral and bone metabolism are regulated differently in utero compared with the adult. The fetal kidneys, intestines, and
skeleton are not dominant sources of mineral supply for the fetus. Instead, the placenta
meets the fetal need for mineral by actively transporting calcium, phosphorus, and magnesium
from the maternal circulation. These minerals are maintained in the fetal circulation at higher
concentrations than in the mother and normal adult, and such high levels appear necessary for the
developing skeleton to accrete a normal amount of mineral by term. Parathyroid hormone (PTH)
and calcitriol circulate at low concentrations in the fetal circulation. Fetal bone development and the
regulation of serum minerals are critically dependent on PTH and PTH-related protein, but not
vitamin D/calcitriol, fibroblast growth factor-23, calcitonin, or the sex steroids. After birth, the
serum calcium falls and phosphorus rises before gradually reaching adult values over the subsequent 24 – 48 h. The intestines are the main source of mineral for the neonate, while the kidneys
reabsorb mineral, and bone turnover contributes mineral to the circulation. This switch in the
regulation of mineral homeostasis is triggered by loss of the placenta and a postnatal fall in serum
calcium, and is followed in sequence by a rise in PTH and then an increase in calcitriol. Intestinal
calcium absorption is initially a passive process facilitated by lactose, but later becomes active and
calcitriol-dependent. However, calcitriol’s role can be bypassed by increasing the calcium content
of the diet, or by parenteral administration of calcium.
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
XI.
XII.
XIII.
INTRODUCTION
OVERVIEW OF FETAL MINERAL...
OVERVIEW OF NEONATAL...
PARATHYROID HORMONE-RELATED...
PARATHYROID HORMONE
ROLE OF PTHrP AND PTH...
CALCIUM-SENSING RECEPTOR
CALCITRIOL AND VITAMIN D
CALCITONIN
FIBROBLAST GROWTH FACTOR-23
SEX STEROIDS
FETAL AND NEONATAL RESPONSES...
CONCLUSIONS
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1194
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I. INTRODUCTION
Normal calcium and bone homeostasis in the adult can
be almost fully explained by the interactions of several
regulatory hormones, including parathyroid hormone
(PTH), calcitriol, fibroblast growth factor-23 (FGF23),
calcitonin, and the sex steroids (estradiol and testosterone). Loss of any one of these hormones can have significant consequences for the adult. Hypoparathyroidism
can cause fatalities due to hypocalcemia; it also causes
hyperphosphatemia (leading to ectopic calcifications),
hypercalciuria (leading to nephrolithiasis and nephrocalcinosis), low bone turnover, and possible neurological
sequelae from basal ganglia calcifications. With vitamin
D deficiency, the serum calcium may be normal or reduced (but not as low as in hypoparathyroidism), serum
phosphorus is low, and undermineralization of the skeleton leads to rickets or osteomalacia. Loss of FGF23
causes hyperphosphatemia, extraskeletal calcifications,
and early mortality. Loss of calcitonin increases bone
resorption, an effect that may become most apparent
during lactation. Deficiency of either of the sex steroids
increases bone resorption and will lead to osteoporosis.
The roles that these hormones play in regulating osteoblasts or osteoclasts have led to some of them (or their
synthetic analogs) being used to treat osteoporosis and
other skeletal disorders.
This review will make clear that mineral and bone metabolism are regulated differently in utero such that many of
these hormones do not appear to have the crucial roles that
they have in the adult.
0031-9333/14 Copyright © 2014 the American Physiological Society
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CHRISTOPHER S. KOVACS
The fetus has several developmental goals, which include
the need to actively pump minerals across the placenta
against concentration and electrochemical gradients, to
maintain higher extracellular concentrations of minerals
compared with the maternal circulation, and to adequately
mineralize the skeleton before birth.
During embryonic development the early pattern for the
endochondral skeleton is laid down, but it is during the later
stages of fetal development that rapid bone formation and
mineralization create a significant demand for mineral. The
adequacy of the mineral supply is a limiting factor for how
much mineral can be accreted by the skeleton before birth,
and the availability of mineral also influences the function
and activity of osteoblasts and osteoclasts. A human fetus
typically accumulates ⬃30 g of calcium by term, with 80%
of that mineral content obtained in the third trimester (121,
224, 683, 733, 734). The kidneys, intestines, and skeleton
(through bone turnover) are not dominant sources of mineral supply for the normal fetus as they are for the adult
(FIGURE 1). Instead, the placenta meets the fetal need for
mineral by actively transporting calcium, phosphorus, and
magnesium from the maternal circulation. To obtain 30 g of
calcium by term, the placenta must deliver 120 –150
mg·kg⫺1·day⫺1 of calcium during the third trimester, or
more than 300 mg/day between weeks 35 and 38 (635, 734,
735, 761). The placenta appears capable of providing mineral even when faced with reduced concentrations of those
same minerals in the maternal circulation. The fetus maintains higher mineral concentrations than in the mother or
normal adult, and such high levels appear necessary for the
skeleton to form and mineralize normally. As this review
will demonstrate, fetal bone and mineral metabolism are
critically dependent on PTH and PTH-related protein
(PTHrP), whereas vitamin D/calcitriol, FGF23, calcitonin,
and the sex steroids may not be required.
The intestines become the main source of mineral after birth,
while the kidneys begin to reabsorb mineral, and normal bone
turnover contributes additional mineral to the circulation.
This switch in the regulation of mineral homeostasis is triggered by a postnatal fall in serum calcium, and followed in
sequence by a rise in PTH and then an increase in calcitriol.
FGF23 then begins to play an important role in regulating
serum phosphorus, renal phosphorus excretion, and calcitriol
synthesis and catabolism. Intestinal calcium absorption is initially a passive process facilitated by lactose, but later becomes
active and calcitriol-dependent. However, calcitriol’s role can
be bypassed by increasing the calcium content of the diet, or by
administering parenteral calcium, thereby preventing or correcting the skeletal changes of rickets or osteomalacia, independent of calcitriol or vitamin D sufficiency.
There are only limited human data that directly examine
regulation of mineral and bone homeostasis during fetal
development. Much of those data consist of cord blood
1144
Ca2+ Ca2+
Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
FIGURE 1. Circulation of mineral within the fetal-placental unit.
Calcium is represented here, but these statements apply to phosphorus (phosphate) and magnesium as well. At the top right, the
main flux of mineral is across the placenta and through the fetal
circulation into bone; however, some mineral returns to the maternal circulation (backflux). On the bottom right, the fetal kidneys filter
the blood and excrete mineral into urine, which in turn makes up
much of the volume of amniotic fluid. On the bottom left, amniotic
fluid is swallowed, and its mineral content can be absorbed by the
fetal intestines, thereby restoring it to the circulation. The renalamniotic-intestinal loop is likely a minor component for fetal mineral
homeostasis. On the top left, although the net flux of mineral is into
bone, some mineral is resorbed from the developing skeleton to
reenter the fetal circulation. If placental delivery of mineral is deficient, fetal secondary hyperparathyroidism ensues, which causes
more substantial resorption of mineral from the fetal skeleton, reduced skeletal mineral content, and possible fractures occurring in
utero or during the birthing process.
samples taken from normal and abnormal preterm and
term fetuses and pathological examination of embryos and
fetuses that died due to congenital abnormalities or obstetrical accidents. The main exception is with vitamin D and
calcitriol, for which there have been randomized interventional trials, cohort studies, and associational studies, each
examining possible effects of vitamin D deficiency on fetal
and neonatal bone and mineral metabolism.
In contrast to the limited human data, mineral and bone
homeostasis have been extensively examined in several animal species using surgical, genetic, and pharmacological
approaches. Regulation of fetal mineral homeostasis was
first studied by administering pharmacological doses of
hormones or antibodies to the mother or fetus, by making
the mother severely vitamin D deficient before pregnancy,
or by removing the thyroid or parathyroids from the
mother, fetus, or both. But most calciotropic and phospho-
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FETAL AND NEONATAL MINERAL METABOLISM
tropic hormones cannot be adequately studied by these approaches because they are produced by more than one tissue. Global and conditional gene deletion models have enabled us to study fetuses that lack PTHrP, PTH, calcitriol,
the vitamin D receptor, calcitonin, or FGF23 (see TABLE 1
for a glossary of mouse models to be discussed). It is through
consideration of these animal data that it becomes possible to
theorize how fetal mineral homeostasis is regulated in the human, and how this regulation changes after birth.
In this review, the term phosphorus is used for consistency
and simplicity when referring to its presence in blood or
bone. However, it is recognized that serum contains mainly
inorganic phosphates (dihydrogen and monohydrogen
phosphate), bone contains phosphorus largely in the form
of hydroxyapatite, while the soft tissues and extracellular
fluids contain organic phosphates in complex with carbohydrates, lipids, and proteins (37).
To avoid any confusion between animal and human data,
within each subsection this review will sequentially discuss
animal data followed by the extant human data. The animal
data will be used to infer how mineral and bone homeostasis
are regulated during fetal and neonatal development, and the
sometimes limited human data will reveal whether there is
agreement or disagreement with what the animal models suggest.
II. OVERVIEW OF FETAL MINERAL
METABOLISM AND SKELETAL
FORMATION
This section describes the normal circulating concentrations (FIGURE 2) and sources (FIGURE 1) of minerals and
hormones during fetal development, and the participation
Table 1. Glossary of mouse models
Name
Protein(s) Encoded by the Gene
Casr null
CasrChon, Pre, Ob, Oc, Para null
Calcium sensing receptor (CaSR) (257, 338)
Calcium sensing receptor, conditionally deleted from chondrocytes, preosteoblasts, osteoblasts,
osteocytes, and parathyroids, respectively (109, 169, 549)
Calcitonin and calcitonin gene-related peptide-␣ (119, 260, 430, 744)
Calcitonin receptor (134, 361)
1␣-Hydroxylase/Cyp27b1 (cytochrome P-450, family 27, subfamily B, polypeptide 1), which
converts 25-hydroxyvitamin D into calcitriol (138–140)
Fibroblast growth factor-23 (FGF23) (395, 626)
Transcription factor glial cells missing (Gcm2), absence of which leads to loss of parathyroids
(238, 384, 385, 623, 691)
Glycoprotein 130 is a transmembrane protein and a receptor subunit common to many
cytokines; it interacts with PTH (616, 753)
Homeobox a3 ablation causes abnormalities in tissues derived from the third and fourth
pharyngeal arches: loss of parathyroids and thymus, hypoplasia of the thyroid, loss of hyoid,
and structural weakness in major arteries that lead hemorrhage at birth (114, 336, 341,
411)
Phex (phosphate regulating endopeptidase homolog, X-linked) encodes an endopeptidase; an
inactivating mutation in the gene leads to elevated FGF23 and models X-linked hypophosphatemic
rickets (395, 484, 627). These are also known as Hyp mice due to the hypophosphatemia
Parathyroid hormone (PTH) (310, 439, 440, 623)
Double-mutant lacking PTH and calcitriol (747, 749)
Double-mutant lacking both PTH and Hoxa3 (parathyroids)
PTH1 receptor (PTH1R) also known as PTH/PTHrP receptor (336, 340, 341, 360, 714)
Double mutant lacking PTH1 receptor and CaSR (341)
Parathyroid hormone-related protein (PTHrP) (297, 336, 340, 570)
Double mutant lacking PTHrP and CaSR (338)
Double mutant lacking PTHrP and Hoxa3 (parathyroids) (336)
Double mutant lacking PTHrP and PTH (439)
TRPV6 (transient receptor potential cation channel, subfamily V, member 6) (659)
Ctcgrp null
Ctr null
Cyp27b1 null
Fgf23 null
Gcm2 null
Gp130 null
Hoxa3 null
Phex null
Pth null
Pth/Cyp27b1
Pth/Hoxa3
Pth1r null
Pth1r/Casr
Pthrp null
Pthrp/Casr
Pthrp/Hoxa3
Pthrp/Pth
Trpv6 null
VdrBos null
VdrLeu, Tok, Mun null
Vitamin D receptor (VDR); developed in Boston, the second zinc finger of the receptor DNAbinding domain has been deleted; there is no transcribed or translated receptor (24, 192,
342, 371–373)
Vitamin D receptor (VDR); developed by three separate groups in Leuven, Tokyo, and Munich;
each deleted the first zinc finger of the receptor DNA-binding domain, but a surrogate ATG
downstream from the second zinc finger causes an aberrant VDR to be expressed which has
normal ligand binding affinity but no DNA binding ability (175, 376, 574, 704, 755)
Reference numbers are given in parentheses.
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CHRISTOPHER S. KOVACS
of parathyroids, kidneys, intestines, placenta, and bone in
regulating fetal mineral metabolism. The roles of the individual calciotropic and phosphotropic hormones are discussed in sections IV–XI.
TOTAL Ca
(mmol/l)
2.7
A. Serum Mineral Concentrations
1.6
1. Animal data
IONOZED Ca
(mmol/l)
2.1
A consistent finding among mammalian fetuses is that the
serum calcium concentration is significantly higher than the
simultaneous maternal calcium. Typically the fetal serum
calcium is 0.30 – 0.50 mM or higher than the maternal level
in rhesus monkeys (189, 524), lambs (148, 201, 435, 505),
calves (201, 715), rodents (201, 338, 340, 342, 346, 347,
623), pigs (98, 101, 353), and foals (201). The ionized
calcium is also increased 0.25– 0.50 mM above the maternal value in fetal rodents (148, 340, 422), confirming that
true hypercalcemia is present relative to maternal and normal adult values. Although only ⬃50% of serum calcium is
ionized in the adult rodent, within fetal rodents ⬃80% of
calcium is free or ionized (148, 340). It is unknown how
early in gestation the serum calcium becomes increased
compared with the maternal value, but it has been demonstrated at all time points for which it has been technically
possible to measure it. This includes the 35th day of gestation in lambs (93, 397), day 14 in rats (346, 347, 675), and
day 15 in mice (330). Within rat fetuses, a small but progressive increase in total and ionized calcium occurs over
the final 7 days, which may indicate that at an earlier time
point there is no difference between embryonic/fetal and
maternal calcium concentrations (346, 675).
0.9
PHOSPHATE
(mmol/l)
1.7
0.9
PTH
(pmol/l)
5.0
0.0
1, 25-D
(pmol/l)
300
0.0
CALCITONIN
(ng/l)
100
0.0
PTHrP
(pmol/l)
5.0
0.0
0
1
2
3
4
DAYS POSTNATAL
FIGURE 2. Schematic illustration of the longitudinal changes in
calcium, phosphate, and calciotropic hormone levels that occur
during the fetal and neonatal period in humans. Normal adult ranges
are indicated by the shaded areas. The progression in PTHrP levels
has been depicted by a dashed line to reflect that it is speculative.
[From Kovacs and Kronenberg (339). Copyright 1997 The Endocrine Society; permission conveyed through the Copyright Clearance
Center, Inc.]
1146
This level of blood calcium, both total and ionized, exceeds the
value that the calcium sensing receptor (CaSR) sets in the
adult through its regulation of PTH synthesis and secretion,
and which is represented by the simultaneous maternal serum calcium concentration (338). CaSR likely acts in response to the high serum calcium to suppress release of PTH
(FIGURE 3). When one or both alleles of Casr bear an inactivating mutation, the fetal serum calcium and PTH increase above normal fetal values, confirming that CaSR can
raise the fetal serum calcium by increasing PTH (338). That
CaSR requires PTH to raise the fetal serum calcium was
confirmed by the finding that if the PTH/PTHrP receptor
(PTH1R) is also ablated in Pth1r/Casr double mutants,
thereby blocking the actions of PTH, then inactivating mutations of Casr fail to increase the fetal serum calcium (338).
What sets the fetal blood calcium at its high target level in
normal fetuses? One possibility is that novel calcium-sensing receptors are expressed during fetal development, which
set the calcium concentration at specific values within the
placental or fetal circulations. Alternatively, the active, forward flow of calcium across the placenta may be responsible for maintaining the high fetal blood calcium. If active
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FETAL AND NEONATAL MINERAL METABOLISM
iCa2+ 1.75 mmol/l
Fetal hypercalcemia due to CASR+/–
• PTH with PTHrP
iCa2+ 1.50 mmol/l
Normal fetal ionized calcium
• Combined effects of PTHrP and PTH
iCa2+ 1.25 mmol/l
Fetal hypocalcemia but normal adult level
• Loss of PTHrP with compensatory PTH
• Loss of PTH PTHrP
iCa2+ 1.00 mmol/l
Extreme fetal hypocalcemia
• Loss of parathyroids
• Loss of PTH and PTHrP
• Loss of PTH1R
FIGURE 3. Regulation of fetal ionized calcium. This schematic
illustrates changes in ionized calcium as observed in murine fetuses
and extrapolated to humans; the levels shown are meant as a guide
for relative changes and are not meant to indicate precise values
found in human cord blood. The normal ionized calcium level is higher
in fetuses than in the normal adult, and is maintained by the combined effects of high concentrations of PTHrP and low levels of PTH
in the circulation. CaSR likely suppresses PTH in response to the
normal high level of calcium in fetal blood. The high fetal blood
calcium may be set by a novel calcium-sensing receptor (other than
the known CaSR), which regulates PTHrP release from the placenta
and possibly other fetal tissues. In the absence of PTHrP, serum
calcium falls but is now maintained at the normal adult level through
the normal actions of CaSR to regulate PTH. Loss of PTH leads to a
similar blood calcium value, approximately equal to the maternal
calcium concentration, without a compensatory increase in circulating PTHrP. Combined loss of PTH and PTHrP, loss of parathyroids,
or loss of PTH1R, each results in the lowest fetal ionized calcium
values. Conversely, inactivating mutations of CASR cause PTH and
the blood calcium to increase above normal fetal levels, in turn
causing PTHrP to become decreased.
placental calcium transfer is the explanation, then fetuses
should maintain a relative gradient of serum calcium about
the maternal level, and acute or chronic changes in maternal
serum calcium should alter the fetal blood calcium.
However, the fetus does not maintain a set gradient of serum calcium relative to the maternal serum calcium; instead, it sets a fixed level of calcium independent of the
maternal calcium value. This value can be defended and
maintained despite significant challenges. The evidence includes that fetal rats were normocalcemic despite maternal
hypocalcemia induced by a calcium-restricted diet (377),
vitamin D deficiency (74, 241, 443), or thyroparathyroidectomy (67, 276). Fetal lambs also maintained normal serum calcium levels despite maternal hypocalcemia induced
by parathyroidectomy (564). In fetal mice both Casr (257)
and Vdr (372) deletion models have revealed that fetuses
will maintain the ionized calcium level determined by their
genotype, regardless of whether the mother is normocalcemic, hypercalcemic, or hypocalcemic (FIGURE 4) (338, 342).
In these models, a lower maternal serum calcium means that
the calcium gradient from mother to fetus is increased,
whereas a higher maternal serum calcium means that the
gradient is decreased. But in all of these models the fetal
ionized calcium was unchanged, confirming that it is the
blood level of calcium that is set and not a relative gradient
compared with the mother. These findings are consistent
with calcium-sensing receptors that determine the fetal
blood calcium level, as opposed to the fetal blood calcium
being determined by active placental calcium transport.
Moreover, acute alterations in the maternal blood calcium
of rodents and primates (such as by infusions of calcium,
calcitriol, calcitonin, PTH, or EDTA) had minimal or no
effect on the fetal blood calcium level (87, 214, 345, 478,
547). However, although a high fetal blood calcium level
was maintained in fetal rats between the 12th and 17th days
of gestation after maternal parathyroidectomy, it declined
during the last several days of gestation, the interval when
the skeleton is most rapidly accreting mineral (106, 208,
220).
What is the purpose of the fetus maintaining a higher blood
concentration of calcium? The robust maintenance of this
“fetal hypercalcemia” across numerous mammalian species
suggests that it is physiologically important. But fetal hypocalcemia does not impair survival to the end of gestation,
as shown by study of Pthrp null, Pth1r null, Hoxa3 null,
Trpv6 null, Gp130 null, and Pthrp/Hoxa3 double mutant
fetuses (336, 339, 341, 616, 659). Another possibility is
that a high blood calcium level in utero may improve survival after birth. As discussed in section III, the postnatal
onset of breathing, obligatory rise in pH, and loss of the
placental calcium infusion contribute to a rapid fall in calcium within 12 h after birth. During the next 24 h, the
serum calcium increases to the adult value. In rodents, the
serum calcium drops more markedly by 40% after birth
(201, 347). If the fetal blood calcium is lower than normal,
conceivably the neonatal blood calcium will fall to an even
lower value, thereby conferring an increased risk of hypocalcemic tetany and death. The early postnatal mortality
of Pthrp null, Pth1r null, Pth null, Gp130 null, and Hoxa3
null fetuses is compatible with this possibility (297, 340,
341, 360, 616, 623). The high fetal calcium concentration
also appears to be important for normal skeletal mineralization to be achieved, since hypocalcemia is associated
with reduced ash weight and skeletal mineral content in Pth
null, Hoxa3 null, Pth1r null, and Gcm2 null fetuses (see
sects. VC and VIC) (336, 341, 623).
The serum ionized calcium concentration differs significantly among fetuses obtained from different commercially
available strains of mice; for example, fetuses from the commonly used inbred C57BL/6 strain maintain a significantly
lower ionized calcium compared with fetuses from outbred
Black Swiss (340). Therefore, unknown genes also contribute to the regulation of fetal mineral homeostasis, or specifically the set calcium level maintained by normal fetuses.
Phosphorus plays key roles during endochondral bone development: it induces apoptosis of hypertrophic chondrocytes (158, 579) and is incorporated into osteoid before
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1147
CHRISTOPHER S. KOVACS
A
B
Ionized calcium (mmol/L)
2.0
1.5
Casr +/- mothers
Vdr +/- mothers
WT mothers
1.0
0.5
Vdr null mothers
0.0
WT
Vdr+/-
Vdr null
WT
Casr+/- Casr null
FIGURE 4. Fetal ionized calcium level is set independently of the maternal calcium value. Data have been
merged for simplicity and schematically represented in this illustration. In A, the ionized calcium of Vdr⫹/⫺ and
Vdr null mothers is represented by their respective horizontal lines ⫾ SE. Despite normocalcemia in Vdr⫹/⫺
mothers (who bore WT, Vdr⫹/⫺, and Vdr null fetuses) and hypocalcemia in Vdr null mothers (who bore Vdr⫹/⫺
and Vdr null fetuses), the maternal ionized calcium did not affect the ionized calcium level of their respective
fetuses. The fetuses maintained a set level independent of the maternal ionized calcium, and that value was no
different in Vdr null fetuses compared with WT and Vdr⫹/⫺ littermates. In B, the ionized calcium of WT and
Casr⫹/⫺ mothers is represented by the respective horizontal lines (⫾ SE). Despite normocalcemia in WT
mothers (who bore WT and Casr⫹/⫺ fetuses) and hypercalcemia in Casr⫹/⫺ mothers (who bore WT, Casr⫹/⫺,
and Casr null fetuses), the maternal ionized calcium did not affect the ionized calcium level of their respective
fetuses. The fetuses maintained a set level independent of the maternal ionized calcium, and that value was
increased in Casr⫹/⫺ and Casr null fetuses compared with their WT littermates. The background strain was
Black Swiss for all mice represented in this figure. [Original data in A were published as two separate panels
in Kovacs et al. (342). Original data in B were also published as two separate panels in Kovacs et al. (338) and
adapted with permission of the American Society for Clinical Investigation, copyright 1998.]
calcium binds to it (487, 579, 759). Consequently, serum
phosphorus is an important determinant of the formation
and mineralization of bone during fetal development. It is
typically 0.5–1.0 mM higher in the fetus than in the mother,
as observed in rats (211, 213, 315, 346), mice (623), lambs
(377, 403, 435), pigs (98, 353), calves (201), and foals
(201). Fetuses maintained normal phosphorus levels despite
hypophosphatemia in pregnant rats and mice (207, 395).
Conversely, hyperphosphatemia in pregnant rats and sheep
caused fetal hyperphosphatemia (41, 199, 206, 208).
Serum magnesium also appears to be maintained independent of the maternal value, but variable results have been
seen in animal models, including slightly increased (42, 100,
330, 435) and decreased (201, 377) concentrations compared with the maternal value in fetal lambs, modest increases (202) or no change (201) in fetal rats, a modestly
increased level in foals (201), and small but statistically
significant increases in fetal mice (430, 623). Maternal hypermagnesemia did not alter the fetal magnesium level in
fetal lambs or rats (42, 200), and maternal hypomagnesemia did not alter the fetal magnesium concentration in
fetal lambs (42).
2. Human data
Human fetuses share the finding of an elevated serum calcium and ionized calcium, typically 0.30 – 0.50 mM above
the maternal level (143, 148, 463, 523, 594, 595). This has
1148
been demonstrated at 15–20 wk of gestation by fetoscopy
(453), and at delivery of preterm singleton and twin pregnancies (mean gestational age 33 wk) (152).
Serum phosphorus is typically ⬃0.5 mM higher in fetal than
maternal blood (143, 523, 544, 594). Since calcium and
phosphorus are each raised above adult normal values, fetal
blood has a high calcium x phosphorus product (Ca x P)
that may facilitate spontaneous calcium-phosphate crystals
to form and cause soft tissue calcifications. In adults, a Ca x
P value ⬎5.6 mmol2/l2 or 70 mg2/dl2 is associated with
calciphylaxis, coronary artery calcifications, and increased
mortality (58, 115, 126, 724). However, this level may be
needed to mineralize the fetal skeleton and may be nonhazardous in utero due to its relatively short duration.
Serum magnesium is also set independently of the mother’s
concentration but is typically raised only 0.05 mM higher
than the maternal value (60, 143, 523, 544, 594).
3. Summary of key points
Serum calcium, ionized calcium, and phosphorus concentrations are set at significantly higher concentration in fetuses than in the mother and can generally be maintained
despite abnormal concentrations in the maternal circulation. Putative calcium-sensing receptors (other than the
known CaSR) may set the fetal or placental calcium concentration at its high target value. This “fetal hypercalce-
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FETAL AND NEONATAL MINERAL METABOLISM
mia” is not necessary for fetal viability, but may reduce the
occurrence of neonatal hypocalcemia, and is required to
mineralize the fetal skeleton.
B. Calciotropic and Phosphotropic Hormone
Concentrations
1. PTH
A) ANIMAL DATA.
PTH circulates at lower levels in the fetal
circulation compared with the maternal value in rodents,
lambs, and calves (118, 623, 675, 685, 715). These low
values likely derive from the fetal sources because intact
PTH does not cross the placenta of non-human primates,
sheep, and rodents (196, 205, 341, 345, 478). The fetal
parathyroids begin to express PTH by mid-gestation in rats
and lambs (390, 602), and must be its dominant source
because genetic ablation of the parathyroids (in Hoxa3 null
fetal mice) results in undetectable serum PTH (336, 341).
An additional source of PTH is the placenta, which in the
mouse has been shown to express the Pth gene and may
contribute a small amount of PTH to the circulation (623).
The normally low concentration of PTH in fetal blood remains important for maintaining the blood calcium concentration because fetal mice lacking PTH or the PTH/PTHrP
receptor are each hypocalcemic compared with their WT
littermates (340, 341, 623).
Why is PTH suppressed in fetal blood? As noted above, the
high concentrations of total and ionized calcium in fetal
blood likely activate CaSR on fetal parathyroids to suppress
PTH synthesis and release (FIGURE 3). Several lines of evidence from fetal mice support this conclusion. Ablation of
one or both alleles of Casr results in a progressive increase
in PTH in fetal mice and an increase in the fetal blood
calcium compared with WT fetal littermates (338). Conversely, in the absence of PTHrP (Pthrp null fetuses), the
circulating PTH level rises but the fetal blood calcium is
maintained at a level equal to the maternal value, rather
A
Casr+/- mothers
B
100
80
80
PTH (pg/ml)
PTH (pg/ml)
100
40
20
60
p<0.02
40
20
0
0
WT
(4)
Casr+/- Casr-/(10)
(4)
The low circulating level of PTH does not mean that the
fetal parathyroids are unable to produce greater amounts of
PTH. PTH increases not only with ablation of one or both
alleles of Casr (336, 340), but in response to EDTA-induced
hypocalcemia in fetal lambs (631), calves (715), and rhesus
monkeys (524). However, a second study in rhesus monkeys found no increase in fetal PTH in response to EDTAinduced hypocalcemia (189).
Active placental transport of calcium may contribute to the
suppression of PTH by bringing calcium into the circulation
through a route that does not require PTH. Furthermore,
maternal hypercalcemia seems to enhance the forward flow
of calcium to the fetus and further suppress the fetal PTH
level, even if the fetal serum calcium does not change. For
example, when wild-type (WT) and Casr⫹/⫺ sister females
are mated to the same Casr⫹/⫺ males, the fetal PTH concentration is suppressed in offspring of hypercalcemic
Casr⫹/⫺ females compared with their genetic counterparts
from normocalcemic WT females, even though the fetal
blood calcium remains unaffected by the mother’s genotype
or serum calcium (FIGURE 5) (338). Conversely, maternal
hypocalcemia may impair the forward flow of calcium from
mother to fetus, and force the fetal-placental unit to upregulate the mechanisms that maintain the fetal blood calcium
and placental calcium transport. Consistent with this, maternal parathyroidectomy or calcitonin infusions in pregnant rats have been shown to cause a marked increase in
fetal PTH, parathyroid gland hyperplasia, and bone resorption; the skeleton is undermineralized (104, 206,
541, 542, 625).
WT mothers
p<0.001
60
than above it (336, 340). These data suggest that the fetal
parathyroids are constrained by CaSR to maintain the
blood calcium no higher than the adult level. Furthermore,
when Casr is also ablated in Pthrp null fetuses to create
Pthrp/Casr double-mutants, the fetal blood calcium rises
well above the maternal level despite the absence of PTHrP
(338).
WT
(6)
FIGURE 5. Maternal hypercalcemia suppresses fetal
PTH while fetal expression of CaSR determines the relative
fetal PTH level. Ablation of one or both copies of Casr
results in a stepwise increase in PTH concentrations in the
fetal circulation (A and B), confirming the ability of CaSR to
regulate PTH release from the fetal parathyroids. However,
fetuses obtained from hypercalcemic Casr⫹/⫺ mothers (A)
have lower PTH levels than in fetuses of the corresponding
genotype obtained from normocalcemic WT mothers (B).
Since maternal PTH cannot cross the placenta, the suppression of fetal PTH must be mediated by maternal serum
calcium. The number of observations for each genotype is
indicated in parentheses. [From Kovacs et al. (338). Copyright 1998 American Society for Clinical Investigation.]
Casr+/(6)
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CHRISTOPHER S. KOVACS
B) HUMAN DATA. In human babies, PTH also circulates at low
levels compared with the maternal circulation and normal
adult values; very low (⬍0.5 pM) concentrations may be
observed (20, 143, 168, 177, 255, 306, 501, 544, 571, 580,
598 – 600, 671, 736). This suppression of PTH has been
observed as early as 19 wk of gestation in preterm babies
(501, 571, 580, 599), although one study found unsuppressed PTH values in cord blood of infants aged 31–36 wk
(152). The suppression of fetal PTH is considerable when it
is realized that the maternal PTH value is also usually suppressed during pregnancy compared with normal adult values, at least in women from North America and Europe
(328, 339). The parathyroids produce immunoreactive
PTH beginning at 10 wk of gestation (367); whether the
human placenta produces PTH has not been determined.
Maternal hypercalcemia can result in a normal or slightly
increased cord blood calcium (131, 278, 610, 700) and
suppression of the fetal parathyroids, which will later be
realized as neonatal hypoparathyroidism, hypocalcemia,
and tetany (see sect. XII, A, B, and G). These findings have
been observed in babies born of mothers whose hypercalcemia during pregnancy was caused by primary hyperparathyroidism (79, 149, 304, 393, 609, 718), inactivating mutations of Casr (529), or hypercalcemia of malignancy (9,
131, 278, 454, 529, 673, 674, 700). Conversely, maternal
hypocalcemia caused by hypoparathyroidism (11, 76, 389,
586, 652, 710) or pseudohypoparathyroidism (226, 710)
has been associated with fetal parathyroid gland hyperplasia, normal cord blood calcium, increased PTH, and effects
on the fetal skeleton that include increased resorption, demineralization, and fractures occurring in utero or during
delivery (see sect. XII, C and D).
2. Calcitriol
A) ANIMAL DATA.
Rodents and lambs have been most frequently used to study fetal mineral homeostasis, and in the
data discussed thus far, they have yielded similar results.
However, with respect to vitamin D physiology, there are
some differences between fetal rodents and lambs. This may
be in part due to the differences in placental structure and
function that are discussed in section IIF2; in brief, rodents
have hemochorial placentas (with similar structure and diffusional characteristics as human placentas), while lambs
have epitheliochorial placentas (536).
Calcitriol typically circulates at ⬍50% of the maternal
value in fetal rodents (309, 342, 369, 709) and pigs (352,
353); in contrast, calcitriol significantly exceeds the maternal value in fetal lambs (4, 101, 155, 377, 505, 565). Radiolabeled calcitriol does not cross the rat placenta (475),
which means that the low concentrations of calcitriol must
be synthesized within the fetus or placenta. Placentas of
lambs differ in that radiolabeled calcitriol crosses in both
directions (155), and a high metabolic clearance rate of
calcitriol within fetal lambs indicates that calcitriol produc-
1150
tion is markedly upregulated compared with their ewes
(566). Maternal nephrectomy in rats did not alter fetal calcitriol levels, confirming that these metabolites are independently synthesized in the fetal-placental unit (369). Fetal
kidneys and placenta each express the 1␣-hydroxylase
(Cyp27b1), which converts 25-hydroxyvitamin D to calcitriol (234, 663, 729). Placental trophoblasts, yolk sac, and
decidua also express 24-hydroxylase (Cyp24a1), which catabolizes 25-hydroxyvitamin D and calcitriol into inactive
forms (30, 135, 728).
The fetal kidneys contribute a significant amount of calcitriol to the fetal circulation because fetal nephrectomy
reduced the fetal calcitriol level by 60% in fetal lambs
(564). This may mean that ⬃40% of calcitriol in the fetal
circulation is produced by the placenta, at least in lambs.
Although the data from lambs indicate that calcitriol can
cross the placenta from the feta to maternal circulations
(155), comparatively little calcitriol produced by the placenta may reach the maternal circulation in rodents. This
conclusion is supported by the finding that maternal kidneys in pregnant mice have 35-fold higher expression of
Cyp27b1 than in the placentas (310).
25-Hydroxyvitamin D readily crosses the rat placenta; consequently, the low level of calcitriol cannot be due to inadequate supply of its precursor (240). It is likely that renal
1␣-hydroxylase expressed by fetal kidneys is suppressed by
the high ionized calcium, high phosphorus, and low PTH
concentrations typically observed in fetal blood. The 1␣hydroxylase is capable of responding to PTH in utero because Casr⫹/⫺ and Casr null fetuses demonstrated a stepwise increase in both PTH and calcitriol levels (338), but the
calcitriol levels of Casr null fetuses remained lower than in
adult WT females (338, 342). Calcitriol levels did reach the
adult normal range in Vdr null fetuses which lacked the
vitamin D receptor (VDR) (192). As noted below, PTHrP
circulates at high levels in fetal blood and through its mimicry of PTH action on the PTH/PTHrP receptor it might
stimulate high fetal calcitriol concentrations. However, systematic studies of PTH1R binding and signaling induced by
NH2-terminal PTH and PTHrP, comparison of their crystal
structures, and clinical studies of the effect of NH2-terminal
PTH and PTHrP infusions, suggest that PTHrP is much less
potent than PTH at stimulating the 1␣-hydroxylase (195,
269, 270, 740; reviewed in detail in Ref. 333).
FGF23 conceivably contributes to low calcitriol levels because it inhibits 1␣-hydroxylase and stimulates the catabolic enzyme 24-hydroxylase. However, its effect during
fetal development appears relatively insignificant because
Fgf23 null fetuses had no change in serum calcitriol or the
renal and placental expression of Cyp27b1 (395); however,
Fgf23 null fetuses did have modest but significantly reduced
renal expression of Cyp24a1 (395). Conversely, 11-fold
increased FGF23 in Phex null male fetuses did cause a small
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FETAL AND NEONATAL MINERAL METABOLISM
but statistically significant decrease in serum calcitriol, and
this was accompanied by significantly increased expression
of Cyp24a1 in placenta and fetal kidneys but no change in
the expression of Cyp27b1 (395).
As has been demonstrated in the fetal rat, low serum calcitriol in WT fetuses may also be due to preferential catabolism of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin
D, instead of synthesis of calcitriol from 25-hydroxyvitamin D (369, 475). This results in fetal levels of 24-hydroxylated forms that are up to 40-fold higher than calcitriol in
fetal rats and sheep (369, 475, 506). The high expression of
Cyp24a1 in murine placentas is approximately equal to that
of the maternal kidneys (310), confirming that the placenta
has significant capacity to catabolize 25-hydroxyvitamin D
and calcitriol into 24-hydroxylated forms.
B) HUMAN DATA.
The human data are largely consistent with
the rodent data mentioned above, and differ from data obtained from fetal lambs. Calcitriol circulates at low levels in
fetal blood, typically ⬍50% of the maternal value (190,
264, 599, 642, 736). Umbilical artery values are higher than
those obtained from the umbilical vein (736), which suggests that it is the fetal kidneys and not the placenta that
contribute much calcitriol to the fetal circulation. As in the
animal models, 25-hydroxyvitamin D readily crosses the
placenta (254) and achieves cord blood levels that are typically 75–100% of the maternal value at term with a fetalmaternal correlation coefficient of 0.8 – 0.9 (190, 264, 599,
713, 736). Calcitriol synthesis in the fetus is likely suppressed by the high serum calcium, high phosphorus, and
low PTH concentrations typically observed in cord blood.
Human trophoblasts and maternal decidua express 1␣-hydroxylase and convert 25-hydroxyvitamin D to calcitriol
(156, 728, 758). Trophoblasts, yolk sac, and decidua also
express 24-hydroxylase (30, 728). In one study of human
placentas, CYP27B1 mRNA expression was higher than in
adjacent decidua while CYP24A1 mRNA was lower (176),
and there is evidence that CYP24A1 is methylated in human
placenta (479). These findings have prompted other researchers to conclude that trophoblast synthesis of calcitriol
is unopposed or “unfettered” by catabolism into 24-hydroxylated forms (383, 479). However, the available functional data suggest the opposite conclusion, that human
placentas preferentially metabolize 25-hydroxyvitamin D
to 24,25-dihydroxyvitamin D instead of calcitriol (572),
resulting in up to 40-fold higher concentrations of 24-hydroxylated forms compared with calcitriol in cord blood
(152, 253). These findings are the same as in fetal rodents,
as noted above.
Maternal calcitriol increases two- to threefold during pregnancy, and the placenta has often been assumed to be the
source of this increase. However, the placenta contributes
little or no calcitriol to the maternal circulation, as revealed
by an anephric woman on dialysis whose serum calcitriol
remained low before, during, and after a pregnancy (698).
Instead, it is the maternal kidneys that upregulate the synthesis of calcitriol during pregnancy. This is also similar to
the rodent data mentioned earlier.
3. PTHrP
A) ANIMAL DATA. Cytochemical assays have revealed that fetal
blood contains high PTH-like bioactivity, which cannot be
accounted for by the low immunoreactive levels of PTH (3,
20, 67). Following the discovery of PTHrP and the development of specific radioimmunoassays for it, studies in fetal
pigs (7) and sheep (94, 396) determined that high immunoreactive levels of PTHrP correlated closely with, and likely
accounted for, the high PTH-like bioactivity in fetal blood.
PTHrP has a full length of PTHrP1–139 in rodents compared
with splice variants PTHrP1–139, PTHrP1–141, or PTHrP1–173
in humans. But PTHrP is a prohormone that is processed
into several circulating peptides. In the human these include
PTHrP1–36, PTHrP38 –94, PTHrP38 –101, PTHrP107–139, and
possibly PTHrP141–173, each of which may have its own
receptor and distinct role (333, 490, 517, 745). The structures of these peptides were originally determined through
study of tumor cell lines transfected with the human PTHrP
gene. Which of these PTHrP peptides circulate in fetal life of
animals or humans has not been determined. Most available assays have been designed to detect fragments that
contain PTHrP1–34 or PTHrP1– 86. Notably, a PTHrP1–34
assay used in rodents or lambs will detect NH2-terminal
PTHrP1–36 and full-length PTHrP1–139, whereas the
PTHrP1– 86 assay will only detect full-length PTHrP1–139, a
form that may not be abundant due to rapid cleavage into
PTHrP1–36.
Immunoreactive forms of PTHrP detected by a PTHrP1– 86
assay did not cross the placentas of sheep and goats (537),
but whether smaller fragments can cross the placenta has
not been determined. Full-length PTHrP1–139 has about
twice the molecular weight of PTH, and since PTH does not
cross the placenta (196, 205, 341, 345, 478), full-length
PTHrP is also unlikely to cross.
Numerous tissue sources may contribute to the circulating
concentration of PTHrP, because in the embryo and fetus
the gene is expressed by skeletal growth plates (299, 365),
cardiac and vascular smooth muscle (89), trophoblasts (5,
336, 602), intraplacental yolk sac (336), amnion (68, 184),
chorion (68), umbilical cord (185), and numerous other
tissues and cell types. Venous umbilical PTHrP levels are
higher than umbilical arterial levels in pigs, which suggests
that the placenta may be an important source of circulating
PTHrP in the fetus (7). As discussed in section IIC, the fetal
parathyroids were originally proposed to be the source of
PTHrP in fetal sheep, but the effect of fetal parathyroidectomy on the plasma PTHrP concentration was not reported.
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CHRISTOPHER S. KOVACS
Loss of fetal parathyroids did not alter the plasma
PTHrP1–34 concentration in Hoxa3 null and Gcm2 null
fetuses (623). Pth1r null fetuses, which lack the PTH1R,
have an 11-fold increase in PTHrP1–34 and increased placental expression of PTHrP mRNA and protein (341).
These findings are compatible with the placenta being the
dominant source of PTHrP in the circulation of fetal rodents. Whether fetal parathyroid glands in rodents produce
PTHrP remains unresolved and is discussed in section IIC.
B) HUMAN DATA.
As with animal fetuses, prior to the discovery of PTHrP it was recognized that human cord blood
contained low immunoreactive intact PTH concentrations
but high levels of PTH-like bioactivity as measured in cytochemical bioassays (20, 21, 571). The discrepancy is now
explained by high circulating levels of PTHrP (600, 671),
which mimics many of the actions of PTH on the PTH1R.
The concentrations of PTH and PTHrP are often reported
in unrelated units, but when expressed in equivalent units
(pM), human cord blood PTHrP levels are 2– 4 pM and up
to 15-fold higher than the simultaneous levels of PTH (0.2–
0.5 pM) (168, 306, 671).
A few studies have provided conflicting data by finding
undetectable levels of PTHrP1– 86 in cord blood (592) or
very low levels of PTHrP1– 86 that were no different among
pregnant women, fetal samples obtained by cordocentesis,
and cord blood samples (501). These results likely reflect
several problems that are commonly found among clinical
studies that included PTHrP measurements. First, PTHrP is
subject to rapid cleavage and degradation in serum; consequently, blood samples are optimally collected as EDTA
plasma with aprotinin (a protease inhibitor), kept chilled on
ice, and promptly (within 15 min) centrifuged, separated,
and frozen. Even when optimally collected as EDTA-aprotinin plasma and kept chilled on ice, PTHrP begins to degrade within 15 min of sample collection (275). Many studies did not approach this rigor of sample collection and
preparation, and it is common to see use of stored sera that
had been allowed to clot at room temperature for up to 60
min (678). All of these factors will result in failure to detect
the full concentration of PTHrP that was present in the
original blood sample, and may even result in undetectable
PTHrP when it should be present. Second, the most commonly used assays in clinical studies have been those that
purportedly measure PTHrP1– 86. This form is not predicted
to exist in humans (333), but the assay should detect fulllength forms PTHrP1–139, PTHrP1–141, or PTHrP1–173 if
they are present in the circulation. However, it will not
detect PTHrP1–36, which is conceivably the more abundant
NH2-terminal form of PTHrP because it derives from all
full-length forms. Conversely, a PTHrP1–34 assay should
detect each of PTHrP1–36, PTHrP1–139, PTHrP1–141, and
PTHrP1–173. Standardization of sample requirements and
PTHrP assays are sorely needed; in the meantime, it is preferable to use a PTHrP1–34 assay and to use only plasma that
1152
has been rigorously collected and processed under the optimal conditions described here.
4. FGF23
A) ANIMAL DATA. FGF23, produced largely by osteocytes and
osteoblasts, is a phosphorus-regulating hormone that controls the supply of phosphorus at the mineralizing surface of
bone through actions on distal tissues (622). It downregulates the expression of sodium-phosphate cotransporters 2a
and 2c (NaPi2a and NaPi2c) in proximal renal tubules
(614). It also inhibits 1␣-hydroxylase (Cyp27b1), increases
expression of 24-hydroyxlase (Cyp24a1), and inhibits intestinal expression of NaPi2b (614). In adults, increased
effective circulating levels of FGF23 lead to hypophosphatemia and renal phosphate wasting, while loss of FGF23
causes hyperphosphatemia and impaired renal phosphate
excretion (52).
FGF23 is predominantly expressed in rat fetal osteoblasts,
in addition to thymus, liver, and kidney (754). Murine fetuses express FGF23 as early as embryonic day 12.5 in
heart, liver, and somites; it later appears in bone (627).
Using the sensitive method of real-time quantitative RTPCR, comparative study of WT, Fgf23 null, and Phex null
placentas revealed that the murine placenta expresses a low
level of Fgf23 mRNA, which is absent in Fgf23 null placentas but increased in Phex null placentas (395). An independent study used the less sensitive method of conventional
RT-PCR and did not find visible Fgf23 amplification on an
agarose gel (484).
The concentration of intact FGF23 in fetal blood from WT
fetuses was equal to the simultaneous maternal level in mice
from the outbred Black Swiss strain for several mouse models (395), whereas the level was about half the maternal
value in WT fetuses from the inbred C57BL/6 strain (194,
395). The maternal value in turn was approximately double
the nonpregnant level as revealed through longitudinal
studies in both strains of mice (194). The discrepancy in the
fetal FGF23 blood levels between the Black Swiss and
C57BL/6 mice, within the same laboratory and using the
same intact FGF23 assay, indicates that the genetic background of the mice influences fetal FGF23 physiology. Phex
null male fetuses and Phex⫹/⫺ female fetuses had markedly
increased intact FGF23 concentrations that equaled the simultaneous maternal concentration from their Phex⫹/⫺
mothers (395), whereas in another study the FGF23 level in
Phex null male fetuses far exceeded the maternal concentration (484).
Although no formal studies have been done with radiolabeled FGF23, it evidently does not cross the murine placenta because Fgf23 null fetuses had undetectable FGF23
despite high levels present in the maternal circulation (395).
Furthermore, the high FGF23 concentrations observed in
Phex⫹/⫺ mothers did not alter the circulating FGF23 level
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FETAL AND NEONATAL MINERAL METABOLISM
in their WT fetuses compared with WT fetuses born of
Fgf23⫹/⫺ mothers (395).
6. Sex steroids
A) ANIMAL DATA. The dominant sex steroids, testosterone and
B) HUMAN DATA. Scant data are available pertaining to FGF23
in normal human fetuses. In two studies, intact FGF23 levels in cord blood were 0.15- to 0.30-fold the values obtained from unrelated healthy adults or their mothers (483,
661). But COOH-terminal FGF23 was approximately twofold the values of unrelated healthy adults (661). These
divergent results may indicate that FGF23 is abundantly
produced but rapidly cleaved in utero, thereby resulting in
elevated levels of the nonfunctional COOH-terminal fragment. Cord blood levels of the FGF23 coreceptor Klotho
were sixfold adult and neonatal values and may contribute
to a reduction in the circulating intact FGF23 level (483).
Human trophoblasts also express Klotho (483). Use of conventional RT-PCR did not reveal visible evidence of FGF23
expression in human trophoblasts (484).
5. Calcitonin
A) ANIMAL DATA.
Thyroid C-cells and calcitonin are detectable as early as the 30th day of an approximate 145-day
gestation in fetal lambs (292), whereas in fetal mice and rats
C-cells and calcitonin do not appear until day 15.5 or later
out of a gestational period that lasts 19 –22 days, respectively (22, 203, 284, 474, 645, 679). Near term, fetal calcitonin concentrations are maintained at higher concentrations than in the maternal circulation in rodents (201, 430)
and lambs (201, 204). Calcitonin within the fetal circulation must derive from fetal sources because maternal calcitonin does not cross the rat or mouse placenta (212, 430).
Rat and mouse placentas also express calcitonin mRNA
and protein (284, 293, 337). The high circulating concentration of calcitonin may occur in response to the increased
serum and ionized calcium that is normally present in the
fetal circulation. Consistent with this, experimentally induced hypercalcemia in fetal pigs, lambs, calves, and nonhuman primates caused a further, marked increase in calcitonin, the magnitude of which correlated with the change in
serum calcium (101, 380, 548). The relative contribution of
thyroid and placenta to circulating calcitonin has not been
determined. The fetal thyroid is a significant source because
infusion of calcium chloride into the left thyroid artery of
fetal lambs caused a 2- to 10-fold increase in calcitonin
secretion (215).
B) HUMAN DATA. The thyroid C-cells differentiate around the
12th week of gestation (107), and calcitonin is detectable in
those cells from as early as the 15th week of gestation (368).
Serum calcitonin is increased to as much as twice the maternal level in both term and preterm babies (255, 343, 523,
583, 599, 736, 742). Trophoblasts of the placenta also express calcitonin and may, therefore, contribute to the
amount in the fetal circulation (35).
estradiol, are considered as calciotropic hormones within
this review because of their importance in regulating postnatal skeletal development and bone turnover. Testosterone in male fetal rats surges over the last several days of
gestation before declining on the last day (720, 730). It
exceeds the concentration in female fetuses (239, 272, 720,
730) and was higher than the maternal value in three studies
(239, 720, 730), and equal to maternal in another (272).
Testosterone was also higher in male compared with female
fetal lambs (18). Conversely estradiol concentrations are
similar between male and female rat fetuses (239), and exceed the maternal concentration (146, 239). In fetal lambs,
testosterone and estradiol exceeded the maternal concentration at all time points (117, 650, 757).
B) HUMAN DATA.
Human pituitary cells produce luteinizing
hormone and follicle stimulating hormone from as early as
5 wk of gestation (619, 620), and the levels of both gonadotropins are higher in female fetuses throughout gestation
(676). The gonadotropins and placenta-derived human
chorionic gonadotropin drive the Leydig cells within the
developing male testis to produce testosterone (29),
whereas only negligible amounts of testosterone are produced in the ovaries or in the adrenals of both sexes (546).
The ovaries, testes, and adrenals produce little or no estradiol until late in fetal life (91, 546). In male fetuses, testosterone exhibits higher concentrations between 11–17 wk of
fetal age before subsequently declining to trough values
(545, 739); at term, the concentration is modestly higher or
no different than in female fetuses (301, 458, 509, 545, 682,
739). Estradiol levels are also similar between male and
female fetuses (250, 509, 545, 682, 739). It is generally
considered that in fetal humans estradiol and testosterone
are present at low levels in cord blood near term compared
with normal adult values (133, 676). Indeed, in most studies
the fetal levels of estradiol and testosterone were much
lower than simultaneous maternal concentrations at term
(15, 179, 216, 458, 465, 509, 681, 682, 696). However, in
a few studies either or both of testosterone and estradiol
have been approximately equal to (250, 545) or significantly greater than (14, 357, 739) the maternal concentrations.
These discrepancies in relative fetal-to-maternal sex steroid
levels are likely the result of whether the umbilical vein,
artery, or mixed cord blood was sampled. By the third trimester, the placenta accounts for nearly all of the estrogens
(estradiol, estrone, and estriol) in the maternal circulation
(437), and it synthesizes them through aromatization of
androgens that arise about equally from the fetal and maternal adrenals (382, 618). The concentrations of these estrogens progressively increase during pregnancy and reach
peak values in the maternal circulation during the third
trimester (382, 480, 697). If the umbilical vein or mixed
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CHRISTOPHER S. KOVACS
cord blood are sampled, the levels will indicate high concentrations due to placental synthesis of sex steroids,
whereas umbilical artery values reflect systemic levels
within the fetus. Several studies have found that the umbilical artery estradiol or conjugated estrogen levels are significantly lower than the umbilical vein values (589, 617,
696). Such findings confirm that placental production of sex
steroids confounds the cord blood measurements and that
the umbilical artery values alone should be used to indicate
systemic fetal concentrations. In the previously cited studies
that reported elevated sex steroid levels in cord blood, it
was mixed cord blood that had been sampled in two of
them (14, 739), whereas the source was not specified in the
third study (357).
7. Summary of key points
The human fetal circulation is characterized by low concentrations of PTH, calcitriol, and the sex steroids as well as
high levels of PTHrP and calcitonin (FIGURE 2). Parathyroid
CaSR likely suppresses PTH in response to the high fetal
blood calcium. 25-Hydroxyvitamin D readily crosses the
placenta whereas calcitriol does not. The low fetal calcitriol
levels are likely due to suppression of the fetal renal 1␣hydroxylase (CYP27b1) by low PTH and high serum calcium and phosphorus, and increased activity of the catabolic enzyme 24-hydroxylase.
C. Fetal Parathyroids
1. Animal data
In rodents, the thymus and a single pair of parathyroids
derive from the third pharyngeal pouch (56, 384). Parathyroids are friable such that fragments detach during embryonic migration, which likely accounts for parathyroid tissue
within the thymus and adjacent tissues (232, 384, 385, 657,
705). Gcm2 is intensely expressed by the primordium derived from the third pharyngeal pouch in mice; therefore,
deletion of Gcm2 should ablate the parathyroids and result
in undetectable PTH, as occurs when Hoxa3 is deleted
(232, 336, 341, 411). Consistent with this, the normal expression of PTH mRNA and protein in the common parathyroid/thymus primordium on embryonic day 11.5–12.5
is absent in Gcm2 null fetuses (385). However, a low level
of PTH has been measured in the circulation of Gcm2 null
fetuses even though the parathyroids (and parathyroid remnants in the thymus) have been eliminated (623). Pth is also
expressed in normal placenta, and its expression there is
upregulated in Gcm2 null fetuses compared with placentas
from WT littermates (623), and so placental PTH may explain the low but detectable PTH blood levels in Gcm2 null
fetuses. Adult Gcm2 null mice were originally reported to
have normal circulating levels of PTH, a finding that is not
easily explainable unless Gcm2-independent sources of
PTH activate in the absence of parathyroids (238).
1154
PTH mRNA and protein can also be detected by mid-gestation in the parathyroids of fetal rats and lambs (390, 602).
It has been suggested that fetal parathyroids secrete both
PTH and PTHrP. However, the evidence that fetal parathyroids express PTHrP mRNA and protein is inconsistent and
inconclusive. PTHrP was first detected in parathyroids of
fetal lambs by immunocytochemical methods with older
polyclonal antibodies that did not cross react with PTH (5,
390, 396). These studies have not been repeated with newer
and more specific monoclonal antibodies, nor has PTHrP
mRNA expression been examined in parathyroids from fetal lambs. RT-PCR and in situ hybridization failed to detect
PTHrP mRNA in fetal or adult rat parathyroids (601, 695),
whereas PTHrP mRNA was detected by RT-PCR in a third
study of adult rat parathyroids (274). The more sensitive
technique of real time quantitative RT-PCR has not been
used on fetal parathyroids from rats or lambs, while murine
fetal parathyroids have not been directly examined at all for
their ability to express PTHrP.
If the fetal parathyroids are a significant source of PTHrP in
the fetal circulation, then fetal parathyroidectomy or genetic ablation of the parathyroids should reduce the circulating PTHrP level. However, the plasma PTHrP level was
not measured in any of the studies in which fetal lambs and
rats were parathyroidectomized. Instead, the parathyroid
secretion of PTHrP was inferred from the finding that parathyroidectomy in fetal lambs caused a reduction in placental calcium transport that could be rescued by infusion of
autologous blood or PTHrP, but not PTH (discussed in
more detail in sects. IVB and VB). In contrast, genetic ablation of parathyroids in Hoxa3 null and Gcm2 null fetuses
did not alter the plasma PTHrP level (341, 623), while an
11-fold upregulation of plasma PTHrP in Pth1r null mice
was associated with upregulation of PTHrP mRNA and
protein expression in the placenta but not in the region of
the neck that contains the parathyroids (341). These findings in rats and mice suggest that the parathyroids are unlikely to be a significant source of PTHrP in the fetal circulation, whereas the data from fetal lambs remain consistent
with parathyroid-localized expression of PTHrP.
Despite a low level of PTH in the fetal circulation, the
evidence is consistent across all animal models that fetal
parathyroids are required to maintain the normally high
level of blood calcium. Loss of parathyroids from any cause
results in fetal hypocalcemia, as demonstrated in fetal lambs
(thyroparathyroidectomy followed by replacement of thyroxine) (1, 2), rats (thyroparathyroidectomy or decapitation) (520, 521, 554), and mice (loss of parathyroids
through genetic ablation of Hoxa3 or Gcm2, or loss of PTH
alone in Pth null fetuses) (336, 341, 623).
PTH and PTHrP both participate in the normal regulation
of the fetal blood calcium, such that loss of either hormone
results in fetal hypocalcemia, and loss of both leads to the
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FETAL AND NEONATAL MINERAL METABOLISM
greatest decrease in blood calcium (336, 340, 341, 623).
This is discussed in more detail in section VIA.
Fetal parathyroids are also required to regulate magnesium
and phosphorus, since thyroparathyroidectomized fetal
lambs, and Hoxa3 null, Gcm2 null, and Pth null fetal mice
have hypomagnesemia and hyperphosphatemia (47, 341,
397, 398, 623). Loss of PTHrP itself causes hyperphosphatemia with a normal serum magnesium concentration
(341, and unpublished data).
In sections IVB and VB, the evidence that the parathyroids
regulate placental calcium transport is discussed.
2. Human data
In humans, parathyroids and thymus share a common developmental origin within the third pharyngeal pouch,
while a second pair of parathyroids originate from the
fourth pouch (56, 222, 518, 669). As in the animal models,
the friable nature of developing parathyroids leads to thymic and other ectopic locations for fragments that detach
during embryonic migration.
PTH is detectable by immunochemical methods within
parathyroids beginning at 10 wk of gestation (367). Expression of PTHrP has not been sought in human fetal parathyroids, but PTHrP mRNA and protein have been detected in
the oxyphil cells of adult normal, hyperplastic, and adenomatous parathyroids by a variety of methods (28, 137, 157,
277, 313, 314, 424, 552). Since adult parathyroids express
PTHrP, it is conceivable that the fetal parathyroids do as
well.
3. Summary of key points
Fetal parathyroids produce low amounts of PTH, but
whether or not they produce PTHrP is unclear. The evidence is stronger for PTHrP production by parathyroids of
fetal lambs than it is for fetal rodents or humans; species
differences may explain these discrepancies.
D. Renal Mineral Reabsorption, Excretion,
and the Amniotic Fluid
1. Animal data
In the adult, the kidneys play key roles in the regulation of
mineral homeostasis: 1) by adjusting the relative reabsorption and excretion of minerals in response to PTH, calcitriol, calcitonin, FGF23, and CaSR; and 2) as the dominant site of expression of 1␣-hydroxylase and synthesis of
calcitriol.
The fetal kidneys may be of lesser importance because the
placenta assumes control over mineral handling and synthe-
sizes calcitriol. Another reason is that excreted mineral is
not lost to the organism, because urine forms a large component of the amniotic fluid which is swallowed (autourophagia), absorbed, and thereby recycled (563). Renal
blood flow and glomerular filtration rate are also much
lower during fetal development compared with after birth
(237, 378, 605). Nevertheless, alterations in serum mineral
concentrations and renal excretion of minerals have been
observed in response to alterations in kidney function, or
from manipulations in the factors that normally control
renal tubular function.
In fetal lambs, bilateral nephrectomy led to hypocalcemia,
hyperphosphatemia, increased PTH, and a 60% fall in the
circulating level of calcitriol (455, 564). Additional fetal
lambs underwent a control procedure that consisted of bilateral ureteral sectioning to permit fetal urine to drain into
the fetal peritoneal cavity while retaining functional kidneys in situ. Ureteral sectioning had no effect on fetal calcium or calcitropic hormone levels (455). The investigators
concluded that loss of renal 1␣-hydroxylase and production
of calcitriol resulted in altered fetal mineral homeostasis,
rather than the loss of renal function to reabsorb and excrete minerals. However, other investigators performed bilateral nephrectomy in fetal lambs and observed an increase
in serum phosphorus, no change in serum calcium, and a
nonsignificant reduction in the excretion of calcium into the
amniotic fluid and allantois (459). Bilateral nephrectomy in
fetal rats did not alter the serum calcium or phosphorus
either; calcitriol and PTH were not measured in those experiments (105).
With respect to hormonal control of renal mineral handling, thyroparathyroidectomy in fetal lambs caused hypocalcemia, hyperphosphatemia, increased renal fractional
excretion of calcium, and reduced renal phosphorus excretion (398). Pharmacological administration of NH2-terminal fragments of PTH or PTHrP increased serum calcium
and reduced renal calcium excretion, but did not alter serum phosphorus or urine phosphorus excretion (142, 398).
These results may indicate that thyroparathyroidectomy
causes hypocalcemia in part through loss of the effects of
parathyroid-derived PTH and PTHrP on the renal tubules.
However, in contrast to fetal lambs, absence of parathyroids in Hoxa3 null and Gcm2 null fetal mice, loss of
PTH1R in Pth1r null fetuses, and absence of PTH in Pth
null fetuses, each caused hypocalcemia, hyperphosphatemia, and reduced amniotic fluid calcium, which is a
measure of renal calcium excretion (341, 623; and unpublished data). The low blood calcium in these murine fetuses
must cause a reduced renal filtered load of calcium, which in
turn may explain reduced excretion of calcium into the
amniotic fluid. Conversely, fetal mice with high blood calcium have increased excretion of calcium into the amniotic
fluid, which is likely the result of an increased renal filtered
load of calcium (338).
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CHRISTOPHER S. KOVACS
Whether calcitriol itself plays any important role in fetal
kidney function is uncertain. Data have already been cited
about how circulating calcitriol levels are low in fetal rats
but increased in fetal sheep. As already noted, fetal nephrectomy in lambs reduced the serum calcitriol level by 60%
and was accompanied by a fall in serum calcium and an
increase in serum phosphorus in one study (455), but serum
calcium remained normal in another study of nephrectomized fetal lambs (459), and serum calcium and phosphorus were normal in nephrectomized fetal rats (105). The
investigators in the first study assumed that loss of renal
production of calcitriol caused the hypocalcemia and hyperphosphatemia and were able to show that pharmacological treatment with calcitriol reversed these abnormalities
(455). However, in Vdr null fetal mice, ionized calcium and
phosphorus, and amniotic fluid calcium and phosphorus,
were not altered compared with WT littermates (342). It
appears likely that renal production of calcitriol is relatively
unimportant for fetal calcium homeostasis in most mammals, although it is possible that the situation is different for
fetal sheep because of high circulating levels of calcitriol.
FGF23 increases renal phosphate excretion in the adult by
downregulating expression of NaPi2a and NaPi2c in the
renal tubules. Fetal kidneys show abundant expression of
the FGF23 targets Klotho, NaPi2a and NaPi2c, Cyp27b1,
Cyp24a1, and Fgfr1-Fgfr4 (395). But in Fgf23 null fetal
mice, loss of FGF23 did not alter serum phosphorus or
amniotic fluid phosphorus excretion, and there was no effect on expression of NaPi2 and NaPi2c in fetal kidneys
(395). Conversely, in Phex null male fetuses, which have
markedly increased circulating concentrations of FGF23,
there was also no effect on serum phosphorus, amniotic
fluid phosphorus excretion, and NaPi2 and NaPi2c expression in fetal kidneys (395). These results suggest that fetal
kidneys do not share the key role in renal phosphate handling or responsiveness to FGF23 that the adult kidneys
have.
2. Human data
There are no available mineral or calciotropic hormone
measurements or skeletal data from anephric human fetuses, apart from noting that affected babies can have scoliosis, rib anomalies, and absent toes, radii, or thumbs
(528). Those skeletal abnormalities may reflect the underlying genetic anomalies rather than an effect of absent kidney function. Bilateral renal agenesis presents with very low
amniotic fluid volumes (oligohydramnios or anhydramnios) due to the lack of fetal urine production (147, 528),
confirming that urine comprises much of the normal volume of amniotic fluid. In turn, the lack of amniotic fluid
causes pulmonary hypoplasia, which has been confirmed by
noting that a monoamniotic twin with bilateral renal agenesis was born with normal pulmonary function, presumably
because the twin’s kidneys provided the normal volume of
amniotic fluid (511). The absent kidney function and pul-
1156
monary hypoplasia contribute to the uniform fatality of this
condition in the newborn period.
3. Summary of key points
The fetal kidneys are a likely source of the low level of
calcitriol in the fetal circulation and are capable of excreting
and reabsorbing minerals. Whether fetal kidneys contribute
importantly to fetal mineral homeostasis is uncertain, especially since urine enters the amniotic fluid and can then be
recycled through the process of autourophagia.
E. Intestinal Mineral Absorption
1. Animal data
Adult intestines are the main route of mineral entry into the
organism, with calcium being absorbed in part through passive processes, and actively through calcitriol-dependent
mechanisms. Fetal intestines must play a minor role in mineral homeostasis because the only material available to be
swallowed and absorbed is the amniotic fluid. No studies
have examined the relative contribution of the fetal intestines to mineral homeostasis. However, data from newborns likely reflect the function of the intestines in utero.
Intestinal calcium absorption in newborn rats is largely passive, nonsaturable, and not dependent on calcitriol (218,
219, 243). The vitamin D receptor is undetectable within
enterocytes at days 7 and 14 after birth (242), which explains its lack of responsiveness to calcitriol at earlier stages
(243).
2. Human data
Although the intestines are considered a minor player in the
regulation of fetal mineral homeostasis, a significant volume of amniotic fluid (fetal urine) is normally swallowed
and absorbed. When swallowing or absorption are not possible, such as in gastrointestinal atresias, an increased volume of amniotic fluid (polyhydramnios) is present at term
(8, 141).
Intestinal calcium absorption has not been studied in human fetuses. However, a preterm baby at birth is the equivalent of a fetus of the same gestational age, and the functional capacity of a preterm neonate’s intestines may inform
about the functional capacity of the fetal intestines. Studies
in preterm neonates have shown that calcium absorption is
proportional to intake and does not reach a maximum (i.e.,
is nonsaturable); these findings suggest that passive absorption of calcium is the dominant mechanism by which calcium is absorbed in preterm fetuses (44, 221, 581). Calcium
absorption did not vary by vitamin D intake in preterm
infants, consistent with passive absorption being the dominant mechanism of absorption rather than active, calcitrioldependent mechanisms as seen in older infants and children
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FETAL AND NEONATAL MINERAL METABOLISM
(75). The calcium requirement of the preterm neonate is
very high at birth because the fetal skeleton is typically
accreting ⬃300 mg or 120 –150 mg/kg of calcium daily at
that age (761). Current guidelines recommend a daily calcium intake of 120 –200 mg/kg for preterm babies, which
exceeds what the intestines should be capable of absorbing,
so parenteral administration of calcium is needed (445).
The postnatal changes in intestinal calcium absorption are
discussed in more detail in section IIIE.
3. Summary of key points
The fetal intestines are likely a minor player in fetal mineral
homeostasis because only amniotic fluid is available to be
ingested, but their precise contribution has not been determined. Study of preterm babies have suggested that calcium
absorption in the fetal intestines is likely passive and not
regulated by calcitriol.
F. Placental Mineral Transport
This section reviews the mechanisms through which minerals are thought to be transported across the placenta, the
methods used to study placental mineral transport, and the
evidence that maternal factors influence the rate of transport. The effect of fetal sources of the calciotropic and phosphotropic hormones on placental calcium transport is discussed later within the relevant portions of sections IV–X.
1. Overview of mechanisms
A) ANIMAL DATA. Most of the calcium content of the newborn
skeleton is obtained from the maternal circulation during
the last third of gestation. In calves, 95% of the 673 g of
calcium in the fetus is accreted during the last 3 mo of a
9-mo gestation, while in fetal rats 95% of the required 12.3
mg of calcium is accreted during the last 5 days of a 22-day
gestation (121). Active placental calcium transport is necessary because diffusional flow is insufficient and the calcium exchange occurs against a substantial electrochemical
gradient (95, 198). Additional studies have confirmed that
it is an active process, dependent on Mg2⫹ and ATP (188).
It has been estimated from study of perfused sheep placentas that active transport of calcium comprises two-thirds of
the forward flow from mother to fetus (331).
Calcium transport is thought to occur via mechanisms and
pathways that are similar to calcium transport across intestinal cells. Gated calcium channels [such as transient receptor potential cation channel, subfamily V, member 6
(TRPV6)] open within maternal-facing basement membranes to allow calcium entry into the relevant cells, calcium then shuttles to the opposite basement membrane
bound to binding proteins, and then Ca2⫹-ATPase at the
fetal-facing basement membranes actively pumps calcium
into the fetal circulation (95, 659). The relevant calcium
transporting cells are the trophoblasts and probably the
intraplacental yolk sac cells within rodent placentas (63,
188, 337). The flow of calcium is not unidirectional, but the
magnitude of backflux is difficult to measure; it may be
⬍1% of the forward flow in sheep, rats, and mice (340,
507, 535, 660, 721), but 80% of the forward flow in primates (535).
The calcium binding protein calbindin-D9k transports calcium within renal and intestinal cells, and likely plays a
similar role in trophoblasts and yolk sac cells (288). It is
expressed as early as day 10 of gestation in rodent placentas
(607), and its expression increases markedly over the last
week of gestation in rats (81, 151, 227) and mice (150,
659). Other calcium binding proteins are also expressed
within trophoblasts and intraplacental yolk sac cells and
show increased expression over the same interval (251, 252,
692). Ca2⫹-ATPase expression doubles during the last 7
days of gestation (227, 693). TRPV6 expression increases
14-fold during the last 4 days of gestation in the mouse
(659). All three of these calciotropic genes are expressed by
trophoblasts, but within rodent placentas, they are most
intensely expressed within the intraplacental yolk sac, a
unique structure that also expresses other calciotropic genes
at much higher intensity than in the surrounding trophoblasts (FIGURE 6) (659).
Varying levels of evidence support the role of calbindinD9k, Ca2⫹-ATPase, and TRPV6 in placental calcium transport. Pthrp null and Trpv6 null fetuses exhibit reduced
placental calcium transport, and this is accompanied by
reduced expression of calbindin-D9k within the intraplacental yolk sac of each model (337, 659), but whether the
reduced expression caused or resulted from a lower rate of
placental calcium transfer is unknown. Deletion of calbindin-D9k does not alter serum calcium or intestinal calcium
absorption in adult mice, but fetal minerals and placental
calcium transport have not been measured (51, 351, 364).
Ca2⫹-ATPase has been shown to be key because inhibition
of its activity by dinitrophenol, ouabain, quercetin, or a
neutralizing antibody leads to a reduction in the rate of
calcium transport in perfused placentas from rats (63, 95).
Pmca1 encodes the dominant placental Ca2⫹-ATPase, but
ablation of it results in embryonic lethality (486, 530); consequently, placental calcium transport cannot be measured.
Pmca2 or Pmca4 are isoforms that are also expressed
within trophoblasts (456, 649), and the knockout mice each
have mild phenotypes, but the fetal skeleton and placenta
have not been examined (486, 530). The lack of an adult
phenotype does not preclude a significant fetal phenotype,
as seen by Trpv6 null adult mice that have no impairment of
intestinal calcium absorption (51), whereas Trpv6 null fetuses have severe hypocalcemia, a 40% reduction in placental calcium transfer, and a 50% reduction in skeletal ash
weight (659). Overall, the available data are consistent with
important roles for Ca2⫹-ATPase and TRPV6 in regulating
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CHRISTOPHER S. KOVACS
A
Giant trophoblasts
Amnion
Labyrinthine
trophoblasts
mated to be ⬃7% of the forward (maternal to fetal) flow in
lambs (660). The placenta expresses numerous phosphateregulating genes that are present in the intestines and kidneys, including the sodium-phosphate channels NaPi2a,
NaPi2b, NaPi2c; the FGF23 coreceptor Klotho, and all four
FGF receptors (395, 483, 484). A low level of FGF23 expression has also been demonstrated (395).
28
Visceral
Yolk Sac
Intraplacental
Yolk Sac
Parietal Yolk Sac Endoderm
B
Trophoblasts
Maternal Vessels
Reichert’s Membrane
Parietal
Yolk Sac
Mg has been used to study active placental magnesium
transport in lambs (47, 96), wherein the back-flow is ⬃30%
of the forward flow (100). The factors that control magnesium transport are unknown.
B) HUMAN DATA.
The human skeleton accretes more than
80% of its 30 g of calcium during the third trimester (121,
224, 683, 733, 734). Human placentas have been perfused
in vitro, and this has led to estimates that active transport of
calcium comprises about one-third of the forward flow
from mother to fetus (655). Human trophoblasts express
the same genes (TRPV6, calbindin-D9k, and Ca2⫹-ATPase)
and are expected to transport calcium in a manner similar
to rodent placentas.
C) SUMMARY OF KEY POINTS.
Sinus of Duval
Visceral Yolk Sac
Fetal Vessels
FIGURE 6. Anatomy of the intraplacental yolk sac. By the time the
mature placenta has formed (A), compressed portions of the yolk
sac line the uterine cavity (decidua) that is not in contact with the
placenta, and it overlays the dome of the placenta. The parietal layer
and Reichert’s membrane of the yolk sac are in contact with the
decidua and the dome of the placenta, while the columnar or visceral
yolk sac layer is in apposition to this layer, separated by the yolk sac
cavity. The yolk sac bilayer that overlays the dome of the placenta
forms fingerlike projections into the placenta near the insertion of
the fetal vessels (boxed area in B, the intraplacental yolk sac). The
intraplacental yolk sac is positioned between maternal and fetal
blood spaces, as visualized in detail in B. Within this structure, the
parietal layer and Reichert’s membrane overlay maternal blood
spaces and vessels, while the columnar layer overlays fetal blood
vessels. Between these layers is the sinus of Duval, which also
communicates with the yolk sac cavity and the uterine lumen. The
two layers of the intraplacental yolk sac have the most intense
expression of calciotropic genes compared with the rest of the
murine placenta and are thought to be a site of maternal-fetal
calcium exchange. [From Kovacs et al. (337).]
placental calcium transfer, while it remains unknown
whether calbindin-D9k is essential.
Fewer studies have examined placental transport of other
minerals. 32P transport across the rat placenta increases
until term, paralleling the increase in placental calcium
transport (315, 701, 702). Back-flux of 32P has been esti-
1158
Active transport of calcium is
required to meet the fetal requirement for mineral, and it is
transported through mechanisms that are similar to those
active within intestinal cells. The mechanisms governing
phosphorus and magnesium absorption are not established.
2. Placental structure and expression of calciotropic
and phosphotropic genes
A) ANIMAL DATA. The placentas of monkeys, lambs, pigs,
goats, rats, and mice have each been studied as models of
human placental structure and function, but there are significant differences among their macro- and microstructures that must be considered (174, 311, 536, 553, 575,
644).
Lambs, goats, and pigs have cotyledonary placentas, in
which the structure is organized into 60 –70 cotyledons that
spread out over the entire uterine wall. Microscopically the
placenta is epitheliochorial, in which the maternal and fetal
circulations remain separated by full thicknesses of maternal and fetal tissues (536, 575, 644). Calbindin-D9K and
Ca2⫹-ATPase expression are concentrated in the interplacentomal region (457, 472, 743), which is thought to be an
important site of calcium exchange between mother and
fetus (291).
Monkeys, rats, and mice have discoid placentas in which
the tissue is confined to a single plate (575, 644). Microscopically the structure is hemochorial, in which maternal
blood directly bathes the fetal chorion because the fetal
trophoblasts have fully invaded the maternal (uterine) tissue. Consequently, structural barriers between the maternal
and fetal circulations are significantly reduced in hemo-
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FETAL AND NEONATAL MINERAL METABOLISM
chorial compared with epitheliochorial placentas (575,
644). Rodent placentas are hemotrichorial with three layers
of trophoblasts interposed between the maternal and fetal
circulations (404, 536, 644). Maternal blood circulates in a
labyrinthine series of small channels created by the trophoblasts (129). Rodent placentas also contain the intraplacental yolk sac, a bilayered membrane formed when part of the
yolk sac becomes enfolded within the placenta; it is positioned directly between fetal and maternal blood vessels
(FIGURE 6) (166, 167, 289). This structure shows more intense expression of many calciotropic genes compared with
the surrounding trophoblasts, including calbindin-D9k,
Ca2⫹-ATPase, PTHrP, PTH1R, TRPV6, vitamin D receptor, calcitonin, calcitonin receptor, and CaSR (35, 63, 81,
83, 84, 95, 150, 151, 293, 337, 338, 356, 423, 471, 608,
656, 659). Its location between the maternal and fetal vessels is optimal for it to be a site of maternal-fetal calcium
and other nutrient transport, independent of the trophoblast labyrinth (330, 337, 659). This route may enable rodents to meet proportionately larger calcium demands created by their large litters and short gestational interval.
It has not been possible to determine the extent to which the
intraplacental yolk sac may contribute to placental calcium
transport. Platelet-derived growth factor-␣ encodes the intraplacental yolk sac, but deletion of this gene leads to embryonic lethality (482). However, Pthrp null and Trpv6 null
fetuses have reduced placental calcium transfer (340, 659),
and this is accompanied by reduced expression of calbindinD9k and Ca2⫹-ATPase within the intraplacental yolk sac of
the Pthrp null, reduced expression of calbindin-D9k within
the Trpv6 null, but normal expression of calbindin-D9k
within the trophoblasts of the Pthrp null (337, 340, 659).
The mouse placenta expresses numerous FGF23 target
genes including Klotho, NaPi2a, NaPi2b, NaPi2c,
Cyp27b1, Cyp24a1, Fgfr1, Fgfr2, Fgfr3 and Fgfr4; there
was also low-level expression of Fgf23 (395). Cellular localization of these genes has not been determined.
B) HUMAN DATA. Human placentas are discoid and hemochorial and are considered to have similar structure, function, and diffusional characteristics to placentas of mice and
rats (174, 311, 536, 553). They are hemomonochorial,
meaning that a single trophoblast cell layer separates maternal and fetal blood. Fingerlike projections (villi) protrude
into a large intervillous maternal blood-filled space. There is
no labyrinth as in the rodent placenta, nor is the intraplacental yolk sac present.
The trophoblasts express numerous calciotropic and phosphotropic genes including calbindin-D9k, Ca2⫹-ATPase
(isoforms 1 and 4), and TRPV6 (63, 95, 273), PTHrP (82,
162, 184), PTH1R (82, 183), CaSR (69), VDR (664), 1␣hydroxylase (156, 728, 758), calcitonin (35), calcitonin receptor (356, 471), and Klotho (483).
C) SUMMARY OF KEY POINTS.
The epitheliochorial placentas of
sheep are structurally and functionally different from the
hemochorial placentas of humans and rodents, but there are
also differences between human and rodent placentas. The
rodent placenta is considered a close functional match to
the human placenta. All in vivo data about placental function come from study of non-human placentas, whereas
there are limited in vitro human data of placental gene
expression and mineral transport.
3. Methods for studying placental mineral transport
Several methods have been used to assess placental mineral
transport. There are significant differences among them
that need to be reviewed to understand the inherent limitations in the data obtained from them.
A) IN SITU PLACENTAL PERFUSION IN RATS AND LAMBS. The most
rigorous technique is in situ placental perfusion, which has
been used in lambs, goats, guinea pigs, and rats (97, 433,
554, 654, 723). The procedure begins by removing the fetus
and connecting the placenta to a semi-closed circuit via the
umbilical vessels (FIGURE 7). Autologous fetal blood or a
blood substitute is used to perfuse the placenta in situ (i.e.,
within the mother), but the flow rate and perfusion pressure
are maintained at arbitrary set levels. 45Ca and 51Cr-EDTA
are infused together into the maternal circulation (32P or
28
Mg are substituted for 45Ca to measure placental phosphorus or magnesium transport, respectively). Repeated
blood samples are taken from the maternal circulation and
umbilical vein to calculate the radioisotope clearance rates.
51
Cr-EDTA is not actively transported but diffuses across
these placentas, so its appearance within the umbilical vein
is considered to reflect diffusion of isotope. The rate of
active calcium transfer is calculated from formulas that incorporate the speed at which the 45Ca concentration declines in the maternal circulation, and the rate at which the
concentration of 45Ca increases relative to 51Cr-EDTA in
the umbilical vein (554). Hormones, drugs, or antibodies
can be infused into the umbilical artery (placental in-flow)
to test their effects on fetal regulation of placental calcium
transport, or into the maternal circulation to determine if
there are maternal effects on the regulation of placental
calcium transport (723). Significant limitations of this technique include that it is done in the absence of fetal control,
the mother is deeply anesthetized with potential effects on
uterine and placental blood flow, a single placenta is measured with no simultaneous matched control, and the perfusion is done at a set pressure and rate that may not reflect
physiological values. Indeed, when 32P accumulation in the
placenta has been assessed before and up to 10 days after
removal of the fetus and compared with an intact placenta
and fetus within the same uterus, significant differences in
phosphate concentration are presented at 1 h after the fetus
is removed, are four to five times normal at 6 h, and become
profound as the days pass (703). Significant advantages of
this technique include that frequent sampling from the pla-
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CHRISTOPHER S. KOVACS
Venous
sampling
Pressure
gauge
Reservoir
from
umbilical
veins
to umbilical
arteries
Perfusion
(in-flow)
Pump
FIGURE 7. Placental perfusion studies in fetal lambs and rats. The fetus is removed from the uterus
sometime before the experiment begins, and the umbilical vessels are connected to a semi-closed circuit to
enable perfusion of the placenta in situ. 45Ca (alternatively, 32P or 28Mg) and 51Cr-EDTA are infused in the
anesthetized mother, and continuous measurements are made of the declining concentration of radioactivity
in the maternal circulation, and the increasing concentration of radioactivity in the umbilical vein portion of the
circuit. 51Cr-EDTA controls for diffusion of the isotopes and for blood flow differences from one placenta to the
next. Test peptides or hormones are infused via the umbilical artery portion of the circuit, and changes in
subsequent maternal-fetal transfer of 45Ca, 32P, or 28Mg are detected as an increase in the rate at which the
isotope concentration increases within the umbilical vein portion of the circuit. The reservoir contains autologous fetal blood or a blood substitute. The same procedure has been miniaturized for use in murine placentas,
but due to their small size it is possible to take only a single blood sample from the mother and the fetal circuit,
it is necessary to dilate the placental and umbilical vasculature first, and the experiment is done without
51
Cr-EDTA. [From Kovacs (330), with permission from Elsevier.]
cental circuit and the mother enable similar experimental
conditions to be maintained from one pregnant dam to the
next. The same method has been used to study placental
phosphorus and magnesium transport. Studies with magnesium have been very limited due to the short half-life of the
isotope, hence the need to have it custom-synthesized locally and at high cost.
B) PLACENTAL TRANSPORT WITHIN INTACT MICE. The second
method was developed for use in mice (FIGURE 8). An intracardiac injection of 45Ca and 51Cr-EDTA is administered to
a pregnant mouse that remains anesthetized from isoflurane
for ⬍30 s. Between 5 and 30 min after the injection, a
C-section is done to remove the fetuses, and the radioactivity within each fetus is quantified. 51Cr-EDTA controls for
differences in diffusion, blood flow, or size of the individual
placentas because it crosses the placenta by passive diffusion only. The relative rate of placental calcium transfer is
calculated by normalizing the 45Ca activity to 51Cr-EDTA
within each fetus; the 45Ca activity alone can also be compared (340). Additional adjustments can be made for placental weight, but these do not add significantly to the adjustment for 51Cr-EDTA. The utility of 51Cr-EDTA has
been shown by a two- to threefold variation in its accumulation that can be present among WT fetuses of the same
mother, while the 45Ca/51Cr activity of the WT fetuses remains unchanged (unpublished data). This likely indicates
that blood flow to any one placenta within the same murine
uterus is constantly changing and will not be simultaneously equal among all placentas in a litter. Significant limitations of this procedure include that a relative and not an
1160
absolute rate of calcium transfer is obtained among the
fetuses within each litter, and that after a blind intracardiac
injection the amount of isotope reaching the maternal circulation may differ somewhat between experiments. However, each fetus within a litter is exposed to the same ratio
of 45Ca to 51Cr-EDTA in the maternal blood. Significant
strengths include that the dam is only briefly anesthetized, the fetuses remain intact, placental blood flow or
perfusion pressure has not been changed, all placentas
within a litter received the same concentrations of isotopes, and the 8 –12 normal, heterozygous, and null fetuses within each litter serve as controls for each other, as
well as enabling comparisons from one litter to the next.
The fetuses can also be treated with hormones prior to
the intracardiac injection of isotopes (FIGURE 8). The
procedure has also been adapted to study placental phosphorus transport (395).
C) IN SITU PLACENTAL PERFUSION ADAPTED TO MICE. The third
method has adapted the in situ placental perfusion technique for the small scale of murine placentas. Mouse placentas are ⬍1 cm in diameter with tiny umbilical vessels,
which represents substantial technical challenges for a perfusion experiment. One placenta is selected, and its fetus is
removed. The placental and umbilical vasculature are first
maximally vasodilated with nitroglycerin in order that the
umbilical vessels can be successfully catheterized and
hooked up to a semi-closed circuit (61, 62). 45Ca is infused
alone, without 51Cr-EDTA or another isotope to control for
diffusional differences or placental size. Numerous maternal or umbilical blood samples cannot be collected because
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FETAL AND NEONATAL MINERAL METABOLISM
A
B
FIGURE 8. Placental mineral transfer studies in fetal mice. The
pregnant mother receives an intracardiac injection of isotopes [45Ca
or 32P combined with 51Cr-EDTA] (A). After 5, 15, or 30 min, the
fetuses are removed from the uterus, and the activity of each isotope is measured within each fetus. Net transfer of 45Ca or 32P is
expressed relative to the accumulation of 51Cr-EDTA for each pup.
To test the effect of hormones and drugs to regulate the rate of
placental mineral transfer, a laparotomy is performed and selected
fetuses are given an intra-abdominal injection of the test substance
or diluent (B). After a time course of 60 min or longer (to allow the
hormone or drug to have its effects), the placental calcium transfer
experiment proceeds with the intracardiac injection of isotopes administered to the mother as in A. [From Kovacs (330), with permission from Elsevier.]
the recoverable blood volumes of adult and fetal mice are
too small, so maternal-fetal clearance of 45Ca cannot be
directly calculated. Instead, the rate of disappearance of
45
Ca from the maternal circulation was determined by sampling a single time point from different mothers, and creating a disappearance curve from the aggregate results (61,
62). This curve allows the maternal-fetal 45Ca clearance to
be estimated under the assumption that experimental conditions remain identical from mouse to mouse. Significant
strengths of this procedure are that it provides a quantitative estimate of the rate of placental calcium transfer in
mice, and that it may be possible to study the effects of
different hormones or drugs. This technique shares the significant weaknesses mentioned earlier for the in situ perfusion technique in fetal lambs and rats. Additional significant
concerns include that an artifactual result may occur from
dilating the placental vessels with nitroglycerin, especially if
placental vasculature, blood pressure, or responsiveness to
vasodilators differ between WT and mutant placentas. Furthermore, the lack of repeated blood sampling and the use
of a derived clearance curve mean that the amount of isotope in the maternal and placental circulations are assumed
rather than directly measured. As noted above, blood flow
can vary two- to threefold from one murine placenta to the
next within the same uterus based on the 51Cr activity in
each fetus after its administration to the mother, but this
technique requires a single placenta and an arbitrary perfusion pressure and rate.
D) INDIRECT MEASURES. A fourth technique has involved measuring the calcium or phosphorus content of the fetal skeleton and inferring the rate of placental mineral transport
from that. In one approach, the pregnant ewe receives a
single infusion of 45Ca or 32P 7 days before slaughter followed by daily treatment with a calciotropic hormone, and
at the end of the experiment, the 45Ca or 32P content of the
fetal skeleton is measured (38, 165). In a second approach,
the pregnant ewe is immobilized and catheterized, and calciotropic hormones or diluent are injected three times daily
into fetal lambs for 12 days. At the end of the experiment,
the ash weight and mineral content of the fetal skeleton are
determined (40). At best, these two approaches are measures of net accretion of mineral by the fetal skeleton, and
only very indirect measures of what the rate of placental
calcium or phosphorus transport might have been during
the experiment. The amount of mineral in the skeleton is a
composite result from cycling within several compartments
of the fetus (blood, amniotic fluid, soft tissues, and bone),
the effect of the hormones to directly stimulate or inhibit
bone formation and mineralization independent of placental mineral transport, and back-flux of mineral across the
placenta into the maternal circulation (330).
Some investigators have interpreted a fall in fetal blood
calcium to the maternal level or below (so-called “reversal
of the maternal-fetal gradient”) to indicate that placental
calcium transport has decreased. This is not a valid approach; as results will show in sections IV–X, the blood
calcium level is regulated independently of the rate of placental calcium transport.
E) ISOLATED PLACENTAL MEMBRANES AND VESICLES.
The fetalfacing basement membranes of human syncytiotrophoblasts have been isolated and induced to form vesicles in
vitro, with the fetal side facing inward. These can then be
used to study the effects of calciotropic hormones and inhibitors to induce signal transduction or stimulate 45Ca
accumulation within the vesicle (649). In such studies, accumulation of 45Ca within the cultured vesicles reflects the
transport of 45Ca across the basement membrane, so it is a
surrogate for measurement of placental calcium transport
in the intact animal.
Other investigators have created vesicles from either maternal-facing or fetal-facing basement membranes, but with
each membrane oriented so that calcium will be transported
outward into the media. The vesicle is loaded with 45Ca and
then efflux is measured under the influence of PTHrP, PTH,
competitive antagonists of PTH or PTHrP, and other hormones (178).
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Alternatively, other investigators have used isolated maternalfacing brush-border membranes, and fetal-facing basement
plasma membranes of syncitiotrophoblasts, to study the expression of calciotropic hormone and their receptors, and the
effects of calciotropic hormones and inhibitors on signal transduction within these cells (354, 355). No estimate of placental
calcium transport is provided with these approaches.
F) SUMMARY OF KEY POINTS.
Several methods exist for estimating the rate of placental mineral transport, and each has
advantages and disadvantages that must be considered in
interpreting the data. The placental perfusion technique in
rats and lambs provides the most quantitative data but occurs in the absence of fetal control, and placental function
begins to deteriorate within an hour after removal of the
fetus. The placental transport technique within intact mice
enables relative transport among fetal genotypes within a
litter to be accurately determined while each fetus remains
undisturbed and in control of placental function.
4. Maternal regulation of placental mineral transport
A) ANIMAL DATA. The fetal-placental unit regulates placental
mineral transport, but it remains unclear to what extent (if
any) maternal hormones or other factors provide any direct
regulation of placental mineral transport. Indirect maternal
regulation may occur because the concentration of minerals
within the maternal circulation represents the supply from
which the placenta must extract the necessary minerals. For
example, if the maternal blood calcium is reduced by diet or
altered maternal hormone levels, it might be anticipated to
reduce the rate of placental calcium transport.
However, several studies suggest that placental calcium
transport is normal even in the presence of maternal hypocalcemia or specific hormone deficiencies. For example,
calcium transport across perfused placentas of sheep remained normal despite significant maternal hypocalcemia
induced by parathyroidectomy or a severely calcium-deficient diet (99, 723). Similarly, hypocalcemia in Vdr null and
Pth null mothers did not impair placental calcium transfer
or reduce the skeletal mineral content in their WT or mutant
fetuses (342, 623). Loss of calcitonin in pregnant Ctcgrp null
mice did not alter placental calcium transfer or the ash weight
and calcium content of the fetal skeleton (430). Excess FGF23
and hypophosphatemia in Phex⫹/⫺ females did not alter placental phosphorus transport to any of the fetuses nor the phosphorus content of the skeleton (395).
On the other hand, when pregnant ewes were made hypocalcemic by a very low calcium diet, their fetal lambs had
slightly delayed ossification and ash weight, despite no
change in fetal levels of calcium, phosphorus, PTH, calcitriol, and a bone resorption marker (377). Similarly, when
pregnant rats were hypocalcemic due to thyroparathyroidectomy or calcitonin infusions, the fetal bones showed increased resorption when subsequently cultured in vitro
1162
(541, 542), the fetal parathyroids enlarged (206, 625), and
fetal femur length and mineral ash content were reduced
(104). These findings in lambs and rats are compatible with
a reduction in placental calcium transport that is sufficient
to delay primary mineralization of bone or induce secondary hyperparathyroidism in the fetuses, while enabling the
fetuses to maintain normal concentrations of minerals
within the circulation. Conversely, acute (295) and chronic
(338) maternal hypercalcemia in rodents have been shown
to suppress the fetal PTH level (FIGURE 5). This suggests
that maternal hypercalcemia increases the flow of calcium
across the placenta, thereby suppressing the fetal parathyroids. Furthermore, when the perfusate calcium concentration is increased on the fetal side of a perfused placenta, the
rate of active transport is not decreased in the perfused
guinea pig placenta (433). This means that hypercalcemia in
the fetus does not suppress placental calcium transport,
which implies that placental calcium transport is regulated
independently of the fetal blood calcium. With placental
calcium transport being nonsuppressible by fetal hypercalcemia, this may explain why maternal hypercalcemia can
have adverse consequences on fetal parathyroid function
(see sect. XII, A, B, and G).
The impact that maternal hypercalcemia and hypocalcemia have on fetal bone metabolism in intact fetuses contradicts the normal placental calcium transport results
obtained in the study of isolated, perfused placentas of
sheep and rats. The problem may be that the placental
perfusion model is not sensitive enough to detect a small
reduction in placental calcium transport, one that may be
sufficient over the course of gestation to cause fetal secondary hyperparathyroidism and skeletal resorption. Alternatively, the absence of the fetus may lead to an artifactually normal result that does not reflect what occurs
when the fetus remains in control of placental function.
When the mother is hypocalcemic, placental mechanisms
may upregulate to extract the needed mineral from a lower
maternal concentration. Consistent with this, when a maternal calcium infusion was administered to pregnant rats,
it caused an acute and marked rise in the serum calcium of
fetuses whose mothers were hypocalcemic from thyroparathyroidectomy, but caused no increase in serum calcium of
fetuses from normal pregnant rats (106).
The effect of maternal hormone deficiencies on placental
mineral transport has also been inferred by determining the
net skeletal calcium content of vitamin D-deficient rats
(228), thyroidectomized, thyroxine-supplemented (“calcitonin-deficient”) sheep (38, 163), and sheep that received
daily administration of prolactin or bromocriptine (39). In
each study, the fetal skeleton had normal mineral content,
implying that placental mineral transport had been normal
in utero. The lack of an effect of maternal vitamin D or
calcitonin deficiency has been confirmed by direct measure-
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FETAL AND NEONATAL MINERAL METABOLISM
ment of placental calcium transport in Vdr null and Ctcgrp
null mothers compared with their WT sisters, as discussed
below in sections VIIIB and IXB (342, 430).
supply context for the subsequent sections that describe
specific skeletal actions of the calciotropic and phosphotropic hormones.
A competitive but weak PTHrP antagonist, PTHrP7–34, was
infused via osmotic mini-pumps into the circulation of pregnant rats between day 8 and 15 of gestation and was found
to decrease fetal weight, inconsistently reduce placental
weight, and to increase the number of apoptotic cells within
the placenta (677). The investigators inferred that administration of PTHrP7–34 causes fetal-placental growth restriction, but these findings have not been independently replicated. Since the Pthrp null fetal mouse does not show evidence of growth restriction or an alteration in placental size
or weight (62, 340), and PTHrP7–34 is unlikely to cross the
placenta, these purported actions of PTHrP7–34 must be
exerted on the maternal side of the placenta.
Patterning of the early embryonic skeleton has been studied
in most detail within the mouse. Numerous genes and signaling pathways are required, include Hox genes, Wnts,
Hedgehogs, bone morphogenetic proteins, fibroblast
growth factors, Notch/Delta, and others (751). Mesenchymal cells are first laid down where bones of the axial and
appendicular skeletons will form. In a few places, such as
the flat bones of the skull, these mesenchymal cells differentiate directly into osteoblasts that then form intramembranous bones. But most of the skeleton forms through the
multistep process of endochondral bone formation, in
which mesenchymal progenitors differentiate first into
chondroblasts and chondrocytes. A relatively avascular cartilaginous template of each bone is formed, with proliferating chondrocytes at each end that lengthen the template
away from the center. The proliferating and early differentiating cells respond to growth factors and hormonal signals
such as PTHrP, which prevent chondrocytes from terminally differentiating. As the distance increases from the zone
of proliferating chondrocytes, older chondrocytes no longer
receive the signals that prevent them from undergoing terminal differentiation, so they become hypertrophic and
then undergo apoptosis. Dead chondrocytes and matrix are
removed by chondroclasts, new blood vessels form, and
osteoblasts begin to lay down the primary spongiosa of
bone. Primary ossification centers are present in most endochondral bones of the mouse at term (an exception being
small bones within the digits), while most secondary ossification centers develop after birth.
B) HUMAN DATA.
There are, of course, no direct measurements in human babies of placental mineral transport.
However, there are indirect indications that placental calcium transport may be altered when maternal serum calcium is significantly increased or decreased. Maternal hypercalcemia due to primary hyperparathyroidism or familial hypocalciuric hyercalcemia causes suppression of the
fetal parathyroid glands that can last for months after birth
or be permanent (54, 79, 393, 417, 493, 529, 609, 674).
Maternal hypercalcemia likely increases the flow of calcium
across the placenta, thereby suppressing the fetal parathyroids. Conversely, maternal hypocalcemia caused by hypoparathyroidism or pseudohypoparathyroidism has been associated with intrauterine fetal hyperparathyroidism, skeletal demineralization, intrauterine fractures, and bowing of
the long bones (226, 389, 652, 710). This is compatible
with reduced placental calcium transfer that prompts secondary hyperparathyroidism to develop in the fetus.
C) SUMMARY OF KEY POINTS. There is no direct evidence that
maternal hormones regulate placental mineral transport.
Study of isolated perfused placentas of sheep suggest that
maternal hypocalcemia does not reduce placenta calcium
transport. However, the functional outcomes in animals
and humans (hypoparathyroidism in fetuses from hypercalcemic mothers, and secondary hyperparathyroidism in fetuses from hypocalcemic mothers) indicate that significant
disturbances in maternal serum mineral levels must alter
placental mineral transport despite failure of the perfused
placentas to demonstrate it.
G. Endochondral Bone Formation,
Mineralization, and Remodeling
1. Animal data
The details of fetal skeletal formation are well beyond the
scope of this review; instead, a brief overview is provided to
It is not until primary ossification centers have formed that
the skeleton begins to accrete substantial mineral content;
simultaneous with this, the placenta ramps up the expression of calciotropic genes and the rate of mineral delivery.
As previously mentioned, the fetal rat skeleton accretes
95% of its mineral content during the last 5 days of a
22-day gestation (121).
While the net flow of mineral is into bone to support bone
modeling, some turnover or remodeling of the newly
formed bone occurs in utero, which allows calcium to reenter the circulation and maintain the serum calcium level.
This concept is supported by the presence of osteoclasts and
expression of osteoclast-specific genes within fetal bones
(336, 359) and the presence of bone resorption markers in
serum and amniotic fluid (338). As described in detail in
sections IVA and VIA, Pthrp null and Pth1r null fetuses are
hypocalcemic, which may be due in part to loss of normal
skeletal responsiveness or bone turnover. In support of this,
expression of a constitutively active PTH1R (Jansen receptor) within the endochondral skeleton increased the fetal
serum calcium, including in double-mutants that also
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CHRISTOPHER S. KOVACS
lacked Pthrp (330, 596). Furthermore, the fetal skeleton
can undergo increased osteoclast-mediated resorption in response to such factors as maternal hypocalcemia (104, 377)
or an inactivating mutation of CaSR (338), and present
with an undermineralized skeleton or fractures at birth.
2. Human data
In human fetuses, a complete cartilaginous scaffold of the
endochondral skeleton forms by the 8th week of gestation.
Primary ossification centers form in vertebrae and long
bones between the 8th to 12th wk, but most mineralization
does not occur until the third trimester when 80% of the ash
weight and mineral content is accreted (121, 224, 683, 733,
734). Secondary ossification centers form in the femurs by
the 34th week. The rate of mineral accretion accelerates
from ⬃60 mg/day at week 25 to ⬎300 mg/day from the
35th through 38th weeks, before tapering off slightly in the
last 2 wk (635, 734, 735, 761). Normal skeletal turnover
likely contributes to maintaining the high level of calcium
within the fetal circulation, and skeletal resorption will increase in response to severe maternal hypocalcemia caused
by hypoparathyroidism.
calcium then rises steadily to reach adult levels by 7–14
days after birth (201, 422). Ionized calcium displays a
bimodal pattern, increasing to ⬃80% of the high fetal
value during the subsequent 12–36 h, and then declining
to normal adult levels over the subsequent 7 days (422).
Albumin increases steadily such that the albumin-bound
fraction will account for ⬃50% of the serum calcium, in
contrast to only 20% of the total during fetal life (422).
Neonatal lambs differ significantly by maintaining a high
serum calcium, similar to the fetal value, until 3– 4 mo of
age (94, 201).
Phosphorus follows a similar time course in rodents,
dropping significantly in the first 2– 4 h after birth, increasing significantly by 6 h after birth, and declining
again over the subsequent day (201). The early decline is
consistent with the loss of the placental phosphorus infusion, while the subsequent rise corresponds to a transient hypoparathyroid state of the newborn. Conversely
in lambs, and similar to what occurs with calcium, serum
phosphorus increases over the first 24 h and stays elevated for at least a week (403).
3. Summary of key points
2. Human data
The skeleton is developing and mineralizing during fetal
development, but still participates in the normal regulation
of serum mineral levels, and will be adversely resorbed if the
mineral supply from the placenta is insufficient.
After birth, the decline in serum and ionized calcium are
typically 20 –30% over the first 12–24 h (32, 143, 387,
594). In one study, the ionized calcium fell from the mean
umbilical cord level of 1.45 mM to a mean of 1.20 mM by
24 h after birth (387). After that, the serum and ionized
calcium follow a pattern similar to the rodent data,
achieving normal adult values after several days. A single
study reported that delivery by elective C-section resulted
in a lower blood calcium and higher PTH levels at birth
compared with babies delivered vaginally (31). Most
studies have not distinguished mode of delivery among
the neonatal blood values. The use of formula or breast
milk influences the postnatal time course of calcium as
well. The fall is greater in infants fed formula (32, 686)
because the high phosphorus content of formula causes
hyperphosphatemia and complexes calcium within the
circulation.
III. OVERVIEW OF NEONATAL MINERAL
HOMEOSTASIS
The alterations in circulating concentrations of minerals
and hormones that occur during the neonatal period are
depicted in FIGURE 2. This section describes the changes in
function and roles that are invoked at birth in parathyroids,
kidneys, intestines, and endochondral skeleton.
A. Serum Mineral Concentrations
1. Animal data
The regulation of bone and mineral metabolism changes
significantly after birth. Severing the umbilical cord means
that the placental mineral pump is lost, and the neonate
must initiate mechanisms to absorb mineral from the intestines and reabsorb mineral in the kidney tubules. The onset
of breathing induces a rise in pH from relatively acidotic
levels in utero, and this will contribute to a fall in the ionized
calcium.
The serum calcium and ionized calcium fall 40% during
the first 6 –12 h after birth in rodents (201, 347). Total
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Phosphorus rises over the first 24 – 48 h after delivery (32,
582, 594); after that, it declines toward adult values, consistent with resolution of transient hypoparathyroidism in
the newborn.
3. Summary of key points
The early neonatal period in humans and rodents is characterized by a significant fall in ionized and total calcium and
a rise in phosphorus over the first 12 h, which largely corrects over the succeeding 24 h.
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FETAL AND NEONATAL MINERAL METABOLISM
B. Calciotropic and Phosphotropic Hormone
Concentrations
1. Animal data
In newborn rats, PTH increases markedly by 6 –12 h after
birth (199, 675), with the peak level corresponding to the
trough level in serum calcium (675). After that, PTH declines toward adult values over the subsequent 2 wk (199,
675).
Calcitriol does not increase until during the third week after
birth (near weaning); during that time, 24-hydroxylase actively produces the inactive metabolite 24,25-dihydroxyvitamin D while little or no calcitriol is produced (201, 729).
Similar findings were found in newborn lambs up to 11 days
after birth with preferential catabolism of 25-hydroxyvitamin D and no production of calcitriol, not even in response
to PTH administration (201, 326). These studies suggest
that the neonatal kidney is initially resistant to PTH and
sluggish in its ability to produce calcitriol.
PTHrP becomes undetectable in the adult circulation, but
exactly when this occurs after birth has not been rigorously
determined. The placenta and extraembryonic membranes
are sources of PTHrP secretion that are lost at birth; if these
are the main sources, then a prompt fall in PTHrP may be
another trigger that causes the parathyroids to upregulate
PTH synthesis and secretion. However, neonatal rat pups
were found to have persistently high PTHrP1–34 concentrations, in the range of 20 – 40 pM (401). Neonatal lambs also
have persistent PTHrP immunoreactivity in the parathyroid
glands, and serum bioactivity attributable to PTHrP, over
the first several months after birth (94, 396); however, circulating PTHrP measurements were not done to confirm
this. Data cited earlier from fetal lambs indicated that the
parathyroid gland may be a key source of PTHrP; the persistently elevated serum calcium and bioactivity attributable to PTHrP in neonates may be an indication of persistent secretion of PTHrP by the parathyroids.
Calcitonin increases during the newborn period in rats
(209), calves (201), and foals (201, 210). In the neonatal
lamb it increases and persists for at least 6 wk after birth
before falling to adult levels (204, 215).
There is a surge in testosterone level of neonatal rats and
mice within the first 2–3 h after birth, which rapidly declines to baseline by 6 h (124, 461). After this, testosterone
is equivalent between male and female mice for the rest of
the newborn period (498, 499, 730). Estradiol is also low in
both sexes (498).
2. Human data
The postnatal time course of PTH was originally delineated
using older COOH-terminal assays, and it was found that
PTH remained low over the first 12–24 h, and did not reach
peak levels until 48 h or later (32, 143, 177, 595, 598, 685).
Subsequent studies used intact PTH assays but obtained
measurements solely at birth and 24 h later, and demonstrated that PTH rose significantly (31, 386, 388, 416, 444,
473, 580). Those studies did not determine when the PTH
value increased or whether the peak level was achieved at
24 h or later.
Calcitriol is low in cord blood and increases to adult values
during the first 2 days after birth, likely in response to the
rise in PTH (144, 642). A similar increase to peak levels by
the fourth day after birth occurs in preterm infants supplemented with vitamin D (231, 582).
Milk contains very high concentrations of PTHrP which, if
absorbed into the neonatal circulation, could also explain
persistently high circulating levels of PTHrP. Neonatal
goats that consumed mother’s milk had significantly higher
PTHrP compared with kids that received only formula,
which may imply that PTHrP had been absorbed into the
circulation (559). No study has examined a time course of
PTHrP levels in neonatal blood after suckling, or used radiolabeled PTHrP in milk to determine if it reaches the
neonatal bloodstream.
It is not known whether the neonatal parathyroids synthesize PTHrP, or how soon after birth the circulating PTHrP
level becomes undetectable. A single study reported that the
plasma PTHrP1–34 level was reduced 50% between birth
and day 1 in healthy newborns, but it then increased back to
the cord blood level by day 2 (72). As in the animal models,
human milk is a very potent source of PTHrP that could
support the circulating level if absorbed; by comparison,
PTHrP concentrations are low to absent in infant formulas
(306, 392, 488). The breast milk content of PTHrP doubled
between 2 and 10 days after delivery, but serum PTHrP was
very low in the neonates and did not change between those
time points (678). The investigators concluded that PTHrP
was not absorbed from milk; however, the blood collections
were not timed to breastfeeding, and the authors did not
consider that PTHrP has a short half-life in blood. Moreover, the assay requires plasma, but the authors measured
PTHrP1– 86 in stored sera, so the low PTHrP values may
reflect the inevitable degradation of PTHrP in sera, and use
of an assay that does not detect PTHrP1–34.
Scant data are available pertaining to serum FGF23 levels
soon after birth. In WT mice, FGF23 was 10-fold higher
than adult values at 12 h after birth (34).
Intact FGF23 increased fourfold between birth and 4 –5
days of age (483, 661), whereas COOH-terminal FGF23
maintained the high values of fetal life (661).
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CHRISTOPHER S. KOVACS
Calcitonin may increase up to 10-fold over cord blood levels during the first 48 h after birth (32, 53, 444, 595), before
gradually declining over the subsequent days or weeks (32,
583).
Estradiol remains very low in male and female newborns
and stays that way until puberty (91, 739). Testosterone
remains very low in females, but in males it surges within
the first 24 h (124), and again in the second week after birth
until about 4 mo of age, after which it declines to very low
levels and remains that way until puberty (133, 739).
first 24 h after birth and contributes to the development of
postnatal hypocalcemia and hyperphosphatemia. The postnatal awakening of parathyroid function explains the subsequent increase in calcium and calcitriol, and decline in
serum phosphorus.
D. Renal Mineral Reabsorption and
Excretion
1. Animal data
3. Summary of key points
Calciotropic hormones levels rapidly progress from fetal
values to levels that are similar to the adult (FIGURE 2).
PTH, calcitriol, and intact FGF23 increase; PTHrP disappears from the circulation after an uncertain time course;
and calcitonin may stay elevated for several weeks.
C. Neonatal Parathyroid Function
1. Animal data
The parathyroids are important for control of serum mineral concentrations in the neonate. Parathyroidectomy in
the newborn rat pup caused a fall in serum calcium and an
increase in serum phosphorus (199, 344). Notably the
length, morphology, trabecular content, and calcium and
phosphorus content of the neonatal skeleton were unaffected by parathyroidectomy in the neonate compared with
sham controls, indicating that the development of the skeleton and accretion of mineral is not dependent on PTH
during this time period (344). Whether the neonatal parathyroids produce PTHrP is uncertain, but suggestive evidence for it comes from neonatal lambs which maintain
increased serum calcium and high PTH-like bioactivity,
similar to what is present during fetal development.
2. Human data
The increase in PTH follows the early postnatal drop in the
ionized calcium, and precedes (and is likely responsible for)
the subsequent rise in ionized calcium and calcitriol, and
decline in phosphorus. Consistent with the concept that the
neonatal parathyroids recover from suppression experienced during fetal development, in the 48 h after birth the
parathyroids are sluggish in their response to acute hypocalcemia, such as due to exchange transfusion with citrated
blood (143, 595, 685, 686). This sluggishness resolves with
increasing postnatal age (685, 686) but becomes prolonged
after maternal hypercalcemia during pregnancy (see sect.
XII, A and B).
3. Summary of key points
A transient phase of relative hypoparathyroidism or sluggish responsiveness by the parathyroids occurs during the
1166
The neonatal kidneys begin to actively reabsorb and excrete
minerals. Over the first 24 h there is reduced cAMP responsiveness to PTH in vitro compared with the response obtained during fetal life (675). The rat kidney does not show
expression of VDR or responsiveness to calcitriol until the
third week after birth (633). TRPV6 and calbindin-D9k
show a progressive increase in expression, reaching a maximum at 3 wk after birth (634). There is low level of expression of CaSR at birth that increases after the first postnatal
day in mice (112).
2. Human data
Urine calcium excretion is low at birth despite the high level
of serum calcium in cord blood and the suppressed PTH
level. After the first 5–7 days, urine calcium excretion increases over the subsequent weeks despite a lower serum
calcium and higher PTH concentration (298, 473). The
change in renal calcium excretion is likely due to the developmental increase in renal blood flow and glomerular filtration rate that occurs over the same interval (171, 237,
298). Additional factors include loss of the effects of PTHrP
to reabsorb calcium, increased expression of CaSR, and
increased responsiveness to calcitriol.
Phosphorus excretion is also low at birth and increases with
postnatal age (378, 605). This likely reflects not only the
increasing level of PTH in the circulation, but (as in the
rodent) a developmental increase in renal responsiveness to
PTH that has been demonstrated in term and preterm infants (378, 406).
The rapid rise in calcitriol after birth likely arises from
synthesis in the kidneys, which are stimulated by PTH.
3. Summary of key points
The neonatal kidneys undergo developmental changes that
progressively increase their responsiveness to PTH, calcitriol, and CaSR. Sluggish kidney function over the first
several days after birth likely contributes to the development of neonatal hypocalcemia.
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FETAL AND NEONATAL MINERAL METABOLISM
E. Intestinal Mineral Absorption
1. Animal data
In newborn rodent pups, calcium absorption occurs mainly
through passive, nonsaturable processes that are not dependent on calcitriol (218, 219, 243, 510 –512). Milk contains
a high content of lactose, which directly increases paracellular calcium absorption and net absorption of dietary calcium (86, 366, 500). As the days pass, intestinal cells begin
to express VDR (242), increase their expression of calbindin-D9k (80, 634) and TRPV6 (634), and display evidence
of calcitriol-dependent active absorption of calcium (243).
Passive transport of calcium progressively declines, such
that by the time of weaning (3 wk of age) calcitriol-dependent active transport of calcium is the dominant means of
intestinal calcium absorption (218, 219, 243), while both
calbindin-D9k and TRPV6 have reached maximally upregulated expression compared with the day of birth (634).
2. Human data
As noted earlier, preterm human infants demonstrate calcium absorption that is proportional to intake, with a linear
relationship between intake and absorption; this is consistent with passive absorption of calcium (44, 46, 221, 581).
Absorption did not vary by vitamin D intake in preterm
infants, which is also consistent with passive, non-vitamin
D-dependent absorption (75). An interventional study
found that preterm infants showed no calcemic response to
short courses of supraphysiological doses of calcitriol (0.1–
3.0 ␮g/kg), which suggests that the preterm intestines are
refractory to calcitriol (708). For perspective, the adult requirement in hypoparathyroidism is typically a total daily
dose of 0.25–1.0 ␮g. A second interventional study used 4.0
␮g/kg of calcitriol administered intravenously for three consecutive days and found a significant increase in serum calcium (325). However, that effect was not the result of an
alteration in intestinal calcium absorption but likely reflected an increase in bone resorption: oral intake was minimal, osteocalcin increased significantly, and urine calcium/
creatinine ratio did not change (325).
A small study of preterm neonates compared human milk,
human milk plus 1,200 IU vitamin D, and human milk plus
1,200 IU vitamin D and phosphorus, and found that only
the group receiving phosphorus supplementation had a significant increase in calcium retention (603). This is also
consistent with a lack of effect of vitamin D/calcitriol to
increase calcium absorption and retention in the preterm
intestines. The preterm infant is also limited by reduced
intestinal absorption of vitamin D that increases with postnatal age (263). Another study supplemented the preterm
neonate with 1,200 IU vitamin D or 0.5 ␮g calcitriol, both
of which increased intestinal absorption of calcium but required up to 4 wk to achieve the full effect, with use of
calcitriol being more effective than vitamin D (604).
Similar to the data from rodents, calcium absorption in
neonates is enhanced by the lactose content of milk (317,
318). Intestinal absorption and retention of calcium increase with both postnatal age and gestational age, with
additional variations superimposed by the composition of
the milk (45, 221, 256, 485, 604, 613). These findings suggest a maturational effect toward an active, calcitriol-dependent, saturable mechanism of intestinal calcium absorption as seen in older children and adults. It may be inferred
from these data that, similar to the rodent intestine, the
human neonate’s intestines undergo progressive upregulation in expression of the vitamin D receptor. However,
longitudinal changes in expression of VDR have not been
studied in human neonatal intestines. Similarly, it is unclear
when the neonatal intestines change from passive to active
absorption, although the responsiveness to calcitriol at 4
wk of age in one clinical study may be an indication that
active absorption has become more prominent by that age
(604).
The preterm infant must supply a high rate of calcium to the
skeleton as if it were in utero, but this exceeds what can be
achieved with breast milk or standard infant formulas. In
the first week after birth, most nutrition is provided parenterally because the intestines are not able to absorb a sufficient amount (445). As oral feeds are implemented, special
formulas high in calcium are needed to meet the preterm
neonate’s need for mineral. In the very low birth weight
preterm neonate, a daily calcium intake of 120 –200 mg/kg
is usually recommended with much of that being given parenterally (445). As the postnatal days pass for the preterm
baby, the skeletal demand for mineral declines from the
high fetal rate and can eventually be met with breast milk or
formula.
The efficiency of intestinal calcium absorption is influenced
greatly by the nutrition that the neonate receives. Absorption of calcium is more efficient from breast milk than from
infant formula regardless of vitamin D supplementation
(223, 604, 732). More than 90% of the phosphate content
of breast milk and formula is absorbed (604), but the higher
phosphate content of formula can lead to double the serum
phosphorus level compared with neonates that consume
breast milk (32, 223, 604). High serum phosphate lowers
serum calcium and is a cause of neonatal hypocalcemia
(686). The differences between breast milk and formula
have had clinical effects, with breast-fed infants having
higher serum calcium levels and a lower risk of hypocalcemia compared with formula-fed infants (32, 223).
3. Summary of key points
A developmentally programmed maturation alters intestine
calcium absorption from passive, nonsaturable mechanisms
to an active, calcitriol-dependent process. This has been
well established in the neonatal rodent, but there is sufficient evidence to accept that it probably occurs in human
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CHRISTOPHER S. KOVACS
neonates as well. This explains why preterm babies are
dependent on passive absorption of mineral until the intestines are capable of responding to calcitriol.
F. Skeletal Metabolism
1. Animal data
The efficiency of intestinal mineral absorption has significant effects on the neonatal skeleton. Neonatal rodents are
largely protected from abnormalities due to the effects of
lactose to enhance absorption of calcium. Consequently,
despite significant abnormalities such as hypoparathyroidism, vitamin D deficiency, or loss of VDR, the neonatal
rodent maintains normal bone mineralization until the time
of weaning. It is at that point in time, when the intestines are
dependent on calcitriol-regulated active intestinal calcium
absorption, that the skeleton begins to develop deficits in
mineral content and altered development (see sects. VD and
VIIID).
Normal peak bone density and skeletal calcium content
differ markedly among mouse strains (48), with the commonly used inbred C57BL/6 strain having a significantly
lower bone mass than the outbred Black Swiss strains (192,
309, 310, 744). Serum and ionized calcium are also significantly lower in C57BL/6 compared with Black Swiss mice
(340), but there are no obvious differences in calciotropic
and phosphotropic hormone levels between the two strains.
Unidentified genes and proteins that affect bone metabolism and development likely explain this interstrain variability.
2. Human data
The late term fetus accumulates skeletal calcium at a rate of
over 300 mg/day or ⬃150 mg·kg⫺1·day⫺1 (635, 734, 735,
761), whereas this drops to ⬃30 – 40 mg·kg⫺1·day⫺1 in the
normal neonate (683). The skeleton is highly dependent on
the intestines to supply needed mineral; any deficiency in the
diet or in the ability to absorb mineral will cause impaired
bone mineralization and increased resorption of the skeleton. This is especially true for the preterm neonate for
whom breast milk or standard formula would provide insufficient amount of calcium. However, the ability of the
intestines to passively absorb mineral in proportion to intake enables special formulas that are high in calcium to
provide the needed mineral, after an initial phase in which
most of the nutrition is administered parenterally.
A consequence of inadequate intestinal delivery of calcium
and phosphorus is rickets of prematurity. This is not due to
vitamin D deficiency because it is not prevented by supplementation of the mother during pregnancy, nor is it responsive to treatment of the neonate with vitamin D or calcitriol
(10, 92, 323, 432, 510). It is precipitated by loss of the
1168
placental calcium pump at a time when skeletal demand for
calcium is very high, normal oral calcium intake is insufficient to meet the demand, and the intestines are developmentally immature, and likely lack expression of VDRs, to
deliver calcium through active absorption (92, 510). Bone
mineral content is initially appropriate for gestational age at
birth, but it fails to increase appropriately if left untreated
(235, 236, 324, 446, 640, 641, 690). Physical and radiological signs of rickets of prematurity may develop, including
rachitic rosary, chest deformity, osteopenia, pathological
(especially rib) fractures, metaphyseal stippling, and flaring
and widening of the epiphyses of long bones (92, 513, 514).
Special oral formulas or parenteral preparations containing
high calcium and phosphorus content will correct the bone
demineralization and allow the skeleton to develop normally (10, 92, 323, 432, 641, 643). Follow-up at 3– 4 yr of
age has shown normal bone mineral content in children
who earlier had rickets or low bone mineral content of
prematurity (266).
3. Summary of key points
The neonatal skeleton accrues mineral at a rapid rate and
but will quickly become demineralized if intestinal mineral
delivery is inadequate. Infants that are preterm, severely
vitamin D deficient, lack calcitriol or the VDR, or are hypoparathyroid/aparathyroid, are especially prone to this
complication.
IV. PARATHYROID HORMONE-RELATED
PROTEIN
In the decades that followed the development of specific
PTH assays, it was recognized that fetal blood contains high
PTH-like bioactivity but low immunoreactive levels of PTH
(3, 20, 21, 67, 571). PTHrP was cloned in 1987 (410, 646,
658), and subsequent work confirmed that it mimics many
of the biological actions of PTH (333), circulates at high
levels in fetal blood compared with PTH (168, 306, 600,
671), and accounts for the high PTH-like bioactivity in fetal
blood (7, 94, 396). The relative difference in circulating
levels of PTHrP and PTH may even be underestimated because the commonly used PTHrP1– 86 assay does not detect
biologically active PTHrP1–36. These data led many to hypothesize that PTHrP is the main calcium-and-bone-regulating hormone of fetal life, and that the suppressed levels of
PTH indicate that it is relatively unimportant until after
birth. Given this context, PTHrP’s role in fetal and neonatal
mineral metabolism will be discussed first, followed by PTH
in section V.
A. Regulation of Serum Minerals
Comparison of Pthrp null fetuses to their WT and Pthrp⫹/⫺
littermates, which share the same intrauterine environment,
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FETAL AND NEONATAL MINERAL METABOLISM
has confirmed the importance of PTHrP in regulating aspects of fetal mineral and bone metabolism. Pthrp null fetuses have a hypoparathyroid-like phenotype characterized
by hypocalcemia (both total and ionized levels), and a serum phosphorus that is raised even further above the normally high fetal phosphorus level (62, 340, 341, 694). Serum magnesium remains normal (330). Notably the fetal
ionized calcium falls to a level equal to the maternal ionized
calcium (340, 694). Serum PTH increases threefold in Pthrp
null fetuses (336), likely through CaSR-mediated effects to
increase serum calcium to the normal adult level but no
higher. This conclusion is supported by additional observations in double-mutant fetuses. When one or both alleles of
CaSR are inactivated in Pthrp null fetuses (i.e., Pthrp null/
Casr⫹/⫺ fetuses and Pthrp/Casr double mutants), serum
PTH and ionized calcium levels increase significantly (338).
However, when the NH2-terminal actions of PTH and
PTHrP are blocked by deletion of PTH1R (Pth1r null fetuses), simultaneous ablation of one or both alleles of CaSR
does not alter the ionized calcium (i.e., Pth1r null/Casr⫹/⫺
fetuses and Pth1r/Casr double mutants) (338).
Section V that follows notes that hypocalcemia occurs in
several models lacking PTH or parathyroids, but within
each there is no compensatory increase in plasma PTHrP
(341, 623). Therefore, PTHrP is unresponsive to falls in the
ambient fetal blood calcium, which may imply that it is
autonomously produced and regulated by, and in response
to, local factors within the placenta such as the flow of
calcium.
The source of PTHrP in the fetal circulation cannot be determined from the Pthrp null model because PTHrP is absent in all fetal tissues.
B. Regulation of Placental Mineral
Transport
Several lines of evidence indicate that PTHrP is an important regulator of the active transport of calcium and magnesium across the placenta from mother to fetus.
Fetal lambs were thyroparathyroidectomized and treated
with thyroid hormone; several days later, the fetus was
removed from the uterus and its placenta was studied using
the in situ placental perfusion model (see details in section
IIF3). The rate of increase in the concentration of 45Ca/
51
Cr-EDTA in the umbilical vein portion of the circuit (placental outflow) was significantly reduced after prior fetal
thyroparathyroidectomy, compared with the rate observed
in placentas from separate ewes whose fetuses had been
intact (97, 723). Furthermore, infusion of autologous blood
from intact fetal lambs (sometimes the twin from the same
ewe) restored the rate of increase in 45Ca/51Cr-EDTA to
normal values (97). These results showed that prior fetal
thyroparathyroidectomy reduced the basal rate of placental
calcium transport when measured several days later but,
importantly, in the absence of the fetus which had been
removed hours before. The investigators concluded that
fetal parathyroids produce a factor that stimulates placental
calcium transport, such that its absence causes the rate of
transport to fall.
The same investigators went on to infuse specific peptide
sequences of PTHrP or PTH into the umbilical artery, to
determine their effects on placental calcium transport. Peptides that contained a mid-region portion of PTHrP
(PTHrP1–141, PTHrP1– 86, and PTHrP67– 86) increased the
rate of change in the concentration of 45Ca/51Cr-EDTA in
the umbilical vein, whereas PTHrP1–34 and PTH1–34 had no
effect (6, 96, 556). When PTHrP38 –94 was later determined
to be the structure of mid-regional PTHrP (490, 517), these
investigators repeated the experiments and confirmed that
PTHrP38 –94 also significantly increased the rate of placental
calcium transport in perfused placentas from previously
thyroparathyroidectomized fetal lambs (745). This approach has also been used to show that mid-molecular
PTHrP stimulates placental magnesium transport, but not
phosphate transport (43, 47, 96, 653).
The conclusion of these experiments was twofold: 1) that
PTHrP stimulates placental calcium and magnesium transport and 2) that the relevant source of PTHrP is the fetal
parathyroids. However, no measurements of plasma
PTHrP were done in thyroparathyroidectomized lambs, so
it remains unknown whether fetal parathyroidectomy reduced the circulating level of PTHrP.
The in situ placental perfusion model has also been used in
rats, and at first glance the evidence obtained seems inconsistent with the data obtained from perfused placentas of
lambs. Decapitation was done to crudely mimic parathyroidectomy, and (similar to the effects of thyroparathyroidectomy in fetal lambs) this resulted in a fall in placenta
calcium transport (554). That deficit in placental calcium
transport was increased slightly (but remained well below
the normal value) after infusion of PTH1–34 (554); no
PTHrP peptides were tested in the decapitation model.
However, in perfused placentas from intact fetuses, PTH1–
1–34
, and PTHrP67– 86 each had no significant ef34, PTHrP
fect on placental calcium transport (554, 611). These data
do not contradict the findings in ovine placentas because
mid-molecular PTHrP was not tested in the decapitated
fetal rat model, and the efficacy of PTH1–34 in the decapitation model was very modest compared with that of midmolecular PTHrP in placentas from thyroparathyroidectomized fetal sheep. Furthermore, the failure of mid-molecular PTHrP to stimulate placental calcium transport in
placentas from intact rats may be the result of PTHrP-mediated effects already being maximally exerted. Data described in the next paragraph corroborate this conclusion.
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CHRISTOPHER S. KOVACS
The creation of Pthrp null fetuses (297) enabled a different
approach to measure placental calcium transport within
intact fetuses using the methodology described in section
IIF3. 45Ca and 51Cr-EDTA were administered by intracardiac injection to pregnant mice, and the intact fetuses were
removed 5, 15, or 30 min later. Compared with their WT
and Pthrp⫹/⫺ littermates, 45Ca/51Cr-EDTA accumulation
in Pthrp null fetuses was 25% lower after 5 min and 40%
lower after 30 min (340). To confirm that this reduction
was specific to loss of PTHrP, a laparotomy was done to
administer an intra-abominal injection of a specific peptide
form of PTHrP, PTH, or diluent to several fetuses. Sixty
minutes later, 45Ca and 51Cr-EDTA were administered to
the mother by intracardiac injection, and the fetuses were
removed 30 min after that. PTHrP1– 86 and PTHrP67– 86
increased the accumulation of 45Ca/51Cr-EDTA to a value
not different from WT littermates, whereas PTH1– 84 and
PTHrP1–34 had no significant effect (340). Importantly, injections of mid-molecular PTHrP had no effect in WT fetuses, which suggests that the WT fetus may already have
maximally stimulated PTHrP responses on the placenta,
such that pharmacological treatment had no additional effect. This is consistent with the data cited in the preceding
paragraph, in which mid-molecular PTHrP failed to stimulate placental calcium transport in placentas from intact
rats.
These results from intact Pthrp null fetuses, and the aforementioned data from in situ perfused placentas from thyroparathyroidectomized fetal lambs, both showed that midmolecular forms of PTHrP stimulate placental calcium
transport, whereas peptides of PTHrP that contain only the
NH2-terminal form, and PTH itself, have no significant
effect. Analysis of Pthrp null placentas revealed reduced
mRNA and protein levels of Ca2⫹-ATPase and calbindinD9k within the intraplacental yolk sac compared with placentas from WT littermates (337), which may indicate that
PTHrP acts in part through the stimulation of Ca2⫹-ATPase
and calbindin-D9k expression. Alternatively, the reductions
in Ca2⫹-ATPase and calbindin-D9k expression could be a
consequence of reduced placental calcium transport rather
than its cause. That these reductions in Ca2⫹-ATPase and
calbindin-D9k expression may be physiologically significant
is supported by Trpv6 null fetuses, which have markedly
reduced placental calcium transport, and loss of Trpv6,
Ca2⫹-ATPase, and calbindin-D9k expression within the intraplacental yolk sac (659). Notably, the reduction of Ca2⫹ATPase and calbindin-D9k expression within the Pthrp null
placenta was limited to the intraplacental yolk sac which
normally expresses PTHrP (337). No difference in Ca2⫹ATPase and calbindin-D9k mRNA or protein expression
was found within the adjacent trophoblasts or when the
entire placenta was analyzed by Western blot (62, 337).
The results described so far are consistent in revealing a role
for PTHrP to stimulate placental calcium transport in fetal
1170
lambs and mice. Contrary evidence comes from the in situ
placental perfusion model, miniaturized for use in fetal mice
(see details in sect. IIF3) (61, 62). A fetal mouse was removed, and nitroglycerin was used to dilate the placental
vasculature before cannulating the umbilical vessels and
performing the placental perfusion technique. 45Ca was administered alone, and a single sample was later obtained
from the maternal circulation and the placental circuit.
With the use of this technique, Pthrp null placentas had an
increased rate of placental 45Ca transport compared with
perfused WT placentas from separate mothers. The investigators concluded that PTHrP inhibits rather than stimulates
placental calcium transfer.
There are several possible reasons why the results of these
experiments were the opposite of what the prior studies
found in lambs and mice had revealed. The likeliest is that
the use of nitroglycerin to dilate the placental vasculature
may have created an artifactual result when the vascular
expression and actions of PTHrP are considered. PTHrP is
expressed within vascular smooth muscle wherein it has
actions to relax smooth muscle and thereby cause vasodilation and increased blood flow (517, 551). PTHrP and PTH
share vasodilatory effects when injected into living animals
(120, 127, 247, 464, 497), and overexpression of Pthrp or
Pth1r within vascular smooth muscle cells of mice leads to
lower blood pressure, increased perfusion pressure, and reduced vascoconstriction in response to challenge with angiotensin II (476). When PTHrP was deleted from vascular
smooth muscle cells by using a vascular smooth muscledriven promoter for Cre in Pthrp floxed mice, the mice
showed reduced renal perfusion and glomerular filtration,
consistent with renovascular constriction (533). Therefore,
a similar increase in vascular constriction and reduced
blood flow may be present in the Pthrp null placenta compared with the WT and Pthrp⫹/⫺ placentas. Consequently,
nitroglycerin may have differential vasodilatory effects in
Pthrp null placentas which, combined with placental function being studied well after the fetus has been removed,
may have artifactually increased the apparent transport of
calcium across the perfused placenta.
The expression of PTHrP within vascular smooth muscles
raises the possibility that one of the mechanisms through
which PTHrP controls placental calcium transport may be
through regulating blood flow to and within the placenta.
In the absence of PTHrP, intraplacental blood flow may be
reduced due to increased vasoconstriction, thereby reducing the rate at which maternal and fetal blood can exchange
minerals and nutrients. However, altering blood flow dynamics is at best a partial explanation because PTHrP and
PTH share these vasodilatory effects through their actions
on PTH1R, whereas it is mid-molecular PTHrP, which does
not act on PTH1R, that has been clearly shown to stimulate
placental calcium transport.
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FETAL AND NEONATAL MINERAL METABOLISM
That PTHrP stimulates rather than inhibits placental calcium transport is supported by studies in other mouse models wherein the placental calcium transport technique for
intact fetal mice was used. Pth1r null fetuses (360) have
greater than 10-fold increases in circulating levels of both
PTHrP and PTH (336, 341). They lack PTH1R and so
cannot respond to NH2-terminal actions of PTH or PTHrP,
but should be responsive to actions of mid-molecular
PTHrP on its (as yet uncloned) receptor(s). The placental
calcium transport rate was increased in Pth1r null fetuses to
150% of the value of their WT fetal littermates (340), in
keeping with anticipated effects of high plasma PTHrP concentrations to increase the rate of placental calcium transport. Aparathyroid Hoxa3 null fetuses and Pth null fetuses
each have normal plasma PTHrP concentrations and normal rates of placental calcium transport (336, 341, 623).
Casr null mice have significantly reduced plasma PTHrP
levels and reduced placental calcium transport (330, 338).
The concordance between plasma PTHrP concentrations
and placental calcium transport among these different
mouse models (Pthrp null, Pth1r null, Hoxa3 null, Pth null,
and Casr null fetuses) are consistent with the conclusion
that PTHrP’s role is to stimulate placental calcium transport.
Previously the increased fetal blood mineral concentrations
were considered to be proof of active transport of calcium
across the placenta, and elimination of the maternal-fetal
gradients in the relative concentrations of calcium or magnesium (such as by fetal thyroparathyroidectomy) were
considered to be evidence that active transport had been
eliminated (97, 100, 198). However, study of these murine
gene deletion models have revealed that placental calcium
transport is not a direct determinant of the fetal blood calcium level or skeletal mineral content. The placenta supplies
the mineral which may then be incorporated into bone or
soft tissues, remain in the circulation or extracellular fluids,
be excreted into the urine and amniotic fluid, or be transported back across the placenta and into the maternal circulation. The blood levels of calcium and incorporation of
calcium into bone are regulated separately from the placental delivery of calcium. Consequently, despite hypocalcemia
and reduced placental calcium transport, Pthrp null fetuses
have a normal or even increased ash weight and mineral
content (the reason for this is addressed in the next section)
(62, 336, 694). In contrast, Pth null and aparathyroid
Hoxa3 null fetuses have a normal rate of placental calcium
transport, but are hypocalcemic with significantly undermineralized skeletons. These findings point to the role of
PTH in regulating blood calcium and stimulating mineralization of the fetal skeleton independent of PTHrP and the
rate of placental calcium transport. Casr null fetuses have a
decreased rate of placental calcium transport and a reduced
mineral content of the skeleton, despite a higher than normal serum calcium that should otherwise facilitate mineralization of the developing skeleton (338).
Although the Pthrp null model does not address whether
fetal parathyroids produce PTHrP or even if parathyroidderived PTHrP regulates placental calcium transfer, when
considered together with results from Hox3 null and Pth1r
null fetuses (sects. V and VI), the studies in fetal mice are
more consistent with a placental source of PTHrP. The
studies in fetal sheep and mice both found that a mid-molecular form of PTHrP stimulates placental calcium transfer, but differ on what its source might be. These seemingly
discrepant results between fetal lambs and mice might be
due to species difference; there are no comparable human
data.
An indirect approach to estimating placental calcium transport was carried out in fetal lambs that received thrice-daily
injections of PTHrP1–34 or PTHrP1– 86, with or without calcitonin, for a total of 12 days. Each form of PTHrP significantly increased the mineral content of the fetal skeleton
compared with control (diluent), and the simultaneous use
of calcitonin blocked the effect of PTHrP (40). The authors
inferred that both forms of PTHrP stimulated placental calcium transport and that calcitonin countered PTHrP’s actions on the placenta. However, these results can be explained by the effects of both NH2-terminal forms of
PTHrP to stimulate bone formation and mineralization,
and for calcitonin to inhibit bone resorption (and, secondarily, bone formation), without implicating any direct effect
of calcitonin on placental calcium transport.
Magnesium and phosphorus are each actively transported
across the in situ perfused placentas of rats (612, 653) and
lambs (47, 96, 397). Mid-molecular PTHrP stimulated
magnesium transport across in situ perfused ovine placentas (47, 96, 397) but not rodent placentas (611), and it did
not affect placental phosphorus transfer (43, 397, 653).
Placental transport of these minerals has not been examined
in Pthrp null fetuses.
C. Regulation of Endochondral Bone
Formation
PTHrP is produced by perichondrial cells and proliferating chondrocytes, and its receptor (PTH1R) is expressed
further down the growth plate, within prehypertrophic
chondrocytes, preosteoblasts, and osteoblasts. PTHrP
acts in concert with Indian hedge hog and Wnt signaling
to delay terminal differentiation and hypertrophy of
chondrocytes (300, 714). PTHrP’s role is clearly illustrated by the Pthrp null skeleton, which has premature
chondrocyte hypertrophy, apoptosis, and initiation of
primary bone formation; the result is a chondrodysplasia
with shortened limbs (FIGURE 9) (297, 300, 360). The
normally cartilaginous portions of the ribs turn into
bone, and bones that normally mineralize after birth become mineralized in utero (297). In contrast to the effects
of loss of PTHrP, overexpression of PTHrP or PTH1R
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CHRISTOPHER S. KOVACS
A
B
C
D
E
F
WT
Pthrp null
H
p<0.02
120
Ash Calcium
(Normailzed to Pthrp+/-)
Ash Calcium
(Normailzed to Hoxa3+/-)
G
Hoxa3 null
100
80
60
40
20
0
WT
Hoxa3+/-
Hoxa3-/-
has the opposite effect of delaying chondrocyte hypertrophy, resulting in a cartilaginous skeleton at birth that
slowly undergoes bone formation and mineralization
over the succeeding weeks (596, 726).
Although PTHrP’s anabolic actions on preosteoblasts and
osteoblasts have been lost, Pthrp null fetuses show normal
to increased expression of osteoblast-specific genes and proteins, including collagen ␣1(I), collagenase-3, osteocalcin,
and osteopontin (297, 359). This is likely because the three-
1172
WT
Pthrp+/-
Pthrp-/-
FIGURE 9. Comparison of the effects of
loss of PTH vs. loss of PTHrP on the fetal
skeleton. Hoxa3 null fetuses lack parathyroids and PTH but have normal circulating
PTHrP, while Pthrp null fetuses lack PTHrP
and have upregulated PTH in the circulation. A–C: whole mounts of fetal skeletons
(ED 18.5) stained with alizarin red S (mineral) and alcian blue (cartilage). Crownrump length, lengths of long bones, and
mineralization pattern appear unaltered in
the Hoxa3 null fetus (A) compared with its
WT sibling (B). Note the normal (blue) cartilaginous portions of the ribs and sternum
in the WT and Hoxa3 null. Contrast the
Pthrp null from the same genetic background (C), which is shorter, has a
rounded vault of the skull, shortened mandible (hence protruding tongue), shortened
and thickened long bones, advanced mineralization, and abnormal mineralization of
the cartilaginous portion of the ribs and
sternum. D–F: von Kossa-stained sections
of the fetal tibias counterstained with
methyl green. The overall morphology and
lengths of the upper portions of the tibia
(both chondrocytic and osseous zones) are
normal in the Hoxa3 null (E), compared
with the progressively shortened and disorganized growth plates of the Pthrp null (F).
The double-headed arrows compare the
lengths of the chondrocytic growth plate
from the proliferating through hypertrophic
zones. The amount of mineral (black from
von Kossa stain) appears to be markedly
reduced in the Hoxa3 null but normal in the
Pthrp null. G shows the calcium content
(by atomic absorption spectroscopy) of
Hoxa3 null fetuses compared with WT and
Hoxa⫹/⫺ littermates, while H compares
the mineral content of Pthrp null fetuses
compared with WT and Pthrp⫹/⫺ littermates. The calcium content is significantly
reduced in Hoxa3 nulls, but normal in
Pthrp nulls due to the accelerated mineralization of bone and abnormal mineralization
of cartilaginous structures. Ash weight and
magnesium content were also reduced in
Hoxa3 nulls but normal in Pthrp nulls (not
shown). [Adapted from Kovacs et al.
(336). Copyright 2001 The Endocrine Society; permission conveyed through the
Copyright Clearance Center, Inc.]
fold compensatory increase in circulating PTH (336) stimulates osteoblasts and prevents the deficits in osteoblast
function that loss of PTHrP should otherwise have caused.
This conclusion is borne out by careful comparison of Pthrp
null to Pth1r null fetuses, as described in section VIC. Even
though increased PTH seemingly ameliorates the skeletal
effects of PTHrP deletion prior to birth, mice that lack
PTHrP expression only within preosteoblasts and osteoblasts have reduced bone formation and bone mass by 6 wk
of age (438).
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FETAL AND NEONATAL MINERAL METABOLISM
In striking contrast to the physiological findings of reduced
placental calcium transport and hypocalcemia, the ash
weight and mineral content of the Pthrp null skeleton has
been variably reported to be normal or modestly increased
(FIGURE 9) (62, 336, 340, 694). This seemingly contradictory finding is likely explained by the early, accelerated, and
abnormal calcification that occurs within the Pthrp null
skeleton. As described above, bones that normally do not
mineralize until after birth become prematurely mineralized
in utero; also, normally cartilaginous structures (such as the
medial aspects of the ribs and the sternum) are transformed
into bone (297). Consequently, the Pthrp null skeleton matures faster and accretes more mineral sooner, and in normal and abnormal locations, thereby leading to a seemingly
normal or increased mineral content despite its slightly
smaller size and reduced rate of placental calcium transport.
purpose? Passive immunization of day-old neonatal mice
with antibodies to NH2-terminal PTHrP did not affect serum calcium or the calcium content of their ashed skeletons
at 48 h (348), but a longer duration may have been needed to definitively determine if the skeleton would become
abnormal. Oral administration of PTHrP1–34 to neonatal
mice had no effect, whereas subcutaneously administered
PTHrP1–34 increased serum calcium significantly (348);
from these data, investigators concluded that PTHrP is not
absorbed into the circulation of the neonatal mouse. Deletion of the Pthrp gene from mammary tissue at the onset of
lactation resulted in milk that lacks detectable PTHrP, but
the growth rate of the pups was qualitatively described as
no different than expected (706). Collectively, these results
suggest that eliminating PTHrP from milk is of no consequence to the neonate.
Complete loss of PTHrP causes death often within minutes
of birth, although a few pups have lived for 5 days. The
cause of death may be multifactorial, including severe hypocalcemia with cardiac arrhythmias, a rigid rib cage that
restricts ventilation, and stiff lungs due to abnormal development of alveolar type II cells and low surfactant (297,
340, 570).
However, the same investigators recently examined pups
that nursed from mothers in which none, one, or two of the
Pthrp alleles had been deleted from mammary tissue at the
onset of lactation (409). Deletion of one Pthrp allele reduced the milk PTHrP content, whereas loss of both alleles
eliminated PTHrP, but the calcium content of milk was
unaltered (409). At day 12, there was an inverse dose-response relationship between the PTHrP content of milk and
the ash calcium content of the pups (409), with loss of milk
PTHrP leading to increased skeletal calcium. Does PTHrP
reduce net intestinal absorption of calcium? That seems
unlikely given the previously cited evidence that the calcium
content of milk is more readily bioavailable than from formula, and results in a higher serum calcium in neonates.
Does some functional portion of PTHrP get absorbed into
the neonatal circulation and act on osteoblasts to reduce
mineral accretion? Such a pathway could explain how milk
consumption might lead to a higher serum calcium but a
lower mineral content of the skeleton. It would give PTHrP
the evolutionary role of protecting against neonatal hypocalcemia by preventing the skeleton from accreting too
much of the mineral in the circulation.
D. Role in the Neonate
The abrupt fall in serum calcium experienced by most mammals after birth is consistent with loss of placental PTHrP,
but there are insufficient data to confirm that PTHrP has
been lost from the neonatal circulation. Conversely neonatal lambs do not experience a fall in serum calcium after
birth, but maintain the same high levels that were present
during fetal life until 3– 4 mo of age. When the calcium level
finally declines in lambs, it corresponds to the age when
increased PTH-like bioactivity attributable to PTHrP finally subsides (94). This result seemingly indicates that
parathyroids of neonatal lambs produce PTHrP for some
months after birth.
PTHrP continues to be produced in the growth plates of
endochondral bone wherein it delays terminal differentiation of chondrocytes, and within osteoblasts wherein it directs bone formation and mineralization, and inhibits apoptosis. Its importance has been illustrated by several mouse
models. Posnatally, Pthrp⫹/⫺ mice develop modestly accelerated endochondral ossification and deficits in bone microarchitecture and mass (23). When Pth null mice are made
haploinsufficient for Pthrp, osteoprogenitor cell recruitment decreases, osteoblast apoptosis increases, bone formation diminishes, and trabecular bone mass is reduced (442).
Conditional deletion of Pthrp from osteoblasts leads to an
osteoporotic skeleton by the age of maturity (438, 441).
The very high concentration of PTHrP in milk seemingly
declares that it is important, but for what physiological
E. Human Data
Possible effects of PTHrP on human trophoblast function or
placental calcium transport have been investigated in vitro.
Vesicles prepared from fetal-facing basement membranes of
syncytiotrophoblasts (32–37 wk of gestation) showed a linear increase in ATP-dependent accumulation of 45Ca within
the vesicles (649), which is consistent with a progressively
increasing rate of active placental calcium transport in late
gestation. Mid-molecular PTHrP38 –94 stimulated ATP-dependent uptake of 45Ca calcium in a concentration-dependent fashion starting at the physiological dose of 5 pg/ml
(648), while PTH, PTHrP1–34, PTHrP67– 86, and calcitonin
had only modest effects at pharmacological doses (50 or
250 pg/ml) (648). 45Ca uptake was inhibited by a protein
kinase C inhibitor, while PTHrP38 –94 increased inositol tri-
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CHRISTOPHER S. KOVACS
sphosphate production and protein kinase C phosphorylation. PTHrP may also promote trophoblast survival since
PTHrP1–34 prevented cytotrophoblast apoptosis while
PTHrP67– 86 had no effect (128).
In contrast to those findings, other investigators created
similar vesicles from maternal-facing and fetal-facing basement membranes of human syncytiotrophoblasts, but with
the direction of calcium transport oriented outwards into
the media. The vesicles were loaded with 45Ca and then
efflux of the isotope was measured. PTHrP1–34 was most
potent to increase 45Ca efflux; PTH1–34 required a fourfold
higher dose; PTHrP67–94 was without effect (178). The
NH2-terminal antagonist PTHrP7–34 significantly reduced
the effect of both peptides. These results suggest that NH2terminal PTHrP can stimulate transmembrane calcium
transport while the mid-molecular form cannot.
As noted previously, cord blood NH2-terminal PTHrP concentrations are 15-fold higher than PTH when expressed in
equivalent pM units (330), although this may underestimate the true concentration of PTHrP depending on the
assay and how the blood sample was collected. The plasma
concentration of mid-molecular PTHrP38 –94 was increased
almost twofold in cord blood of intrauterine growth restricted babies compared with healthy neonates at term
(647). When vesicles of fetal-facing basement membranes
of synciotrophoblasts were made from the placentas of
these same growth-restricted babies, 45Ca uptake increased
50% into the vesicles from IUGR babies over those from
healthy babies (647). These data positively correlate an increase in the concentration of mid-molecular PTHrP with
an in vitro measure of placental calcium transport.
The effects of NH2-terminal PTHrP fragments have also been
studied on isolated maternal-facing and fetal-facing basement
membranes from term human placentas. PTHrP1–34 stimulated phosphoinositide turnover, phospholipase C, and
protein kinase C in both membrane fractions (355, 362). It
was equipotent to PTH1–34 on the phospholipase C pathway but more potent than PTH at stimulating protein kinase C activity (362).
An equivalent to the murine Pthrp null state has not yet
been identified in human fetuses, but is expected to cause
intrauterine death. An autosomal dominant microdeletion
in the human PTHrP gene (PTHLH) has been linked to
brachydactyly type E, which is characterized by short stature and shortened metacarpals and metatarsals (316). This
finding is consistent with a role for PTHrP in regulating the
formation of the endochondral skeleton in humans.
Whether the very high concentrations of PTHrP in human
milk have effects in the neonate to alter intestinal calcium
absorption or bone metabolism has not been directly investigated. Studies during the first 6 to 12 mo after birth have
1174
shown variable results of higher, lower, or no difference in
bone mineral content gains achieved by human milk versus
formula-fed babies (90, 145, 636, 722). However, several
studies have found consistent and significant positive correlations between the duration that an infant received human milk versus formula and the bone mass measured by
DXA as a child, adolescent, or young adult (186, 290, 450).
These correlations do not prove causation nor do they directly implicate the PTHrP content of human milk. However, the results are intriguing, especially given that the
PTHrP content is one of the most striking differences in the
composition of human milk versus formula.
F. Summary of Key Points
The bulk of the available data are consistent with PTHrP
playing several key roles in fetal bone and mineral metabolism, including the regulation of serum calcium and phosphorus, stimulation of placental calcium and magnesium
transport, control of endochondral bone development by
delaying terminal differentiation of chondrocytes, and several effects that stimulate formation, recruitment, activity,
and survival of osteoblasts. Although PTHrP is needed to
maintain a normal serum calcium in the fetus, PTHrP does
not increase in response to systemic hypocalcemia. Animal
and human data support the conclusion that it is a midmolecular form of PTHrP, likely PTHrP38 –94, which stimulates placental calcium transfer through actions on an as
yet unidentified receptor. It remains unclear whether placenta, parathyroids, or both are the key source of PTHrP in
the fetal circulation, nor is it certain when PTHrP disappears from the postnatal circulation. Through its local production in chondrocytes and osteoblasts, PTHrP continues
to play key roles in regulating skeletal development in the
neonate, infant, and child. Exactly what role the high content of PTHrP in milk has is unknown, but there are intriguing clues that it may modulate intestinal calcium absorption
or mineral accretion by the skeleton.
V. PARATHYROID HORMONE
The low levels of PTH in late term fetuses, which rise significantly after birth, suggested that PTH may be unimportant for fetal bone and mineral homeostasis. However, this is
now known to be incorrect.
A. Regulation of Serum Minerals
The most specific evidence that PTH regulates fetal blood
calcium is that hypocalcemia occurs when Pth is ablated in
fetal mice (623), and when PTH antiserum is infused into
fetal rats (197). Hypocalcemia also occurs after parathyroidectomy in fetal lambs and rats (1, 2, 97, 105, 520), and
genetic ablation of parathyroids in mice (341); however,
hypocalcemia due to loss of parathyroids may be caused in
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FETAL AND NEONATAL MINERAL METABOLISM
part by loss of other parathyroid-derived factors such as
PTHrP. Consistent with this possibility, in fetal mice loss of
parathyroids due to Hoxa3 ablation causes the ionized calcium level to fall well below the maternal calcium level
(336), whereas loss of PTH alone in Pth null fetuses leads to
a blood calcium equal to the maternal level (623). In thyroparathyroidectomized fetal lambs, the blood calcium also
falls below the maternal level (2), but in thyroparathyroidectomized or decapitated fetal rats the serum calcium remains equal to or above the maternal level (105, 520).
There is no “PTH-null” equivalent in lambs and rats.
In section IVA, it was noted that in Pthrp null fetuses, serum
PTH increases and likely maintains the blood calcium at the
normal adult level. But in several mouse models that are
hypocalcemic due to PTH deficiency (Pth null, Gcm2 null,
and aparathyroid Hoxa3 null fetuses), there is no compensatory increase in plasma PTHrP (341, 623). These findings
confirm that PTH is responsive to falls in the ambient fetal
blood calcium whereas PTHrP is not.
Although thyroparathyroidectomy or decapitation in fetal
rats causes the blood calcium to fall to a level equal to or
above the maternal level, when the mother was also thyroparathyroidectomized, the fetal serum calcium remained
significantly higher than the maternal serum calcium (105,
519). This was interpreted by the investigators to imply
persistence of active transport of calcium in the absence of
maternal and fetal parathyroids. The experiments were
done prior to the discovery of PTHrP and may indicate
persistent actions of placental PTHrP on serum calcium and
placental calcium transport.
The low level of PTH in the fetal circulation also regulates
serum phosphorus and magnesium because hyperphosphatemia and hypomagnesemia occur in Pth null fetuses
(623). Hyperphosphatemia and a modest reduction in serum magnesium also occur in thyroparathyroidectomized
fetal lambs and rats, and aparathyroid fetal mice (105, 341,
397, 398, 520, 623). Notably calcitriol levels do not change
in thyroparathyroidectomized fetal lambs (1, 97), indicating that fetal production of calcitriol is not dependent on
PTH or parathyroids.
Therefore, PTH is a key regulator of fetal blood calcium,
phosphorus, and magnesium, despite the low circulating
levels that originally suggested otherwise. In fact, PTH may
have a greater role in determining the serum calcium level
than PTHrP, as revealed through study of double-mutants
(see section VI).
B. Regulation of Placental Mineral
Transport
The PTH1R is expressed by murine trophoblasts but is most
intensely expressed within the intraplacental yolk sac (337).
That intense expression implies that NH2-terminal PTH
(⫾N-terminal PTHrP) has some function within the placenta, but most in vivo studies have failed to find an effect of
PTH on placental mineral transport. These included studies
of in situ perfused placentas from thyroparathyroidectomized fetal sheep (6, 96, 556), and placental calcium transfer in intact Pthrp null and WT fetuses (340). However,
Pthrp null fetuses have threefold higher PTH which may
have maximized any effect that exogenously administered
PTH could have had.
Exogenous PTH1–34 did modestly stimulate calcium transport in perfused placentas from rat fetuses that had been
decapitated to crudely mimic parathyroidectomy (554), but
it had no effect in perfused placentas from intact fetuses
(554, 611). Placentas from thyroparathyroidectomized rat
fetuses were not studied.
Comparative study of Pth null and Gcm2 null fetuses
within a similar genetic background has provided evidence
of a possible role for PTH in stimulating placental mineral
transport (623). Placental 45Ca transfer appeared normal at
baseline in Pth null fetuses, but microarray and real-time
quantitative RT-PCR analysis of Pth null placentas revealed
significantly reduced expression in Trpv6, Sg100 (Calbindin-D9k), Vdr and other cation transporters (623). Pth null
fetuses treated in utero with PTH1– 84 responded with a
30% increased rate of 45Ca accumulation, confirming that
exogenous PTH can stimulate placental calcium transfer in
vivo when endogenous PTH is absent (623). Gcm2 null
fetuses have a similar phenotype to Pth nulls with hypocalcemia and hyperphosphatemia, but differ by having a low
but detectable concentration of circulating PTH compared
with their WT littermates. They also differ from Pth nulls
by having a significantly increased rate of placental calcium
transfer, and increased placental expression of Trpv6,
Sg100, and Vdr relative to WT littermates (623). These
observations led to the discovery that there is a low level of
expression of Pth mRNA within WT placentas, which is
absent in Pth null placentas, and significantly increased in
Gcm2 null placentas (623). Therefore, PTH may regulate
placental calcium transfer through local expression of PTH
and PTH1R, in addition to PTH having possible effects
from within the systemic circulation.
PTH does not regulate phosphorus transfer in perfused placentas from fetal lambs (330), and it probably does not
regulate magnesium transport either because parathyroid
extracts and PTHrP1–34 failed to have effects (47, 96). Placental phosphorus and magnesium transfer have not been
studied in aparathyroid or Pth null fetal mice.
Overall, PTH may act systemically and locally within placenta to regulate the transfer of calcium and other solutes.
Its effects may be weaker or secondary to that of PTHrP,
since Pthrp null fetuses have reduced placental calcium
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CHRISTOPHER S. KOVACS
transfer despite a threefold increase in endogenous PTH. If
so, then loss of both PTH and PTHrP should cause a greater
decline in placental calcium transport than loss of PTHrP
alone, but this has not been investigated.
0.25 mM higher serum calcium, and adults have higher
bone mass, compared with C57BL/6 mice (192, 309, 310,
340, 744). These and other differences between the background strains may explain why absence of PTH causes
variable effects on bone.
C. Regulation of Endochondral Bone
Formation
Overall, PTH is a key regulator of skeletal mineralization in
the fetus, as observed in lambs and mice. This may occur
indirectly through PTH’s actions to support the fetal blood
calcium; when serum calcium falls, substrate availability is
reduced and mineral accretion is impaired. PTH may also
directly regulate osteoblast development and function during fetal development, with its effects varying by genetic
background.
Thyroparathyroidectomized fetal lambs displayed reduced
mineralization of the fetal skeleton at term (2), but whether
this is due to loss of PTH, PTHrP, or both could not be
determined. Specifically, examination of the fourth lumbar
vertebrae revealed reduced ash weight, eroded surface, and
mineralizing surface, but increased bone volume, trabecular
thickness, osteoid thickness, and osteoid surface. This was
described as a rachitic phenotype by the investigators, but
shows the effect of loss of parathyroids and PTH to reduce
skeletal mineralization, thereby resulting in the accumulation of unmineralized osteoid.
Study of the more recently developed murine models has
shown that PTH is an important determinant of skeletal
mineralization during fetal development, but its effects on
endochondral bone development appear weaker and somewhat variable compared with loss of PTHrP (624).
Hoxa3 null fetuses lack parathyroid glands and PTH and
(within the Black Swiss outbred background) have very low
skeletal mineral content, but normal cartilaginous and osseous components of the endochondral skeleton (FIGURE 9)
(336, 341). These include normal expression of chondrocyte and osteoblast-specific genes, lengths of the long bones,
and cellular morphology and lengths of the tibial growth
plate and trabecular compartment (336, 341). The skeleton
is essentially normal except for a significant deficit in mineral content (FIGURE 9).
In contrast, Pth null fetuses (within the inbred C57BL/6
background) were initially reported to have slightly shorter
tibial metaphyses, shortened metacarpals and metatarsals,
smaller vertebrae, reduced trabecular bone volumes, and
fewer osteoclasts and osteoblasts (439). These structural
changes largely affected osseous and not chondrocytic parameters, including reduced expression of osteoblast-specific genes.
Why should loss of PTH lead to a bone phenotype that loss
of parathyroids does not share? To resolve this, Pth nulls
and PTH-deficient Gcm2 nulls were back-crossed into the
outbred Black Swiss strain to match that of Hoxa3 nulls
(623). In this strain Pth nulls and Gcm2 nulls each had
normal skeletal lengths and morphology, and a modestly
reduced mineral content that was not as severe as in aparathyroid Hoxa3 nulls (336, 623). Therefore, loss of PTH
caused an osteoblast-deficiency phenotype only in the
C57BL/6 strain. Black Swiss fetuses and their mothers have
1176
D. Role in the Neonate
PTH becomes a major controller of mineral and bone homeostasis in the hours after birth. The parathyroids increase
synthesis and secretion of PTH, which acts to raise the
blood calcium, lower phosphorus, stimulate calcitriol synthesis, inhibit calcitriol catabolism, reabsorb calcium in the
kidney tubules, and upregulate bone formation.
The importance of PTH is most clearly evident through the
study of Pth null mice, which are born in the expected
Mendelian ratios at birth. They are hypocalcemic and hyperphosphatemic at birth, but it is around the time of weaning at 3 wk of age that sporadic sudden deaths begin to
occur from presumed hypocalcemia-induced tetany and arrhythmias (310). The timing is consistent with the maturation of intestinal calcium absorption from a passive to an
active, calcitriol-dependent process, and with Pth nulls being unable to normally stimulate calcitriol synthesis. These
deaths are prevented by using a calcium-phosphate-lactoseenriched “rescue diet” that increases passive absorption of
mineral (310). A similar sporadic mortality occurs in Gcm2
null mice when maintained in outbred background strains,
but mortality is 70 –100% in the inbred C57BL/6 strain for
reasons that appear unrelated to loss of PTH (384).
By 2 wk after birth, Pth null mice in the C57BL/6 background are slightly smaller than their WT siblings due to
shortening of the long bones. Histomorphometric assessment has shown that chondrocytic parameters are still
largely normal, but abnormalities within the osseous component include a significant reduction in mineral apposition
rate, trabecular bone volume, and mineralization (as assessed by von Kossa) (748).
The hypocalcemia in Pth nulls contributes to their increased
mortality and reduced skeletal mineralization independent
of any effect of PTH to stimulate the formation of calcitriol.
This has been tested in Pth/Cyp27b1 double mutants that
lack PTH and calcitriol, are hypocalcemic, hyperphosphatemic, and hypercalciuric, have shortened skeletal lengths
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FETAL AND NEONATAL MINERAL METABOLISM
and reduced trabecular bone volumes, and die before weaning at 3 wk of age (748). When PTH or PTHrP injections
were administered daily starting at day 4 after birth to increase the serum calcium, survival improved such that the
pups could be analyzed at 1 mo of age (749). The serum
calcium increased significantly, hyperphosphatemia persisted, urine calcium excretion decreased, and there were
significantly improved skeletal lengths, trabecular bone volumes, and mineral content (749). These results could not be
mediated by calcitriol because the double-mutants lacked
the ability to stimulate calcitriol synthesis in response to
PTH or PTHrP treatment (749). In a separate report, Pth/
Cyp27b1 double mutants were treated with exogenous calcitriol, and the results were quite similar with improved
survival, increased serum calcium, lowered serum phosphorus, reduced urine calcium excretion, and improved skeletal
lengths, trabecular bone volumes, and mineral content
(747). These effects were not mediated by PTH since the
double-mutants lacked it. Since similar outcomes occurred
when the serum calcium of Pth/Cyp27b1 double mutants
was raised by treatment with PTH, PTHrP, or calcitriol,
these results may indicate the importance of serum calcium
to support osteoblast function and skeletal mineralization,
as well as to prevent deaths due to hypocalcemia. Calcium
should exert its effects through the CaSR expressed by
chondrocytes, osteoblasts, and osteocytes (see sect. VIIC).
E. Human Data
Human trophoblasts express PTH1R (82, 183), which
makes it likely that PTH exerts biological effects within the
placenta. PTH1–34 stimulated 45Ca accumulation into vesicles prepared from fetal-facing basement membranes of human syncytiotrophoblasts, but the minimum dose needed
was 10-fold or more than that needed for mid-molecular
PTHrP38 –94 (648). In vesicles oriented to transport calcium
outward, PTH1–34 stimulated 45Ca efflux but required a
fourfold greater dose than PTHrP1–34 (178). These findings
indicate that PTH has the ability to stimulate calcium transport in human trophoblasts when studied in vitro, but that
it is less potent than PTHrP. NH2-terminal PTHrP may be
the endogenous ligand for the PTH1R expressed within
human trophoblasts, unless PTH is produced locally within
the human placenta.
Congenital hypoparathyroidism can occur from genetic deletion of the parathyroids (DiGeorge and other 22q11.2
deletion syndromes, ablation of Gcm2, etc.) and from activating mutations of the calcium sensing receptor (70). The
animal data predict that hypoparathyroid babies should
have hypocalcemia, hyperphosphatemia, and impaired
skeletal mineralization at birth. However, due to the sporadic nature of these conditions and lack of symptoms at
birth, no cord blood or skeletal radiographs are available.
Symptomatic hypocalcemia is not inevitable after genetic
loss of parathyroids: in a large series of 22q11.2 deletions,
only 60% of affected individuals developed hypocalcemia
at some point in their lives (578). Many presented as neonates, but in others, the presentation was in childhood or as
late as 18 years of age (578). In another series of 12 cases
with confirmed hypocalcemia, only 4 were symptomatic; 10
were diagnosed before 1 mo of age, and the remaining 2
presented at 3 mo and 12 yr of age (71). Serum phosphorus
was inconsistently measured and not always elevated. In
another series of 10 cases with confirmed hypoparathyroidism, the age at diagnosis ranged from 9 days to 13 yr (12).
It seems likely that when hypocalcemia begins in utero as a
result of failure of the parathyroids to form, the individual
adapts to the low calcium and is less likely to become symptomatic after birth. This contrasts to the marked symptoms
that occur after surgically induced hypoparathyroidism in
children or adults.
Pseudohypoparathyroidism or PTH resistance also causes
hypocalcemia, but it develops later in childhood and is usually preceded by hyperphosphatemia and elevated PTH
(412, 470, 687). Consequently, pseudohypoparathyroid
newborns should have normal cord blood calcium and are
unlikely to become hypocalcemic as neonates.
Neonatal or infantile primary hyperparathyroidism has
been described in older literature (57, 405, 562), but these
cases likely represented neonatal severe primary hyperparathyroidism mediated by homozygous or compound
heterozygous inactivating mutations of CaSR (see sect.
VIID). The earliest that conventional primary hyperparathyroidism caused by adenomas or 4-gland hyperplasia has
been reported is 4 years of age, and these are usually in the
setting of multiple endocrine neoplasia types 1 or 2, or
familial primary hyperparathyroidism (321).
F. Summary of Key Points
Despite its low levels in the fetal circulation, PTH is an
important determinant of serum calcium and phosphorus.
It may play a modest role in stimulating placental calcium
transport by reaching trophoblasts from the systemic circulation or through localized expression within the placenta.
Alternatively, expression of PTH1R within trophoblasts
may indicate a role for NH2-terminal PTHrP that is only
fulfilled by PTH when secondary hyperparathyroidism occurs in utero. PTH contributes to endochondral bone development in the fetus mainly by supporting the mineralization
of bone. This occurs by PTH maintaining the normal concentration or supply of minerals in the circulation, as well as
through possible direct effects of PTH on osteoblasts that
may be independent of the higher circulating concentrations of PTHrP. Postnatally, PTH increases after the first
12 h and assumes its well-known roles as a dominant regulator of bone and mineral status, through actions in osteoblasts, kidneys, and (indirectly by stimulating formation of
calcitriol) the intestines.
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VI. ROLE OF PTHrP AND PTH
CONSIDERED IN COMBINATION
PTHrP and PTH overlap in their contribution to the regulation of fetal bone and mineral metabolism. This has been
made most clear by comparative study of double mutants
that lack either PTHrP and PTH, or PTHrP and the parathyroids, and by ablation of their common NH2-terminal
receptor, PTH1R.
A. Regulation of Serum Minerals
It is clear that PTH and PTHrP each participates in the
regulation of the fetal blood calcium, such that loss of either
hormone causes fetal hypocalcemia (336, 340, 341, 623).
Their individual effects appear additive as revealed by studies of a double-mutant colony in which PTHrP, parathyroids, or both were deleted, and compared with the findings
in Pth1r null fetuses that lack the PTH1R (FIGURE 10).
Compared with WT littermates, Pthrp null fetuses had a
modestly reduced blood calcium (equal to the maternal
level), aparathyroid Hoxa3 null fetuses had a markedly reduced blood calcium (well below maternal values), and
Pthrp/Hoxa3 double mutants had the lowest blood calcium
(336). The ionized calcium level caused by simultaneous
loss of parathyroids and PTHrP was equivalent to the ionized calcium level of Pth1r null fetuses (336, 340). Taken
together, these data suggest that the ionized calcium level is
largely determined by the combined NH2-terminal effects
p < 0.001
p < 0.01
2.00
Ionized Calcium (mmol/l)
p < 0.05
1.50
1.00
0.50
0.00
WT
Pthrp
null
Hoxa3
null
Double
Mutant
FIGURE 10. PTH and PTHrP additively regulate the fetal blood
calcium. Mean serum ionized calcium is shown for WT, Hoxa3 null,
Pthrp null, and Pthrp/Hoxa3 double mutant fetuses. For clarity, the
other five possible genotypes have been omitted from the figure. The
upper dashed line indicates the mean maternal ionized calcium level
in this genetic background (equal to Pthrp null), while the lower
dashed line indicates the mean ionized calcium level in Pth1r null
fetuses in a similar genetic background. [From Kovacs et al. (336).
Copyright 2001 The Endocrine Society; permission conveyed
through the Copyright Clearance Center, Inc.]
1178
of both PTHrP and PTH, since loss of the PTH1R led to the
same reduction in blood calcium as loss of PTHrP and parathyroids combined (336).
The greater decline in ionized calcium within aparathyroid
Hoxa3 null fetuses (336, 623) might imply that parathyroid-produced PTHrP has been lost; however, plasma
PTHrP is unchanged among WT, Hoxa3⫹/⫺, and Hoxa3
null fetal littermates (336, 341). It is possible that the parathyroids contribute to fetal mineral homeostasis directly or
indirectly through the release of other factors. For example,
the chromogranins have been estimated to represent 50%
of the protein output of the parathyroids (305, 606). Chromogranin expression increases with hypocalcemia and decreases with hypercalcemia (160, 467), and chromogranins
have been shown to inhibit PTH synthesis and release (26,
576); therefore, chromogranins could conceivably have distant effects to regulate the production of PTHrP by the
placenta (248, 319). However, the possible role that nonPTH secretory products of the parathyroids may have on
mineral homeostasis has not been studied in the fetus or
adult. Pth/Hoxa3 double mutants should reveal whether
loss of parathyroids adds to the phenotype caused by loss of
PTH, but that model has not been studied. Similarly, although Pthrp/Pth double mutants have been examined for
their effect on skeletal development (439), no measurements of serum calcium or phosphorus have been reported.
As noted earlier in section VA, Pth null and aparathyroid
Gcm2 null fetuses in the same Black Swiss strain each had
an ionized calcium that was equal to the maternal level, and
not reduced well below it as in aparathyroid Hoxa3 null
fetuses (623). Pth nulls have hyperplastic parathyroids that
conceivably could produce PTHrP and compensate for the
loss of PTH (439); however, no increase in expression of
PTHrP was observed in the Pth null fetuses (623). Upregulation of placental PTH expression was found in aparathyroid Gcm2 null fetuses, and that may have blunted the fall
in blood calcium caused by loss of parathyroids (623). The
fact that the ionized calcium of Pthrp null fetuses in the
same background is equal to the maternal level should not
be taken as evidence that the magnitude of PTHrP’s effect is
to raise the blood calcium above the maternal level. Pthrp
null fetuses have a significant compensatory increase in
PTH; without it, the serum calcium of Pthrp null fetuses
would likely have been much lower and approaching the
level observed in Pthrp/Hoxa3 null double mutants (336).
Pth1r null fetuses have the lowest ionized calcium level, and
the absence of PTH1R is accompanied by markedly increased circulating concentrations of PTH and PTHrP (336,
340, 341). Although the parathyroids of Pth1r null fetuses
were not directly examined, there was no increase in PTHrP
mRNA expression in the neck region that contains the parathyroids (341). However, there was increased expression of
PTHrP mRNA (by Northern blot and in situ hybridization)
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FETAL AND NEONATAL MINERAL METABOLISM
and protein (by immunohistochemistry) within placenta
and liver of Pth1r null fetuses (340, 341). These findings
provide additional evidence that the fetal parathyroids are
unlikely to be a dominant source of PTHrP in the fetus, and
that placenta and possibly liver may provide much of the
PTHrP that is normally present in the fetal circulation.
B. Regulation of Placental Mineral
Transport
Pth1r null fetuses exhibit a significantly increased relative
rate of placental calcium transport compared with their WT
littermates (340). Since PTHrP circulates at 11-fold higher
levels in these fetuses compared with WT (341), and loss of
the PTH1R blocks NH2-terminal actions of PTHrP, it
seems likely that high circulating levels of mid-molecular
PTHrP are acting on a mid-molecular receptor to stimulate
placental calcium transport (336, 340). These results support earlier findings that mid-molecular PTHrP stimulated
placental calcium transport in fetal lambs and mice (sect.
IVB). The mid-molecular receptor for PTHrP has yet to be
cloned.
C. Regulation of Endochondral Bone
Formation
Pthrp/Hoxa3 double mutants have an overall appearance
and skeletal phenotype that combines the Pthrp null chondrodysplasia with the undermineralized skeleton of the
Hoxa3 null; they are also globally smaller in size (336).
Combining these two genetic deletions confirms that loss of
fetal parathyroids leads to undermineralization of the fetal
skeleton without affecting the underlying cartilaginous and
osseous structure. In contrast, loss of PTHrP alone causes
marked acceleration in chondrocyte differentiation and initiation of primary ossification centres, and both accelerated
and abnormal mineralization of bone (336).
Pthrp/Pth double mutants also show the Pthrp null chondrodysplasia combined with undermineralized skeleton
of the Pth null; they too are globally smaller than WT
siblings (439). However, the Pth null has mild defects in
osteoblast parameters that appear only within the
C57BL/6 genetic background and not the Black Swiss
background (see sect. VC).
Pth1r nulls have a similar global phenotype as the Pthrp
nulls, including accelerated endochondral ossification, dysplasia, and ossification of the cartilaginous portions of ribs;
however, they are globally much smaller than their WT
siblings (360). They also have a greater deficit in skeletal
mineral content than that caused by loss of either ligand
alone (336, 340). This may result from their much lower
ionized calcium level, combined with loss of the effects of
PTHrP or PTH to stimulate osteoblasts.
There are other differences in the skeletal phenotypes of
Pth1r null and Pthrp null fetuses. Whereas Pthrp null fetuses have normal expression of osteoblast genes, and normal or increased mineralization (359), Pth1r null fetuses
show reduced expression of collagenase-3, osteocalcin, and
osteopontin and have significantly reduced mineralization
(359). The explanation may be the increased circulating
levels of PTH in Pthrp null fetuses. The chondrocytic portion of the growth plate is avascular, which will prevent
systemic PTH from compensating for loss of PTHrP production and its local actions within chondrocytes. But systemically high PTH levels should easily reach the highly
vascular bone compartment to maintain apparently normal
expression of osteoblast genes. Such actions of PTH are lost
in Pth1r nulls, resulting in downregulation of these osteoblast-specific genes. Furthermore, the Pth1r null skeleton is
undermineralized despite a relative upregulation in placental calcium transfer (as described in sect. VIB). This likely
indicates that, notwithstanding relatively increased delivery
of calcium from the maternal circulation in Pth1r nulls
compared with their WT littermates, calcium cannot be
incorporated into bone without NH2-terminal actions of
PTH or PTHrP, so it returns to the maternal circulation.
An activating mutation of the PTH1R creates the opposite
phenotype of the Pth1r null fetuses, exaggerating what are
likely the normal NH2-terminal actions of PTHrP to delay
chondrocyte hypertrophy. The result is a largely cartilaginous at birth (596). Its appearance is similar to the effect of
PTHrP overexpression within the developing skeleton
(726).
D. Role in the Neonate
Pthrp/Pth double mutants and Pthrp/Hoxa3 double mutants die soon after birth (336, 341, 439). This is likely a
consequence of the lower blood calcium that these double
mutants must have, in addition to the early mortality conferred by ablation of Pthrp or Hoxa3 alone. In the original
C57BL/6 background, Pth1r null fetal mice die at E11.5–
12.5 due to widespread cardiomyocyte apoptosis, which
implies important roles for PTH1R in early embryogenesis
(532). When crossed into the outbred Black Swiss strain,
they survive to the end of gestation before dying promptly
in a manner similar to Pthrp nulls (340, 360). Consequently, there are no neonatal data on the effects of combined loss of PTH and PTHrP.
E. Human Data
As noted earlier, human trophoblasts express PTH1R (82,
183), which implies that PTH or PTHrP exert NH2-terminal biological effects within the placenta. Loss of PTH1R
causes Blomstrand chondrodysplasia, which is characterized by a chondrodysplasia quite similar to what has been
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CHRISTOPHER S. KOVACS
found in Pth1r null fetal mice (296, 489). It is embryonically lethal; therefore, there are no cord blood measurements of calcium, phosphorus, etc.
F. Summary of Key Points
PTH and PTHrP have additive effects on the serum calcium
level, and in turn affect mineralization of the fetal skeleton.
Loss of PTH or PTHrP have equivalent effects on the serum
calcium, whereas loss of parathyroids in Hoxa3 null fetuses
or loss of PTH1R causes more significant hypocalcemia.
PTHrP has a more profound effect on regulating endochondral bone development, primarily by regulating the fate of
chondrocytes and the initiation of osteoblast activity. PTH
has more modest effects on osseous portions of the skeleton,
but these become much more apparent after birth. Combining absence of PTHrP with deletion of PTH results in a
skeleton that bears the Pthrp-null chondrodysplasia but is
also undermineralized as in the Pth null or Hoxa3 null
skeleton. Upregulation of PTH likely compensates for loss
of NH2-terminal actions of PTHrP on bone within Pthrp
null fetuses.
VII. CALCIUM-SENSING RECEPTOR
CaSR is considered here because it plays a pivotal role in
regulating mineral and bone homeostasis in the adult,
through its regulation of PTH synthesis and release, and
renal tubular handling of calcium. Furthermore, calcium
functions like a calciotropic hormone by binding to its receptor in different tissues, including parathyroids, bone,
kidneys, and placenta. Calcium appears to have direct actions that influence the function of osteoblasts and chondrocytes.
A. Regulation of Serum Minerals
As discussed in section II, A and B, CaSR does not set the
high level of calcium found in normal fetuses, but it suppresses PTH in response to it. Ablation of one (Casr⫹/⫺) or
both (Casr null) alleles of CaSR results in an equal increase
in the fetal blood calcium above the WT value (FIGURE 3),
and a dose-dependent increase in circulating PTH (FIGURE
4) and calcitriol (338). The increase in calcium depends on
PTH because simultaneous ablation of the PTH1R in Pth1r/
Casr double mutants prevents Casr ablation from increasing the blood calcium above the low level present in Pth1r
null fetuses (338). It is puzzling that ionized calcium of Casr
null fetuses is not higher than in Casr⫹/⫺ fetuses; the intrauterine environment may constrain Casr null fetuses from
achieving the higher blood calcium level that they reach
after birth. CaSR is likely responsible for the ionized calcium level of Pthrp null fetuses being maintained at the
maternal level rather than falling below it, through its ac-
1180
tions to stimulate PTH to achieve the adult set point of
calcium (see also sects. IVA and VA) (338, 340).
It is evident from data discussed earlier that CaSR does not
stimulate PTHrP to maintain the fetal blood calcium, because in Hoxa null fetuses, the ionized calcium is reduced
well below the maternal concentration, PTH is undetectable, and the plasma PTHrP level does not increase in compensation (336, 341). However, CaSR may have effects to
downregulate the expression of PTHrP, because circulating
plasma PTHrP is significantly lower in Casr null fetal mice
compared with their siblings (334). The increased ionized
calcium in Casr nulls may be a factor, or CaSR may directly
regulate PTHrP through their colocalized expression within
trophoblasts and intraplacental yolk sac.
In the adult, ablation of CaSR decreases renal calcium clearance in a dose-dependent fashion (257), leading to hypercalcemia combined with hypocalciuria. However, Casr⫹/⫺
and Casr null fetal mice each have increased amniotic fluid
calcium with no difference between them (338). This striking difference from the adult phenotype is likely the result of
two factors. First, fetal kidneys express very low levels of
Casr mRNA until the first postnatal day (112); consequently, the renal phenotype potentially caused by Casr
ablation is absent. Second, the renal filtered load of calcium
should be increased equally in both genotypes due to their
similar increments in ionized calcium. Therefore, in the absence of modulation by CaSR, the increased filtered load of
calcium simply leads to increased renal excretion of calcium
into the amniotic fluid.
CaSR has been selectively ablated from parathyroid glands
(CasrPara null), but the effect that this may have on fetal
mineral homeostasis has not been reported (109).
An activating mutation of Casr has been described in Nuf
mice (271). No fetal data have been published, but it is
anticipated that they should have a similar phenotype as Pth
null fetuses, with reduced skeletal mineralization and low
PTH.
B. Regulation of Placental Mineral
Transport
As noted earlier, CaSR is expressed in murine trophoblasts
and intraplacental yolk sac cells (337). Placental transport
of 45Ca is significantly and dose-dependently reduced in
Casr⫹/⫺ and Casr null fetuses compared with their WT
siblings (338). It is unknown why placental calcium transfer
is reduced. If the CaSR senses calcium flow within the placenta, or regulates placental PTHrP synthesis, CaSR ablation could explain a decrease in placental calcium transport.
The lower plasma PTHrP levels in Casr null fetuses could
result from either postulated mechanism. Alternatively, the
elevated serum calcium or secondary hyperparathyroidism
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FETAL AND NEONATAL MINERAL METABOLISM
that occur in Casr null fetuses might also lead to downregulation of placental calcium transfer.
Additional studies of Pthrp/Casr double mutant fetuses revealed that although ablation of Casr reduced placental
calcium transport in fetuses that had two normal Pthrp
alleles, ablation of one or both alleles of Casr had no effect
on placental calcium transport when both Pthrp alleles
were ablated (338). This result is compatible with downregulation of Pthrp explaining reduced placental calcium
transport in both Pthrp null and Casr null fetuses.
There have been no studies of the Casr activating mutation
from Nuf mice of placental calcium transport.
C. Regulation of Endochondral Bone
Formation
CaSR is expressed by chondrocytes and osteoblasts, with its
highest expression seen within the hypertrophic chondrocytes (109, 110). Chondrocyte-specific ablation of CaSR
(CasrChon null) caused embryonic lethality with severely
delayed cartilage and bone development (109), but when
the chondrocyte-specific deletion was induced between embryonic day 16 and 18 by use of a tamoxifen-driven promoter, the fetal skeleton was shortened and undermineralized due to delayed terminal differentiation of chondrocytes
(109). These results indicate that CaSR promotes chondrocyte differentiation and that its absence delays differentiation, actions that oppose those of PTHrP within the growth
plate. This is consistent with the known effect of high extracellular calcium concentrations to promote chondrocyte
differentiation (108, 111, 257); ablating CaSR must block
this effect.
Ablation of CaSR has also been carried out in preosteoblasts, immature osteoblasts, mature osteoblasts, and osteocytes (CasrPre, CasrOb and CasrOc null), and in each the
fetal skeleton is unremarkable, although skeletal abnormalities develop later (see below) (109, 169, 549).
Bone resorption is increased in global Casr-null fetuses, as
suggested by the increased circulating PTH levels, increased
excretion of the bone resorption marker deoxypyridinoline
into amniotic fluid, and a significant reduction in the ash
weight and mineral content of the fetal skeleton (331, 338).
Since Casr ablation reduces placental calcium transport and
increases excretion of calcium into the amniotic fluid, the
increased circulating calcium concentration must be maintained through resorption of mineral from the fetal skeleton, thus compromising bone strength and mineral content
before birth. The endochondral skeleton has normal lengths
and microscopic morphology at birth in Casr null mice
apart from the reduced mineral content, and this contrasts
with the significant abnormalities found in the chondrocyte-specific knockout of Casr. The explanation for this
discrepancy is thought to be that, although exon 5 is deleted
in the global Casr null, a splice variant lacking exon 5 is
produced which is able to form heterodimers and provide
calcium sensing in certain tissues of the global Casr null
(187). The chondrocyte-specific deletion of Casr does not
produce that splice variant.
Casr has also been deleted selectively from parathyroid cells
(CasrPara null), but the fetal phenotype of this model has not
been reported (109). It is unremarkable in size at birth but
will likely be found to have hypercalcemia and a demineralized skeleton in utero.
D. Role in the Neonate
Postnatally, global Casr nulls develop severe hyperparathyroidism with grossly enlarged parathyroids, hypercalcemia,
fluid depletion (calcium diuresis), and progressive growth
failure (257). Most die before 3 wk of age, but surgical or
genetic deletion of the parathyroids will enable them to
survive (327). Casr nulls are the murine equivalent of neonatal severe primary hyperparathyroidism in humans.
Casr⫹/⫺ neonates are hypercalcemic but have normal life
spans and fertility, analogous to familial benign hypercalcemia (familial hypercalciuric hypercalcemia, FHH).
The skeleton is normal at birth when Casr is ablated from
early osteoblasts (CasrPre null), but the mice fail to thrive,
develop multiple fractures, and die at 1 mo (109, 169).
There are severe abnormalities including decreased osteoblasts, osteoblast apoptosis, osteoclasts, and increased osteoid, all of which contribute to a soft and disorganized
bone structure (109, 169). In contrast, deletion of Casr
from mature osteoblasts and osteocytes (CasrOb null and
CasrOc null) lead to viable mice that later develop osteoporosis with increased numbers of osteoclasts (109, 549).
Postnatally the parathyroid-specific knockout of CaSR
(CasrPara null) develops severe hypercalcemia, markedly elevated PTH, growth retardation, multiple fractures, a severely demineralized skeleton, and marked delay in osteoblast differentiation (109). These findings suggest that
much of the phenotype of the global Casr null is mediated
by hyperparathyroidism, with little or no effect from Casr
ablation in chondrocytes, osteoblasts, and kidney tubules.
The neonatal phenotype of Nuf mice has not been reported.
At 4 wk of age (the earliest time point described) they have
hypocalcemia, hyperphosphatemia, low PTH, and ectopic
calcifications in multiple tissues; they are also prone to sudden death (271).
E. Human Data
No case reports have described blood calcium or PTH values in fetuses or newborns with inactivating mutations of
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CHRISTOPHER S. KOVACS
CASR. These births are sporadic, and serum calcium is not
routinely measured in healthy newborns. The findings from
Casr⫹/⫺ fetal mice predict that human babies with FHH
will be hypercalcemic in utero and that the cord blood calcium will be increased. Since hypercalcemia begins during
fetal development, this likely explains why individuals with
FHH are adapted to a high ionized calcium, and feel hypocalcemic if their blood calcium is reduced to “normal”
values. This parallels the findings in disorders causing hypocalcemia during fetal development, in which affected neonates, children, and adults may remain asymptomatic for
hypocalcemia (see sect. VE).
Neonatal severe primary hyperparathyroidism is usually
caused by homozygous or compound heterozygous inactivating mutations of CASR (193, 394), and occasionally by
heterozygous mutations through a presumed dominantnegative effect (33, 481). It is usually not identified at birth,
but the findings from Casr null fetal mice predict that hypercalcemia will be present in utero and in cord blood. The
neonate is at very high risk to develop severe and life-threatening hypercalcemia, dehydration, failure to thrive, neurological disturbances, nephrocalcinosis, and skeletal demineralization with fractures. The presentation can be delayed
as much as a month after birth (557), in part because maternal hypercalcemia has a suppressive or constraining effect on parathyroid function in utero (see sect. XIIB). Parathyroids are grossly enlarged and serum PTH is quite high.
This is an almost invariably fatal condition unless parathyroidectomy is urgently performed (57), although some very
rare cases have been reported to spontaneously resolve into
milder hypercalcemia that did not require surgery (57, 405,
562, 719). Bisphosphonates and the calcimimetic drug Cinacalcet have been used to lower the serum calcium temporarily to enable the newborn to have surgery at an older age
(719, 737).
Activating Casr mutations cause autosomal dominant hypocalcemia with hyperphosphatemia, and normal (inappropriately low) PTH; hypercalciuria is variably present
(508, 526). The presentation is expected to be similar to
disorders causing hypoparathyroidism, as described in section VE. Cord blood calcium was in the adult-normal range
in one newborn, but below the expected fetal value, and
recurrent apneic seizures began shortly after birth (670). In
another affected neonate, the cord blood calcium and ionized calcium were both low, but the baby showed no hypocalcemic symptoms (217). The variable postnatal presentation is exemplified by the development of hypocalcemic
seizures at day 7 of age in a baby carrying an activating
mutation of CaSR (113). Subsequent investigations confirmed that his sister (age 3 yr), mother (age 31), and grandfather (age 63) all had the same activating mutation causing
hypocalcemia, low PTH, hypercalciuria, nephrolithiasis,
and nephrocalcinosis (113). The sister subsequently developed tetany but only during episodes of gastroenteritis. Fur-
1182
thermore, a survey of 25 affected patients found that only 2
had developed hypocalcemia within the first 30 days after
birth (540). The overall mean age that hypocalcemia was
diagnosed was 28 yr and only 50% had ever been symptomatic (540).
F. Summary of Key Points
Global inactivating mutations of the CaSR cause hypercalcemia in utero. If homozygous or compound heterozygous mutations are present, then skeletal demineralization may develop prior to birth. Postnatally, single inactivating mutations of CaSR cause benign life-long
hypercalcemia (FHH), whereas two inactivating mutations (and occasionally a single mutated allele) cause lifethreatening severe hypercalcemia and hyperparathyroidism. The mutations may occur de novo or be inherited, in
which case the mother may also have FHH; the effect of
maternal hypercalcemia on the fetus and neonate is discussed in section XIIB.
Activating mutations resemble hypoparathyroidism with
hypocalcemia, hyperphosphatemia, and inappropriately
normal or low PTH; these individuals are indistinguishable
from those with genetic causes of hypoparathyroidism, unless they are tested to demonstrate their abundant PTH
secretory reserve, or genetic testing reveals CASR mutations.
VIII. CALCITRIOL AND VITAMIN D
A. Regulation of Serum Minerals
Consistent evidence from multiple species and models
confirms that vitamin D, calcitriol, and the vitamin D
receptor are not required for the fetus to be able to maintain normal serum mineral concentrations. This includes
severely vitamin D deficient rats (74, 228, 241, 443),
1␣-hydroxylase-null pigs (352, 353), and VdrBos and
VdrLeu null fetuses (342, 376), in which serum calcium,
ionized calcium, and phosphorus were all normal in
utero (FIGURE 4). PTH was also normal in VdrBos and
VdrLeu null fetuses, indicating that absence of VDR did
not induce compensatory secondary hyperparathyroidism, as occurs after birth (342, 376).
Four groups independently made Vdr null mice, of which
only the VdrBos null truly lacks VDRs. The three other models (VdrLeu, VdrMun, and VdrTok) each deleted the exon that
encodes the first zinc finger of VDR, but a splice variant is
secreted which has normal ligand binding ability but no
DNA binding ability (175, 376, 574, 704, 755). Of these
four models, studies have been performed in VdrBos null and
VdrLeu fetuses. Inconsistent data have been obtained from
the VdrLeu model that expresses an aberrant but functional
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FETAL AND NEONATAL MINERAL METABOLISM
VDR (376, 574), and these data contrast somewhat with
data obtained from VdrBos null fetuses that lack VDRs (342,
372).
In the first fetal study that used VdrLeu mice, the investigators obtained VdrLeu null mothers by mating VdrLeu⫹/⫺ females to VdrLeu null males, and then mated those VdrLeu
null mothers to WT males so that all fetuses were
VdrLeu⫹/⫺. The progeny of those studies were compared
with unrelated WT females mated to WT males. The authors observed the fetal plasma calcium to be paradoxically
increased in VdrLeu⫹/⫺ fetuses compared with WT fetuses
from unrelated mothers, but the serum calcium was normalized (reduced) in VdrLeu⫹/⫺ fetuses whose mothers had
been placed on the calcium-phosphate-lactose-enriched
“rescue diet” (574). The ionized calcium (the physiologically relevant fraction) was not measured, nor were the
unrelated WT control mothers and their fetuses studied on
the enriched diet. It is not possible to determine from that
study whether the maternal genotype or the fetal genotype
led to the apparent changes in serum calcium on the normal
diet and a reduction in serum calcium on the rescue diet,
since WT and VdrLeu⫹/⫺ fetuses each came from different
mothers. However, the VdrLeu⫹/⫺ fetuses may also have
aberrant signaling since the alternatively spliced VDR has
normal ligand binding ability and may be able to form
functional heterodimers with RXR. Consequently, the unexpected phenotype of hypercalcemia in the VdrLeu⫹/⫺ fetuses may be due in part to the addition of an aberrant VDR
as opposed to resulting from loss of one normal VDR allele.
Moreover, the fact that the VdrLeu⫹/⫺ and WT mothers
were unrelated raises the concern that the apparent differences in fetal serum calcium were artifacts resulting from
the comparison of unrelated mice. Such artifactual results
have been seen before in other models when unrelated controls have been used.
These concerns appear to have been reaffirmed by a second
study in which VdrLeu null and WT mothers were properly
generated as first degree relatives of each other (376).
VdrLeu null mothers were mated to VdrLeu⫹/⫺ males while
the WT mothers were mated to WT males. In this new
study, with proper related controls, there was no difference
in serum calcium, phosphorus, or PTH of VdrLeu⫹/⫺ or
VdrLeu null fetuses from VdrLeu⫹/⫺ mothers compared with
WT fetuses from WT mothers (376). The results contradict
the earlier study from the same group because VdrLeu⫹/⫺
fetuses were normocalcemic in the new study (376) but
hypercalcemic in the earlier study (574). These recent results are identical to those obtained with the VdrBos model
as noted above, in which WT, VdrLeu⫹/⫺, and VdrLeu null
fetuses each have normal serum calcium, phosphorus, and
PTH. However, in this recent study the investigators reported that while VdrLeu null pups survived, their VdrLeu⫹/⫺
siblings died soon after birth (376). An increased postnatal
mortality of Vdr⫹/⫺ pups has not been seen in the VdrBos
model (192, 342), and in fact VdrBos null mothers nurse the
same number of pups as their WT sisters (192). Increased
mortality of VdrLeu⫹/⫺ mice was also not reported in the
first study from the same group (574).
If VdrLeu⫹/⫺ neonates have a real increase in mortality, it is
conceivable that aberrant signaling and physiology created
by the mutant VdrLeu is responsible for it. However, the
investigators speculated that high maternal levels of calcitriol cross the placenta and lead to increased calcitriol
levels in the VdrLeu⫹/⫺ and VdrLeu null fetuses, which might
be toxic to VdrLeu⫹/⫺ fetuses but not to VdrLeu null fetuses
that cannot respond to calcitriol. Such speculation is not
consistent with the stepwise increase in fetal calcitriol levels
that were seen when WT, VdrBos⫹/⫺, and VdrBos null fetuses
were compared with each other within the same litters;
notably, calcitriol levels remained below the maternal value
in all except VdrBos null fetuses, and the WT calcitriol level
was no different from the value seen in other WT fetuses
from other knockout models in the same genetic background (342). In short, there was no evidence of transplacental passage of calcitriol in the VdrBos model, and earlier
cited studies determined that calcitriol does not cross the
rodent placenta (see sect. IIB) (475). Therefore, it seems
unlikely that maternal calcitriol caused increased mortality
in VdrLeu⫹/⫺ mice.
These data from VdrLeu fetal mice need to be interpreted
cautiously because of possible aberrant signaling in
VdrLeu⫹/⫺ fetuses, and because unrelated controls were
used in the first study. The studies in VdrBos fetuses are not
affected by aberrant VDRs and have compared WT to
VdrBos null fetuses, and VdrBos⫹/⫺ to VdrBos null fetuses,
within their respective litters.
Other investigators have found possible calcitriol-mediated effects on the fetal serum calcium level. Pharmacological doses of calcitriol given to pregnant guinea pigs
increased the fetal serum calcium and phosphorus (164).
However, the observed effect was likely indirect through
a documented increase in maternal serum calcium and
phosphorus; a direct effect on the fetus is unlikely because calcitriol does not cross the rodent placenta (475).
Surprisingly, vitamin D deficiency in fetal guinea pigs
lowered calcitriol but caused a paradoxical increased serum calcium (573). Their pregnant mothers had 25-hydroxyvitamin D levels of ⬃ 400 nM (573), which is in a
toxic range for humans or rodents and, therefore, is not
relevant for vitamin D physiology in those species. Furthermore, with a maternal 25-hydroxyvitamin D level of
400 nM compared with a level of ⬃20 nM on a vitamin
D-deficient diet, the guinea pig studies should be considered as contrasting the effects of maternal hypervitaminosis D to vitamin D deficiency, as opposed demonstrating
effects of vitamin D sufficiency vs. deficiency.
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CHRISTOPHER S. KOVACS
Infusion of an antiserum to calcitriol into fetal lambs for 72
h reportedly lowered the blood calcium level; however, no
data, controls, or details about the antiserum were shown to
confirm that brief statement (564). Direct injection of calcitriol into decapitated rats from thyroparathyroidectomized mothers caused an increase in serum calcium, a decrease in serum phosphorus, and an increase in ash weight;
there was no effect in intact or decapitated fetuses of normal
mothers (103). What those pharmacological effects of calcitriol in decapitated rat fetuses might mean for normal fetal
physiology is not clear.
Bilateral nephrectomy in fetal sheep reduced serum calcitriol by 60% and also lowered the ionized and total
calcium, and increased serum phosphorus and PTH
(455). The abnormal serum calcium and phosphorus
could be corrected by direct administration of calcitriol
to the fetus (455). The investigators inferred that loss of
renal production of calcitriol explained the results rather
than uremia from loss of kidney function. However, the
calcitriol-induced changes in serum calcium and phosphorus are pharmacological effects that were not tested
in intact lambs. Furthermore, the control procedure involved leaving the fetal kidneys in situ while diverting the
ureters so that the urine drained into the fetal peritoneal
cavity instead of the amniotic fluid. Consequently, it is
difficult to interpret these results and the suitability of the
renal diversion procedure as a control for bilateral nephrectomy. In the control procedure, the pooling of urine
in the peritoneum means that fluid, solute, and waste
products are retained in a compartment that may not lead
to the equivalent effects that uremia or absent renal function may have on the fetus.
The results in nephrectomized fetal lambs contrast with
those from nephrectomized fetal rats, in which the serum
calcitriol level fell, but there was no effect on the serum
calcium (105). Species differences may explain why nephrectomy causes hypocalcemia in lambs but not rats.
In summary, vitamin D deficiency in fetal rats, loss of 1␣hydroxylase in fetal pigs, loss of VDR in VdrBos null fetal
mice, and loss of VDR in a recent study from VdrLeu null
fetal mice, have shown that the serum calcium, phosphorus,
PTH, and renal excretion of calcium and phosphorus are
unchanged despite the absence of vitamin D, calcitriol, or
VDR. Data from VdrLeu⫹/⫺ fetal mice are inconsistent between the two studies reported by the same group (376,
574), but it is only their most recent study that provided
data from VdrLeu null fetuses (376). Some of the problems
with the VdrLeu⫹/⫺ fetal data include the presence of a mutant VDR that may cause aberrant signaling, and use of
unrelated control mice in the first study. The remaining
animal data provide evidence that pharmacological doses of
calcitriol can have effects in fetuses that may not be relevant
to normal physiology, while the suitability of a control pro-
1184
cedure for bilateral nephrectomy in fetal lambs clouds the
interpretation of the data from that study.
B. Regulation of Placental Mineral
Transport
Where placental calcium transport has been directly measured, it is clear that calcitriol is not required because the
calcium transport rate was normal in severely vitamin D
deficient rats (228), and increased in both VdrBos and
VdrLeu null fetal mice (342, 376). The placentas of severely
vitamin D deficient rats and both VdrBos and VdrLeu null
fetuses displayed normal expression of calbindinD-9k and
Ca2⫹-ATPase (228, 342, 413, 574, 709), while PTHrP and
TRPV6 were upregulated in both VdrBos and VdrLeu null
placentas (342, 376). These findings indicate that the mechanisms that regulate transplacental movement of calcium
do not require calcitriol. Furthermore, the findings in VdrBos
and VdrLeu null fetuses may indicate that the normal role of
calcitriol is to inhibit placental calcium transport, such that
the rate increases when VDR has been ablated.
In contrast, Care (95) nephrectomized five fetal lambs and
judged that the rate of placental calcium transport fell
across their in situ perfused placentas compared with the
placentas from three intact lambs. A pharmacological dose
of calcitriol was reported to increase the rate of calcium
transfer in three placentas from the nephrectomized lambs
(95). Surprisingly, the data were published only in a review
article and without a statistical analysis, but this author’s
assessment of the raw data has revealed that there was no
statistically significant difference among the three groups
(P ⫽ 0.28), nor in the comparison of the placental calcium
transfer rates before and after treatment (P ⫽ 0.14). The
effect of calcitriol was not assessed in the placentas of intact
lambs. Therefore, the conclusion that nephrectomy reduced
placental calcium transport and that it was rescued by calcitriol is not justified by the available data.
An indirect approach to measuring placental mineral transport was carried out in pregnant ewes. A single infusion of
45
Ca or 32P was followed by daily treatment with pharmacological doses of 1␣-hydroxycholecalciferol or diluent. At
the end of a week-long treatment period, the amount of
45
Ca and 32P was higher in fetal tissues from ewes that had
received the calcitriol treatments (165). The same investigators tested pharmacological doses of calcitriol or diluent
administered to pregnant guinea pigs for 10 days, after
which it was found that the mineral content of the fetuses
was higher in the offspring of calcitriol-treated animals than
in those from diluent-treated animals (164). A dose of 45Ca
was also given to the pregnant guinea pigs, and the amount
of radioactivity in fetal blood samples was increased 30 –90
min later in the fetuses whose mothers had previously received treatment with calcitriol. In both reports, the investigators inferred that placental calcium and phosphorus
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FETAL AND NEONATAL MINERAL METABOLISM
transport had been increased, but the approaches were too
indirect to support that conclusion. Both treatments caused
a significant increase in maternal serum calcium and phosphorus, and in turn that would have increased the availability of mineral to be transferred to the fetuses without invoking any direct actions of 1␣-hydroxycholecalciferol or calcitriol on trophoblasts. A simple calcium infusion has been
shown to acutely increase the flow of calcium from mother
to fetus (106), so a role for calcitriol in stimulating placental
calcium transport cannot be inferred when use of calcitriol
raises the maternal blood calcium.
C. Regulation of Endochondral Bone
Formation
Calcitriol is not required for the formation and mineralization of the endochondral skeleton during fetal development. Severely vitamin D-deficient rats (74, 241, 443)
and both VdrBos and VdrLeu null fetuses (342, 376) have
normal skeletal lengths, morphology, ash weight, and
mineral content at term (FIGURE 11). In the two remaining models, VdrMun and VdrTok null, the fetuses were also
described as normal at birth and until the end of weaning,
but no fetal data have been published (175, 755). Chondrocyte and osteoblast gene expression pertinent to endochondral bone development were also normal within
the growth plates and trabecular compartment of VdrBos
null fetal tibias (342).
Additional studies mated VdrBos⫹/⫺ and VdrBos null sisters
to the same VdrBos⫹/⫺ males to determine whether the maternal Vdr null status influenced the fetal phenotype. WT,
VdrBos⫹/⫺, and VdrBos null fetuses born of VdrBos⫹/⫺ mothers were indistinguishable from each other, but had a
slightly larger size, weight, and skeletal mineral content
than their VdrBos⫹/⫺ and VdrBos null counterparts born of
VdrBos null mothers (342). When adjusted for their smaller
size or weight, the ash weight and mineral content of the
VdrBos null mothers’ offspring were no different from that
of VdrBos⫹/⫺ mothers (342). VdrBos null mothers were also
placed on the calcium-phosphate-lactose-enriched “rescue
diet” and compared with VdrBos null mothers that consumed the regular diet, and this had no effect on the size,
weight, or mineral content of the offspring. Thus fetal loss
of VDR in the VdrBos model had no effect on skeletal development or mineral content compared with respective
littermates, maternal loss of VDR resulted in a slightly
smaller fetal size (but proportionately normal mineral content) that was independent of the fetal genotype, and the
maternal diet had no influence on fetal size or skeletal ash
weight and mineral content (342).
These results contrast with those obtained from the VdrLeu
null model (574), which, as described in section VIIIA, in
the first study mated VdrLeu null mothers to WT males so
that all fetuses were VdrLeu⫹/⫺, and compared them with
fetuses from unrelated WT females mated to WT males.
Fetal skeletal mineralization was reduced in VdrLeu⫹/⫺ fetuses compared with unrelated WT fetuses, but improved
when their mothers were treated during pregnancy with the
“rescue diet” (574). This change was in the opposite direction of the fetal serum calcium, which was reduced when
the VdrLeu null mother was placed on the enriched diet. But
in the second study, the authors mated VdrLeu null mothers
to VdrLeu⫹/⫺males so that the offspring were VdrLeu null
and VdrLeu⫹/⫺, and compared them to WT fetuses from
related WT females mated to WT males. Fetal skeletal mineralization was normal in VdrLeu null fetuses but reduced in
VdrLeu⫹/⫺ fetuses from VdrLeu null mothers, while the previous finding of reduced serum calcium was not seen. When
the mothers were placed on the “rescue diet” during pregnancy, the skeletal mineralization was normal in VdrLeu⫹/⫺
fetuses. As previously postulated in section VIIIA, it is possible that the reduced mineralization of VdrLeu⫹/⫺ compared with VdrLeu null and WT fetuses, which was not seen
in the VdrBos null model, is due to aberrant VDR signaling
introduced by the alternately spliced receptor that is expressed by VdrLeu⫹/⫺ mice. It seems a likely explanation
since the Vdr null skeleton was normal in both the VdrBos
and VdrLeu null null fetuses; it was only the VdrLeu⫹/⫺ skeleton that differed between the two models (342, 376). This
would be made clearer if VdrLeu⫹/⫺ ⫻ VdrLeu⫹/⫺ matings
were done so that WT, VdrLeu⫹/⫺, and VdrLeu null fetuses
can be compared within the same litters. If abnormal VDR
signaling explains the phenotype of VdrLeu⫹/⫺ fetuses, then
WT and VdrLeu null fetuses will have normal skeletons and
mineral content while VdrLeu⫹/⫺ fetuses will continue to be
abnormal. Furthermore, to test the investigators’ hypothesis that the VdrLeu⫹/⫺ skeletal phenotype is caused by increased calcitriol in the circulation of VdrLeu null mothers,
VdrLeu⫹/⫺ fetuses obtained from VdrLeu null mothers
should be compared with VdrLeu⫹/⫺ fetuses obtained from
WT mothers.
Fetal guinea pigs differ from the results described for fetal
rats and mice, in that 8 wk of a vitamin D-deficient diet
caused reduced fetal body weight and total body bone mineral content, and increased osteoid with expansion of the
hypertrophic zone of chondrocytes (573). However, as
noted earlier, the comparison may not be physiologically
relevant because the maternal 25-hydroxyvitamin D was
400 nM on the vitamin D-replete diet and ⬃20 nM on the
D-deficient diet, with fetuses having a 25-hydroxyvitamin
D level of ⬃200 nM when their mothers were on the vitamin D-replete diet. It is a comparison of the skeletal effects
of hypervitaminosis D to vitamin D deficiency, rather than
vitamin D sufficiency to vitamin D deficiency. Furthermore,
the vitamin D-replete and vitamin D-deficient diets were
made by different manufacturers and differed in more than
just vitamin D content, including higher vitamin C and
lower phosphorus content in the vitamin D-deficient diet.
Since the standard and control diet differed in more than
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CHRISTOPHER S. KOVACS
just the nutrient being studied, it is possible that dietary
differences influenced the phenotypes of vitamin D “replete” and deficient guinea pigs.
A
Overall, while there are some differences among the models, vitamin D deficient rats and VdrBos and VdrLeu null null
fetal mice show normal endochondral bone development
and mineralization by term. This is very convincing evidence that calcitriol is not required during fetal development to regulate skeletal development and mineralization.
B
D. Role in the Neonate
C
Multiple models of disrupted vitamin D physiology have
been studied during the neonatal period, including severely
vitamin D-deficient rats (74, 241, 443), four different Vdr
null mice (175, 372, 704, 755), and two different models of
1␣-hydroxylase null (Cyp27b1 null) mice (138, 496). All of
these appear normal at birth, although chemistries and ash
weights have yet to be reported in any of the Cyp27b1 null
models at birth. All models remain phenotypically normal
through most of the succeeding 3 wk that the mother is
lactating. As described earlier, near the time of weaning the
intestines become less permeable to passive absorption of
calcium, and more dependent on active, calcitriol-dependent intestinal calcium absorption. It is at this point in time
that all of these models begin to develop hypocalcemia,
hypophosphatemia, secondary hyperparathyroidism, and
skeletal evidence of rickets (138, 241, 371, 372, 443, 496).
However, a calcium-phosphate-lactose-enriched “rescue
diet” bypasses the need for vitamin D/calcitriol or its receptor and allows each of these models to maintain normal
serum chemistries, skeletal morphology, and skeletal mineral content (24, 64, 65, 140, 154, 175, 371, 372, 496, 638,
639, 704, 755).
D
E
These animal data clearly demonstrate that vitamin D, calcitriol, and its receptor are not required for normal fetal
calcium homeostasis, skeletal development, and mineralization. It is at the time of weaning, when the intestines become
the route of mineral delivery, that calcitriol becomes required. Furthermore, since arguably all of the skeletal man-
100
Calcium (mg/g ash)
90
80
70
60
50
40
30
20
10
0
F
WT
(14)
Vdr+/(23)
Vdr null
(17)
WT
(14)
Vdr+/(23)
Vdr null
(17)
Magnesium (mg/g ash)
20
18
16
14
12
10
8
6
4
2
0
1186
FIGURE 11. Skeletal morphology and mineral content in Vdr null
fetuses. A and B show images of fetal skeletons (ED 18.5) stained
with alizarin red (for mineral) and alcian blue (for cartilage). Skeletal
morphology of the Vdr null was consistently normal, as shown by the
normal crown-rump length, lengths of long bones, and mineralization pattern of the Vdr null (B) and its WT sibling (A). C and D are von
Kossa preparations (counterstained with methyl green) of the upper
halves of fetal tibias (ED 18.5), which include the growth plates and
part of the tibial shafts. The overall morphology of the tibias, the
lengths of the growth plates, and periosteal thickness were normal
in Vdr null (D) compared with WT (C), and a normal amount of
mineral (black) was present in the shafts of the tibias. E shows the
calcium content (by atomic absorption spectroscopy) of Vdr null
fetuses compared with WT and Vdr⫹/⫺ littermates, while F compares the magnesium content among the littermates. The calcium
and magnesium content were normal in Vdr nulls. Ash weight was
also normal (not shown). Scale bar indicates 100 ␮m for C and D.
[Adapted from Kovacs et al. (342).]
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FETAL AND NEONATAL MINERAL METABOLISM
ifestations of vitamin D-related rickets can be prevented by
use of a high-calcium diet, these findings suggest that the
main role of calcitriol with respect to the skeleton is not
through direct actions on chondrocytes or osteoblasts, but
indirect through upregulation of intestinal mineral delivery
to provide mineral for chondrocytes and osteoblasts to use.
This point has been confirmed by the observation that selective expression of Vdr in intestinal cells rescued the skeletal phenotype of Vdr null mice (375, 746), whereas after
selective ablation of Vdr from intestinal cells a rachitic phenotype develops (375), and that phenotype can also be produced by dietary calcium restriction (375). Moreover, ablating Vdr or Cyp27b1 from cells within the skeleton does
not cause rickets either. The skeleton is normal if Vdr is
selectively ablated from chondrocytes, mature osteoblasts,
or osteocytes (375, 421), and a high bone mass phenotype
with reduced resorptive surfaces occurs when Vdr is ablated
from osteoblasts (750). Ablating Cyp27b1 from chondrocytes does not reproduce the rachitic growth plate, whereas
low phosphorus will cause the widened, irregular growth
plate (466). The only evidence from mice supporting a possible direct role for calcitriol or VDR in bone cells is that
Cyp27b1/Vdr null double mutants have a skeletal phenotype that is not completely prevented by the calcium-phosphate-lactose-enriched “rescue diet” (495).
E. Human Data
1. Observational studies and case reports
The most rigorous assessment of the fetal skeleton at birth
involved babies that had died of obstetrical accidents: seven
from mothers with clinically diagnosed osteomalacia and
vitamin D deficiency and eight from otherwise healthy
mothers (427). This 1925 report has been cited as confirming the existence of prenatal rickets, despite the fact that the
paper concluded otherwise. The investigators obtained
plain radiographs of the fetal long bones, and reduced the
femurs to ash to measure the mineral content by atomic
absorption spectroscopy. One “osteomalacic parent” baby
was born prematurely at 7.5 mo and had an ash weight that
was less than half of the other 14 near-term or term fetuses,
which is a normal finding given that more than 80% of the
mineral content of the human skeleton is deposited during
the third trimester (224, 683, 734). But that single fetus’s
low ash weight led to the investigators’ impression that
“osteomalacic parent” babies had softer bone and lower
ash weight compared with normal babies. No statistical
analysis was done, but the raw data were presented in a
table that permits such an analysis by modern methods. The
calcium content (mean ⫾ SE) was 374.2 ⫾ 3.0 per thousand
in “osteomalacic parent” babies versus 371.8 ⫾ 13.9 per
thousand in normal babies. Similarly, the phosphorus content was 189.0 ⫾ 1.1 per thousand in “osteomalacic parent” babies versus 189.6 ⫾ 1.6 per thousand in normal
babies. The radiographs revealed no signs of rickets, and
centers of ossification were normal (427). The authors speculated about “a curious cupping” at the ulnar ends in two
babies born to osteomalacic mothers, but that is now
known to be a normal variant (225). Maxwell et al. (427)
concluded “there is no evidence of prenatal rickets” despite
his initial enthusiasm that he would find such evidence.
Rickets could not be present with normal skeletal mineral
content because unmineralized osteoid is pathognomonic
for the condition.
Epidemiological data show that hypocalcemia and rickets
due to vitamin D deficiency are not usually diagnosed until
weeks to months after birth; the peak incidence is between
6 and 18 mo, even in regions where vitamin D deficiency is
endemic (17, 49, 332, 510). Clinically recognized cases
within the first few weeks after birth are rare and the subject
of individual case reports. Vitamin D deficiency is endemic
in India, so clinical suspicion for its presence is high. Over
16 years Teotia et al. (666) examined 165 babies born of
women with severe osteomalacia. Of these, only six babies
showed abnormalities: three had postnatal hypocalcemia
and three had postnatal rickets without hypocalcemia
(666). In another paper summarizing their experience, Teotia et al. (667) wrote that congenital rickets is “uncommon
and develops only when maternal mineral and vitamin D
stores have been completely exhausted.”
This clinical experience of the natural history of rickets
concurs with the results from the animal models in that the
fetal skeleton should be normal at birth with rickets developing later. As explained earlier, intestinal calcium absorption is largely passive at birth but becomes calcitriol dependent as the baby matures (330, 332, 335); this is why vitamin D-dependent rickets usually develops after calcitriol
becomes required to maintain the supply of calcium for the
developing skeleton. Vitamin D-deficiency rickets also
needs to be distinguished from rickets of prematurity,
which is the result of the preterm skeleton having a much
higher demand for calcium than what the preterm intestines
can absorb from a normal intake of milk or formula (see
sect. IIIE).
A few rare, isolated case reports from the past 100 years
have demonstrated that physical, radiographic, or histological changes consistent with rickets may be detected at birth
(50, 279, 400, 426, 429, 448, 577, 741), although only two
of these reports have biochemical confirmation of low 25hydroxyvitamin D levels (279, 448), while one measured
serum vitamin D which is normally low in cord blood (400).
This is an important distinction because skeletal evidence of
rickets is not synonymous with vitamin D deficiency. Instead, rickets means an undermineralized skeleton that is
primarily caused by deficiency of calcium or phosphorus, of
which vitamin D deficiency or genetic disturbances of vitamin D physiology are but a few of the causes (539). The
differential diagnosis of the rachitic-appearing fetal and
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CHRISTOPHER S. KOVACS
neonatal skeleton encompasses at least 50 different conditions (525), including fetal hypoparathyroidism, skeletal
dysplasias (249), metaphyseal chondrodysplasias (525), hypophosphatasias (555, 584), type V osteogenesis imperfecta (27), and scurvy (73). Consequently, it is possible that
some of these cases of rickets were not due to vitamin D
deficiency. For example, in a 1932 case report, the cord
blood calcium was normal (2.65 mM) in the presence of an
apparently undermineralized skeleton (426). In other cases,
the babies hemorrhaged from multiple orifices before dying,
which suggests other severe disturbances such as scurvy or
vitamin K deficiency (73, 426, 429, 543).
In other reports that described rickets as being present “at
birth,” the diagnosis was actually made within the first or
second week after birth, so the skeletal status at birth remains unknown (50, 59, 191, 451, 502, 585, 586, 666).
“Neonatal rickets” is a more appropriate term for such
cases, as opposed to “congenital rickets,” because the latter
implies that the condition was present at birth. Since the
neonate normally adds ⬃30 – 40 mg/kg of calcium per day
to the skeleton while maintaining appropriate blood calcium and phosphorus levels, any impairment in intestinal
absorption of mineral can quickly lead to progressive demineralization of the skeleton, and hypocalcemia. This is
what happens in rickets of prematurity, for example, which
is not caused by vitamin D deficiency but by the skeletal
demand for calcium being far higher in the preterm baby
than in the normal neonate (see sect. IIIE). In one illustrative case of possible neonatal vitamin D-deficiency rickets,
the clinicians anticipated from the maternal history that
rickets might be present in the infant (585). However, convincing radiographic findings were not present at day 2 but
had developed by day 16, confirming that radiographic evidence can develop rapidly after birth (585).
In many cases of neonatal rickets attributed by the authors
to vitamin D deficiency, the cause was clearly not isolated
vitamin D deficiency. Instead, the mothers had such problems as severe anorexia, malnutrition, malabsorption from
celiac disease or pancreatic insufficiency, lactose intolerance, extreme avoidance of calcium and dairy food sources,
use of drugs that interfere with vitamin D metabolism, or
very low intakes of both calcium and vitamin D (50, 279,
426, 428, 448, 543, 632, 666). This fits with Teotia’s impression that rickets will be present at birth only in extreme
cases of complete nutritional exhaustion (667), or as Maxwell described in 1935, “women who have, in the matter of
diet, been living near the starvation-line.” Vitamin D deficiency itself is normally not sufficient to adversely affect the
development or mineralization of the fetal skeleton. But
combined with other significant maternal nutritional deficiencies, an abnormal skeleton may be present at birth.
Additional case reports and case series have used questionable criteria to make a diagnosis of rickets, whether or not
1188
it is due to vitamin D deficiency. Craniotabes (the subjective
determination that the skull is softer than normal) is a common criterion used alone to make the diagnosis of rickets in
many of these reports, but public health data from several
countries have shown craniotabes to be present in 30 –50%
of healthy infants, and to have no correlation with maternal
or neonatal 25-hydroxyvitamin D levels or skeletal evidence of rickets (55, 122, 136, 320, 515, 516). Consequently, craniotabes is not considered a reliable indicator of
rickets or of vitamin D deficiency; its presentation may be
predicted more by ethnicity and gestational age than vitamin D sufficiency. As mentioned earlier, distal ulnar cupping is a normal variant that should not be used to diagnose
rickets (225). Although a large or slowly closing anterior
fontanelle can be seen with rickets (512, 689), these are
nonspecific changes shared among more than 25 metabolic,
genetic, and skeletal disorders (308, 569). Use of widened
fontanelle alone to indicate vitamin D deficiency led to misdiagnosis and maltreatment of a newborn with hypophosphatasia (449). Among the questionable reports that
claimed vitamin D-deficient rickets to be present at birth are
those that relied on the normal variant of ulnar cupping
(491, 534, 577, 752) or craniotabes (191, 752). One report
noted craniotabes at birth but the skeletal radiographs were
normal, but by 2 wk of age the baby had developed hypocalcemia, hyperphosphatemia, elevated alkaline phosphatase; the hyperphosphatemia indicates hypoparathyroidism and not vitamin D deficiency (191). The biochemical abnormalities normalized in the months following
initiation of vitamin D treatment, but this likely reflected
improvement in the hypoparathyroidism rather than being
proof of vitamin D deficiency.
A single-authored report from Japan (491) is particularly
interesting for the claim that 60 of 255 healthy newborn
infants had radiographic evidence of rickets at the wrist.
The serum calcium, ionized calcium, phosphorus, and alkaline phosphatase were normal in cord blood and in the
newborns over the first 8 days after birth, with no differences between the babies with “rickets” versus those without. The normal chemistries (especially calcium and alkaline phosphatase) are an indication that if the radiographic
findings (ulnar cupping, etc.) represented true abnormalities, they were unlikely to be the result of vitamin D deficiency. Similarly, a single observer reported that rachitic
rosaries and ulnar cupping were present in 75 of 858 consecutive newborn infants in Kuwait, who also had normal
serum calcium, phosphorus, and alkaline phosphatase
(534). Would other observers have agreed with that single
observer’s subjective impression that rachitic rosary was
present? 25-Hydroxyvitamin D was measured only in the
infants with the suspected rib cage abnormality, so it is
unknown whether the postulated rib cage abnormality correlated with objective evidence of vitamin D deficiency. The
normal calcium, phosphorus, and alkaline phosphatase is
evidence that vitamin D deficiency was not the explanation
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FETAL AND NEONATAL MINERAL METABOLISM
for the apparent findings. Another report from Japan suggested that the presence of craniotabes in 22% of consecutive newborns meant rickets, but since larger surveys have
already shown that craniotabes is present in up to 30 –50%
of healthy newborns and to have no association with vitamin D sufficiency, that conclusion is not supportable (46).
A curious report from China examined 124 babies that had
died either before or sometime after birth, with no data
provided about the cause of death, the mothers’ health or
obstetrical history, or the percentage that these babies represented of the catchment population (381). Sixteen fetuses
and 28 neonates were said to have histological evidence of
rickets (381). However, the two main criteria used by the
investigator included an island (apophysis) of chondrocytes
in the diaphysis, and an irregular growth plate (381). Islands or protuberances of cartilage into bone and an irregular growth plate are not standard or defining criteria for
rickets and can be seen in a variety of other congenital bone
disorders, including achondroplasias, osteopetrosis, collagen disorders, and other skeletal dysplasias (16, 88, 268,
503, 550, 680). The defining histological criterion for rickets is increased osteoid (unmineralized bone), but the report
did not describe this. Nor were any data provided to support the hypothesis that the abnormalities were due to vitamin D deficiency and not any of the other conditions on
the differential. Consequently, vitamin D-deficiency rickets
cannot be confirmed to be present with any confidence from
those data.
Overall, out of 100 years of published literature, these reports represent a very uncommon occurrence. For those
cases that may have been truly due to vitamin D deficiency,
it should be clear that these are exceptional cases and not
reflective of vitamin D deficiency during pregnancy that is
commonly seen today. Severe hypocalcemia does not usually occur in vitamin D deficiency because secondary hyperparathyroidism minimizes the fall in serum calcium. But the
descriptions of the mothers in the case reports above underscore how extreme their conditions were, with serum calcium levels often half normal, signs of globally poor nutrition, carpopedal spasms, positive Chvostek’s signs, hypotonia, severe osteomalacia, severe back pain, waddling gait,
etc. The confounding presence of other nutritional deficiencies makes it difficult to be certain the degree to which
vitamin D deficiency contributed to the fetal or neonatal
phenotype. But it seems likely that vitamin D deficiency
alone did not explain these rare presentations.
Additional cohort studies have reported that bone mineral
content in newborns did not differ by measures of vitamin
D sufficiency in the mother or baby (122, 725). Congdon et
al. (122) studied infants born to 45 untreated Asian women,
19 Asian women who had received 1,000 IU vitamin D
during the third trimester, and 12 unsupplemented Caucasian women. Mean cord blood concentrations of 25-hy-
droxyvitamin D were 5.9, 15.2, and 33.4 nM, respectively
(122). There were no significant differences in serum calcium, phosphorus, and alkaline phosphatase, while bone
mineral content of the forearm, measured within 5 days of
birth, did not differ among groups (122). Weiler et al. (725)
measured bone mineral content of the lumbar spine, femur,
and whole body by DXA within 15 days after birth in 50
healthy term infants. Mean cord blood 25-hydroxyvitamin
D was 73 and 36 nM in the sufficient and insufficient Caucasians, 44 nM in Asians, and 27 nM in First Nations infants. Differences in bone mineral content were seen that
were attributable to ethnicity but not vitamin D sufficiency
(725).
Viljakainen et al. (712) divided a cohort of 87 children into
two groups above and below the median cord blood 25hydroxyvitamin D, and measured DXA soon after birth and
at 14 mo. There were no differences in anthropometric
parameters or tibial bone mineral density at either time
point. Tibial bone mineral content and cross-sectional area
of the tibia were slightly lower at baseline in the cohort with
lower 25-hydroxyvitamin D; the difference in bone mineral
content was gone at 14 mo while a difference in crosssectional area persisted. Notably bone mineral content is
affected by bone size or area, whereas bone mineral density
corrects for this. The investigators noted disparities in sex
and size of the newborns above and below the median 25hydroxyvitamin D level, and those disparities may account
for the transient difference in bone mineral content noted at
birth.
If vitamin D deficiency alone could readily cause prenatal
rickets, then even more severe abnormalities would be expected to occur in fetuses and neonates bearing inherited
mutations of vitamin D physiology, including the inability
to synthesize calcitriol (1␣-hydroxylase deficiency; pseudovitamin D deficiency rickets; vitamin D dependent rickets
type I; VDDR-I) or the expression of non-functional vitamin D receptors (hereditary vitamin D-resistant rickets; vitamin D dependent rickets type II; VDDR-II). But these
babies appear normal at birth (apart from alopecia in
VDDR-II) and have normal calcium levels (66, 180, 312,
408, 538, 621, 662, 665). In both conditions the child is
fated to develop hypocalcemia, hypophosphatemia, and
rickets. VDDR-I presents within the first 3–12 mo after
birth; VDDR-II can also present in infancy but has often
been diagnosed during the second year or even later (36, 66,
230, 258, 312, 538, 621, 662, 665, 727). Mortality can also
occur in infancy or childhood due to the severity of the
hypocalcemia (36, 374). Pseudofractures and fractures can
occur in both conditions, but bowing of the long bones and
metaphyseal widening are much more common (36, 66,
180, 230, 258, 312, 408, 538, 621, 662, 665, 727).
Available clinical data from pediatric and older age VDDR
type II children are also consistent with the animal data in
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that calcitriol’s role to regulate intestinal calcium absorption can be bypassed. Repeated intravenous or intracaval
calcium infusions, or high oral dose calcium, have been
shown to normalize calcium, phosphorus, PTH, alkaline
phosphatase; rickets was prevented or healed in these children (36, 259, 312, 727). In one case series, the use of
parenteral calcium caused growth velocity to increase, bone
pain to decrease, and biochemical abnormalities to normalize within a year; it also enabled some affected children to
walk for the first time ever (259). Rickets healed more rapidly in younger patients and when parenteral calcium was
used instead of high dose oral calcium. For VDDR type I,
the high-calcium approach is usually not needed because
physiological doses of calcitriol or 1␣-hydroxyvitamin D
should normalize intestinal calcium absorption (662).
Once weight bearing begins, the natural history of untreated vitamin D-deficiency rickets includes progressive
deformities of the long bones (enlargement of the wrist and
bowing of the distal radius and ulna, and lateral bowing of
the femur and tibia), distorted growth plates, and short
stature (513, 514). These changes are the result of the
softer, malleable structure of rachitic bone which bends in
response to weight-bearing activity, and the resulting varus
and valgus deformities can be very dramatic. Notably, fractures of the long bones are not a dominant feature of rickets,
but when they do occur they are single and not multiple. A
study of 736 Nigerian children aged 18 mo or older found
that previous fracture was present in 9% with active rickets,
7% with early or nearly healed rickets, and 9% of normal
children (668). The other clinical features that predicted
rickets were age ⬍5, height-for-age Z-score ⬍2 standard
deviations, leg pain during walking, wrist enlargement, and
costochondral enlargement (668).
Oddly, despite the evidence that overt rickets is not associated with an increased risk of fracture, vitamin D insufficiency has been proposed to cause marked skeletal fragility
in infants such that it would mimic the multiple fractures
seen in physically abused infants and others with osteogenesis imperfecta (303). Moreover, it has been suggested that
for a 6-mo window of time the infant is transiently susceptible to fragility from vitamin D insufficiency in what has
been termed “temporary brittle bone disease” (504). Since
severe vitamin D deficiency causes slowly developing deformities of the limbs without more fractures than in normal
children (668), how could the more modest condition of
vitamin D insufficiency be a cause of overt skeletal fragility
with multiple atraumatic fractures? This seems a theory
that lacks credible evidence to support it; consequently, it
has been thoroughly discredited by others (181, 182, 286,
287, 414, 629, 637, 651). The Society for Pediatric Radiology and the European Society of Paediatric Radiology
jointly wrote that the diagnosis fails to meet minimum standards of reliable scientific information that can be presented
in the courts of law “because it lacks appropriate grounding
1190
in scientific methods and proceduresѧit is not generally accepted within the field of radiology, but is instead based on
the unsupported speculation and subjective beliefs of a very
small number of medical professionals” (436).
Overall, the human genetic disorders, the population surveillance data on the incidence of rickets, 100 years of case
reports of extreme cases, and much of the clinical observational studies pertaining to vitamin D deficiency, are consistent with the animal models in that hypocalcemia and
rickets are not likely to be present at birth. Instead, hypocalcemia and rickets will develop postnatally in children with
severe vitamin D deficiency or genetic disorders causing loss
of calcitriol or VDR, with the peak age of presentation
occurring not at birth but between 6 and 18 mo of age.
Furthermore, calcitriol’s role in stimulating intestinal calcium absorption can be bypassed by enriching the diet or
administering calcium parenterally. Left untreated, the long
bones are malleable, resulting in limb deformities, short
stature, and eventually osteoarthritic changes from the deformed joints.
2. Interventional studies
One of the earliest randomized trials of vitamin D supplementation still stands as the most definitive because it involved women who were severely vitamin D deficient by
today’s standards (77). Brooke et al. (77) studied 126 Asian
women living in England, 59 who were treated with vitamin
D and 67 controls. The mean 25-hydroxyvitamin D level
was 20 nM at baseline and the treatment group achieved a
level of 168 nM at term; this marked difference was ideal
for determining if correction of vitamin D deficiency resulted in any maternal or fetal benefits. The treatment group
nominally received 1,000 IU vitamin D per day but must
have received 10,000 IU per day or more to have reached
that increment in 25-hydroxyvitamin D. The mean cord
blood 25-hydroxyvitamin D level was 10 nM in control
babies and 138 nM in babies born of the treated mothers.
Cord blood calcium and phosphorus showed no statistically significant differences between the control and treated
babies. Treatment also had no effect on birth weight,
crown-heel length, forearm length, triceps skinfold thickness, and head circumference, but a small decrease in fontanelle diameter was observed (77). There was also a higher
alkaline phosphatase level in control babies, but fractionation confirmed that this was entirely accounted for by the
placental fraction. The babies were examined radiologically, and there were no signs of rickets in either group (77).
Interestingly, at days 3 and 6 after birth, the control group
had a significantly lower blood calcium than in the vitamin
D-replete babies (2.18 ⫾ 0.04 vs. 2.30 ⫾ 0.04 mM, P ⬍
0.05) (77). Five infants in the control group had symptomatic hypocalcemia (irritability and serum calcium ⬍1.8
mM), whereas none of vitamin D-replete babies became
hypocalcemic (77). These results mirror the findings in the
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FETAL AND NEONATAL MINERAL METABOLISM
previously discussed animal studies, in which serum minerals will be normal at birth but fall postnatally when severe
vitamin D deficiency is present. In one of two follow-up
analyses of the same data (which have been mistaken as
separate trials by other reviewers), a slightly greater increment in weight and length over the subsequent 12 mo was
seen in the babies of vitamin D-supplemented mothers (78,
425).
Cockburn et al. (116) randomized 164 women to receive
either 400 IU vitamin D beginning at the 12th week of
pregnancy or nothing. Cord blood 25-hydroxyvitamin D
was 28 versus 20 nM in the treated versus controls, and
there was no significant difference in cord blood calcium or
phosphorus. However, by the sixth day, calcium was significantly higher while phosphorus was significant lower in the
infants whose mothers had received vitamin D compared
with controls. A lower phosphorus is not understandable in
the context of a higher 25-hydroxyvitamin D level.
In a smaller study of 77 women from France, Mallet et al.
(407) administered 1,000 IU vitamin D2 daily during the
third trimester, or a single dose of 200,000 IU vitamin D2 at
the start of the third trimester, or no supplementation (407).
The two treatment arms raised cord blood 25-hydroxyvitamin D to 15.7 and 18.2 nM compared with 5.3 nM in
controls. There were no significant differences in maternal,
cord blood, and day 2 or 6 neonatal calcium levels, hypocalcemic tetany, and skeletal or anthropometric parameters of
the babies.
A randomized study by Delvin of 40 women in France compared 1,000 IU vitamin D3 supplementation daily to placebo starting at 6 mo of pregnancy, and raised the cord
blood 25-hydroxyvitamin D from 17.5 to 45 nM (153).
There were no effects on skeletal parameters or on the cord
blood calcium, phosphorus, or PTH. However, at 5 days of
age, the treated group had a significantly higher serum and
ionized calcium (153).
Marya performed two studies in vitamin D-deficient Asian
women in India (419, 420). In the first study there were
three treatment arms: 800,000 IU vitamin D2 was given in
the 7th and 8th months of pregnancy to 20 women, 1,200
IU vitamin D2 per day were given to 25 women during the
third trimester, and 75 women received nothing. There was
a small increase in uncorrected cord calcium in babies of
high-dose mothers compared with babies of untreated
mothers (2.68 vs. 2.52 mM in untreated), but no change in
serum phosphorus or other benefit (420). The second study
administered 600,000 IU vitamin D2 in the 7th and 8th
months of pregnancy to 100 women and gave nothing to
100 others. A significant increase in uncorrected cord blood
calcium was observed in the treatment group (2.77 vs. 2.57
mM) with no difference in cord blood phosphorus (419). A
slightly greater birth weight, crown-heel length, and head
circumference were also found in the babies from treated
mothers (419). Cord blood 25-hydroxyvitamin D was not
measured in either study. The lack of disclosure of methodological details in the two studies, especially whether and
how the subjects were truly randomized, leaves the results
somewhat questionable.
Yu et al. (756) conducted a randomized unblinded trial in
London of 180 women, with three arms beginning at week
27 of pregnancy: 800 IU vitamin D2 per day, a single dose of
200,000 IU of vitamin D2, or no treatment (756). Mean
cord 25-hydroxyvitamin D was significantly higher in the
two treatment groups compared with the controls (26, 25,
and 17 nM, respectively). However, there were no significant differences in gestational age at delivery, birth weight,
or incidence of low birth weight.
Roth et al. (567) completed a trial in Bangladesh
(NCT01126528) in which 160 women were randomized to
35,000 IU/wk vitamin D3 or placebo beginning at 26 –29
wk until delivery. Achieved cord blood levels of 25-hydroxyvitamin D were 103 versus 30 nM, respectively. There
was no effect on length, weight, head circumference, and
femur length at birth (568). Cord blood calcium levels were
done but have not been reported.
A similar study by Hashemipour et al. (246) in Iran randomized 160 women to receive 50,000 IU vitamin D per
week or 400 IU daily beginning at 26 –28 wk for 8 wk
(246). Cord blood 25-hydroxyvitamin D was 69 versus 27
nM, and cord blood calcium (not corrected for albumin)
was also significantly higher in the vitamin D-treated babies
at 2.48 versus 2.28 mM. There was no difference in the
incidence of hypocalcemia between the groups. Small but
statistically significant increases in newborn weight, length,
and head circumference were seen in the vitamin D-treated
babies.
Grant et al. (233) completed a study in New Zealand (ACTRN12610000483055) that randomized 260 women to
placebo, 1,000 IU vitamin D daily, or 2,000 IU vitamin D
daily. Cord blood 25-hydroxyvitamin D levels were 33, 60,
and 65 nM, respectively. There was no difference in serum
calcium among the three groups at 2.58, 2.62, and 2.60
mM, respectively (233). Postnatally the infants continued in
their treatment arms by receiving placebo, 400 IU, or 800
IU of vitamin D daily. 25-Hydroxyvitamin D rose faster in
the high-dose group, but by 6 mo of age the values were no
different among the three groups of infants (78, 85, and 84
nM, respectively), and there was no difference in serum
calcium at any postnatal time point (233).
Kalra et al. (294) completed a trial in India in which 97
women were randomized to one dose of 60,000 IU vitamin
D in the second trimester or 120,000 IU vitamin D in each
of the second and third trimesters; 43 other women who
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CHRISTOPHER S. KOVACS
received standard care served as controls. Achieved cord
blood 25-hydroxyvitamin D median values were 28.2,
24.1, and 18.5 nM, respectively, and there was no difference in cord blood calcium across the groups (294). Small
increments in length, head circumference, and weight, and a
slight decrease in anterior fonatelle length, were seen in the
both vitamin D groups compared with the nonrandomized
controls (294).
Hollis et al. (262) recently completed two larger trials. The
first (NCT00292591) randomized women at 12–16 wk of
gestation to receive 400, 2,000, or 4,000 IU vitamin D3 per
day; 350 completed the study out of the planned 516 enrollment (262). The primary outcome of the first study was
a combination of the 25-hydroxyvitamin D levels and bone
mineral density of both mother and infant; however, the
BMD values have not been published. Achieved cord blood
25-hydroxyvitamin was 46, 57, and 66 nM, respectively.
The second study (NCT00412087) randomized women at
12–16 wk of gestation to receive 2,000 or 4,000 IU vitamin
D3 per day; 160 completed this study out of the planned
559 (717). Achieved cord blood 25-hydroxyvitamin D was
55 and 68 nM, respectively. The cord blood calcium values
for both studies, and the bone density results for the first
study, have been described verbally by the authors in public
presentations as showing no significant differences (716). In
the first study, 4,000 IU vitamin D had no effect on gestational age at delivery, birth weight, mode of delivery, need
for level II or III neonatal care, cord blood calcium, preterm
birth, preterm labor, preeclampsia, infection, or BMD (261,
262, 716). Post-hoc analyses of both studies have suggested
that some combinations of nonskeletal obstetric or neonatal outcomes may show a trend to improvement with higher
achieved 25-hydroxyvitamin D level, but only after arbitrary exclusion of certain races from the analysis (261, 262,
265, 716).
Whether vitamin D supplementation during pregnancy has
any effect on neonatal bone mass may be determined by the
Maternal Vitamin D Osteoporosis Study (MAVIDOS), in
which more than 1,000 women have been randomized to
receive 1,000 IU vitamin D or placebo daily from 14 wk of
gestation until term (245). The primary outcome is the
whole body bone mineral content of the neonates as assessed by DXA.
Overall, these trials have not shown any compelling evidence that higher dose vitamin D supplementation
achieves any benefit related to calcium and bone metabolism prior to birth, although there is likely a decreased
risk of hypocalcemia after birth when the starting 25hydroxyvitamin D level is very low, as in the Brooke
study. When the basal level of 25-hydroxyvitamin D is
higher than that, there may be no additional benefit from
supplementation.
1192
3. Associational studies
A number of associational studies have recently drawn attention for their inferences that higher intakes of vitamin D
may be needed during pregnancy to ensure optimal bone
health in the fetus, infant, and child. These studies have
examined associations between estimated maternal intake
of vitamin D or single measurements of maternal 25-hydroxyvitamin D during pregnancy, and various skeletal
outcomes in the offspring. In agreement with data from
observational studies and randomized trials discussed
above, there were no significant associations found with
weight, skeletal lengths, or bone mineral density at birth
(281, 285, 363, 399, 460, 713).
A study by Morley et al. (460) concluded that maternal
25-hydroxyvitamin D below 28 nM predicted a slightly
shorter knee-heel length in newborns as assessed by a handheld device. However, left unstated in the abstract was that
the association was not statistically significant after correcting for gestational age of the babies.
Seemingly opposing results were obtained in studies by Mahon et al. and Viljakainen et al. in which increased metaphyseal area of the femur or tibia was associated with lower
(399) or higher (713) maternal 25-hydroxyvitamin D during pregnancy. Curiously, for each study the investigators
concluded that an adverse effect of low 25-hydroxyvitamin
D had been found. Mahon et al. (399) considered greater
cross-sectional area of the femoral metaphysis to be evidence of prenatal rickets in 242 mother-baby pairs assessed
by high-resolution ultrasound, while Viljakainen et al.
(713) considered greater cross-sectional area of the tibial
metaphysis to be an indication of stronger bone in 125
mother-baby pairs assessed by pQCT. More recently Ioannou, together with Mahon and colleagues, adapted their
ultrasound technique to calculate femoral volumes in 357
mother-baby pairs (281). Maternal 25-hydroxyvitamin below 75 nM was associated with slightly smaller femur
length, width, and volume, an association that is in the
opposite direction from the original report by Mahon and
co-workers (281). Although maternal 25-hydroxyvitamin
D level predicted fetal femoral volume in a univariate analysis, there was no association after adjusting for maternal
height and skinfold thickness (280). The seemingly contradictory results in the studies by Mahon, Viljakainen, and
Ioannou could indicate that vitamin D has complex, sitespecific effects on skeletal development, or these could be
chance findings with no true causal link between maternal
25-hydroxyvitamin D during pregnancy and the areas or
volumes of fetal long bones.
A study by Javaid et al. (285) has drawn much attention. In
198 mother-child pairs there were no significant associations between maternal serum 25-hydroxyvitamin D during
pregnancy and birth weight, length, placental weight, abdominal circumference, head circumference, or cord blood
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FETAL AND NEONATAL MINERAL METABOLISM
calcium (285). There were also no associations of maternal
25-hydroxyvitamin D with skeletal and anthropometric parameters in the infants at age 9 mo. But at 9 years of age,
children whose mothers had had a 25(OH)D below 27.5
nM during pregnancy had a slightly but significantly lower
bone mineral content by DXA than children of mothers
whose 25-hydroxyvitamin D had been ⱖ50 nM. These data
confirm the theory that vitamin D exposure during fetal
development programs bone mass attained later in childhood (123). However, a recent study by Lawlor et al. (363)
analyzed ⬃20 times the number of mother-child pairs than
Javaid (3,960 vs. 198) and found no significant association
between maternal 25-hydroxyvitamin D during pregnancy
and offspring bone mass at age 9 years, as assessed by DXA.
Lawlor et al. (363) had almost eight times the number of
women with 25-hydroxyvitamin D below 27.5 nM (220 vs.
28 mothers) and should have had greater power to detect an
association. But the contradictions continue, because Lawlor’s colleagues originally reported from the same database
that estimated maternal UVB exposure in the third trimester
of pregnancy in 6,995 mother-offspring pairs positively
predicted offspring bone mass at age 9 years as assessed by
DXA (593). The difference between the two studies by Lawlor and colleagues may be that estimated UVB exposure is a
less accurate variable than the measurement of 25-hydroxyvitamin D. Overall, it is challenging to conclude anything at present from these contradictory studies, which
could be reporting chance associations rather than indications of causal links.
These associational studies are confounded by factors that
contribute to low maternal 25-hydroxyvitamin D, including maternal obesity, lower socioeconomic status, poorer
nutrition, and lack of exercise, prenatal care, vitamin supplementation, etc. Consequently, a lower value of 25-hydroxyvitamin D may simply predict a less healthy pregnant
woman, rather than revealing cause-and-effect results of
low maternal 25-hydroxyvitamin D on fetal bone health.
Furthermore, the mother’s 25-hydroxyvitamin D value in
pregnancy may predict factors that will remain unchanged
and be shared or held in common with the child (lower
socioeconomic status, poorer nutrition, obesity, etc.), in
turn influencing childhood growth and bone mass at age 9
years without invoking a causative role for 25-hydroxyvitamin D exposure prior to birth.
Some of the associational studies have much larger sample
sizes than in the clinical trials of vitamin D supplementation, and therefore may be detecting real differences that the
trials could not. Alternatively, the associational studies may
simply be reporting “noise” caused by residual confounding, which in turn could explain the contradictory findings.
A basic principle of associational studies is that causation
cannot be proven from the results, but hypotheses may be
raised that warrant further testing.
F. Summary of Key Points
Overall, the available animal and human data indicate that
cord blood calcium, phosphorus, and skeletal morphology
and mineral content should be normal at birth despite vitamin D deficiency, VDDR-I, or VDDR-II. In rare instances
where the mother is more profoundly unwell due to vitamin
D deficiency combined with global malnutrition or other
factors, the baby may develop skeletal signs of rickets that
are detectable at birth. Consistently, it is after birth that
hypocalcemia and rickets will develop if severe vitamin D
deficiency persists, or VDDR-I and VDDR-II are not recognized and treated. The peak incidence of vitamin D deficiency rickets is at 6 –18 mo after birth. Larger-scale clinical
trials of vitamin D supplementation during pregnancy that
systematically measure skeletal mineral content and morphology in newborns, and larger and carefully executed
associational studies, are needed to be certain whether vitamin D deficiency in utero has any effects on skeletal mineralization at term or later in childhood. Several systematic
reviews agree with the conclusion that current data are
insufficient to know if vitamin D supplementation during
pregnancy confers any skeletal or nonskeletal benefits on
fetuses (244, 560, 561).
IX. CALCITONIN
Ctcgrp encodes both calcitonin and calcitonin gene-related
peptide-␣, and ablation of it results in Ctcgrp null fetuses
appearing in the expected Mendelian ratios. In contrast,
ablation of the calcitonin receptor (Ctr) causes embryonic
lethality at mid-gestation for unknown reasons (134, 361).
Ctcgrp null mice live normal lifespans and are fertile, but
show a small but significant reduction in the number of
viable fetuses the day before expected birth compared with
Ctcgrp⫹/⫺ mothers (430). Calcitonin is expressed in the
endometrium during the time of implantation (349), and
attenuation of its expression blocks implantation of the
early embryo (760). Taken together, these findings may
indicate that calcitonin plays critical roles during implantation and during development until mid-gestation. That
Ctcgrp null fetuses do not experience embryonic lethality
may indicate that maternal calcitonin compensates for its
absence in the early embryo before the placental barrier is in
place, whereas such rescue would not be possible in Ctr
nulls due to lack of the receptor. Alternatively, Ctr may also
mediate signaling by unknown ligands that are critical during embryonic development.
A. Regulation of Serum Minerals
There is some evidence for calcitonin having a role in regulating fetal mineral concentrations. Infusion of calcitonin
antiserum into fetal rats at day 21.5 of gestation caused a
0.16 mM increase in blood calcium 1 h later (200), while
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CHRISTOPHER S. KOVACS
pharmacological doses of calcitonin injected into fetal rats
or lambs caused hypocalcemia and hypophosphatemia (41,
211). However, when calcitonin deficiency was created by
fetal thyroidectomy followed by thyroxine replacement, the
blood calcium in fetal lambs was unaltered (97).
The potential role of calcitonin has been investigated in
Ctcgrp null fetal mice. Serum calcium and phosphorus were
normal, but serum magnesium was significantly reduced
compared with the littermates (430). This finding also occurred in Ctcgrp null fetuses from both Ctcgrp⫹/⫺ and
Ctcgrp null mothers (430) and indicates that loss of either
calcitonin or calcitonin gene-related peptide-␣ affects magnesium homeostasis.
Calcium, phosphorus, and magnesium content of amniotic
fluid were normal in Ctcgrp null fetuses. PTH showed a
nonsignificant increase among Ctcgrp⫹/⫺ and Ctcgrp null
fetuses compared with WT.
B. Regulation of Placental Mineral
Transport
A reduction in placental mineral transport has been inferred
from intact lambs in which pharmacological doses of calcitonin blunted the effect of PTHrP1–34 and PTHrP1–36 to
increase the mineral content of the skeleton at term (40).
However, that indirect measurement could have resulted
from reduced incorporation of calcium into bone, as opposed to a specific reduction in placental calcium transport.
In contrast, fetal thyroidectomy with subsequent thyroxine
replacement did not alter placental calcium transport in
sheep (298).
Placental transport of calcium was unaltered in Ctcgrp null
fetuses from both Ctcgrp⫹/⫺ and Ctcgrp null mothers (430).
Magnesium transport has not been measured.
C. Regulation of Endochondral Bone
Formation
Endochondral bone development appeared normal in
Ctcgrp null fetuses as assessed by examination of whole
mount fetuses, tibial histomorphometry, and gene expression within the developing endochondral skeleton (430).
The ash weight and calcium content were also normal, but
there was a modest but statistically significant reduction in
skeletal magnesium content of Ctcgrp null fetuses which
paralleled the reduction in serum magnesium (430).
D. Role in the Neonate
No disturbances in mineral metabolism have been reported
in Ctcgrp null neonates.
1194
E. Human Data
Genetic calcitonin deficiency has not been described in humans. A few early studies suggested that the normal high
calcitonin levels in the neonate could contribute to hypocalcemia in preterm infants (558, 583, 595). However, subsequent studies found that gestational age at birth was a more
significant determinant of neonatal calcitonin levels than
the postnatal fall in serum calcium or the development of
hypocalcemia (444, 558, 707). Therefore, the available human data suggest that calcitonin may have little role in
mineral homeostasis of the neonate.
F. Summary of Key Points
Calcitonin may play critical roles during implantation and
early embryonic development, which could explain the embryonic lethality caused by loss of CTR. Loss of calcitonin
and calcitonin gene-related peptide-␣ caused a selective reduction in serum magnesium and skeletal magnesium content, but no other disturbance in bone and mineral metabolism. Whether loss of calcitonin or calcitonin gene-related
peptide-␣ exerts this effect on magnesium is unknown.
X. FIBROBLAST GROWTH FACTOR-23
Fibroblast growth factor-23 (FGF23) plays a key role in
regulating phosphorus metabolism in postnatal mice and
humans. FGF23 forms a complex with membrane-bound or
soluble forms of its co-receptor Klotho and one of four FGF
receptors (usually FGFR1c), and downregulates expression
of sodium-phosphate cotransporters 2a and 2c (NaPi2a and
NaPi2c) within the proximal renal tubules (350, 614, 699,
731). It also inhibits the renal 1␣-hydroxylase (Cyp27b1),
increases expression of 24-hydroxylase (Cyp24a1), inhibits
PTH, and inhibits intestinal expression of NaPi2b (161,
267, 415, 614). Loss of FGF23 leads to impaired renal
phosphorus excretion, hyperphosphatemia, increased calcitriol, increased intestinal phosphorus absorption, skeletal
abnormalities, extraskeletal calcifications, and early mortality (161, 267, 415). Conversely, excess FGF23 causes
enhanced renal phosphorus excretion, hypophosphatemia,
low calcitriol, reduced intestinal phosphorus absorption,
and rickets or osteomalacia (19, 161, 267).
A. Regulation of Serum Minerals
Intact FGF23 is undetectable in Fgf23 null fetal sera, but
serum phosphorus and calcium, amniotic fluid phosphorus
and calcium, calcitriol, and PTH are all normal (395). Conversely, Phex null male fetuses (also known as Hyp fetuses)
have 7.8-fold increased intact FGF23 in one study (395)
and 1,000-fold increased in another (484). Despite elevated
FGF23, Phex null males have normal serum phosphorus
and calcium, amniotic fluid phosphorus and calcium, and
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FETAL AND NEONATAL MINERAL METABOLISM
PTH (395, 484). Serum calcitriol was halved in Phex null
males compared with WT fetuses (395, 484).
Fetal kidneys showed abundant expression of Klotho,
NaPi2a and NaPi2c, Cyp27b1, Cyp24a1, and Fgfr1-Fgfr4
(395). But in Fgf23 null fetal kidneys, the only change in
expression was downregulation of Cyp24a1 (395). Conversely, in Phex null male fetal kidneys there was significantly increased expression of Cyp24a1 (395, 484) and decreased expression of Klotho and Fgfr2 (395), whereas all
other FGF23 targets showed no changes (395, 484).
These results show that neither loss nor excess of FGF23
disturbs serum phosphorus or renal excretion of phosphorus into amniotic fluid, but excess FGF23 did lower calcitriol, likely through increased Cyp24a1-mediated catabolism. The relative lack of responsiveness to deficiency or
excess of FGF23 was not due to failure of expression of
FGF23 target genes.
B. Regulation of Placental Mineral
Transport
Murine placentas showed abundant expression of Klotho,
NaPi2a, NaPi2b, NaPi2c (NaPi2b expression was greater
than NaPi2a and NaPi2c), Cyp27b1, Cyp24a1, and Fgfr1Fgfr4; there was also low level expression of Fgf23 (395).
But in Fgf23 null fetal placentas the only significant change
was loss of Fgf23 (395). Conversely, in Phex null male
placentas, there was significantly increased expression of
Cyp24a1 (395, 484) and a threefold increase in Fgf23 (P ⫽
0.054) (395), whereas all other FGF23 targets showed unchanged expression (395, 484).
Placental transport of 32P was assessed using the procedure
for intact fetuses. Fgf23 null and Phex null male fetuses
showed a normal rate of placental 32P transport at 5 min,
confirming that neither absence nor excess of FGF23 affected placental phosphorus transport (395).
Therefore, FGF23 does not regulate placental phosphorus
transport, despite expression of the relevant FGF23 target
genes within the placenta.
C. Regulation of Endochondral Bone
Formation
In Fgf23 null and Phex null male fetuses, endochondral
development appears normal the day prior to birth. Tibias
showed no alteration in the length or cellular morphology
of the cartilaginous or osseous compartments, and a normal
distribution of mineral (395). Skeletal ash weight and ash
mineral content (calcium, magnesium, phosphorus) were
also unchanged in Fgf23 null and Phex null male fetuses
compared with their WT siblings (395).
D. Role in the Neonate
Scant data are available pertaining to the role of FGF23 in
phosphorus metabolism of the neonate. In WT mice,
FGF23 spikes to 10-fold higher than adult values within 12
h after birth, concurrent with a transient increase in serum
phosphorus (34). But this increase appears nonessential because Fgf23 null mice have normal serum calcium and phosphorus at delivery (395) and 1 day after birth (615). Hyperphosphatemia and increased calcitriol are evident in Fgf23
nulls at 10 days after birth, and hypercalcemia by 2 wk
(615). Reduced renal phosphorus excretion is present at 3
wk after birth in Fgf23 nulls (the earliest time point studied)
(626). Fgf23 nulls are born with normal length and weight
but are smaller by 2 wk of age (615). By 3 wk of age, the
skeleton is undermineralized and progressive limb deformities develop, but whole body mineral content is increased
due to excessive mineralization of soft tissues, including
heart, lungs, and kidneys (468, 615, 626, 627). Mortality is
100% by 10 –12 wk (468, 615, 626, 627).
Phex null mice are similarly indistinguishable from their
WT siblings at birth, and as adults they are commonly identified by low serum phosphorus rather than genotyped. Although serum phosphorus is normal at birth (395), Phex
null pups surprisingly develop modestly increased serum
phosphorus compared with WT from 2 h until 10 h of age
(34). Hypophosphatemia begins at 24 h and is accompanied
by increased FGF23, low calcitriol, and high PTH (34). By
72 h, the tibias display slight shortening, normal bone volumes, but fewer osteoblasts and osteoclasts within the trabecular compartment (34).
The neonatal findings reveal that absence or excess FGF23
exert powerful effects on mineral metabolism in the neonate, in striking contrast to a lack of effect in the fetus.
E. Human Data
Intact FGF23 rises from the low levels in cord blood to
reach adult values by 5 days after birth (661). Genetic loss
of FGF23 action causes familial hyperphosphatemic tumor
calcinosis, which commonly presents during the first two
decades of life with calcific tumor masses, hyperphosphatemia, elevated calcitriol, and normal or elevated serum
calcium (19, 161, 267, 494, 531). It has presented as early
as 18 days after birth with a calcific mass and hyperphosphatemia (527, 628) and as late as age 65 (494). Specific
causes include missense mutations in FGF23, inactivating
mutations in GALNT3 which normally O-glycosylates
FGF23 to be prevent its premature cleavage and inactivation, and inactivating mutations in KLOTHO which make
FGF23 unable to activate its receptor (19, 161, 267).
Hereditary disorders of excessive FGF23 action include Xlinked hypophosphatemic rickets, autosomal dominant hy-
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CHRISTOPHER S. KOVACS
pophosphatemic rickets, autosomal recessive hypophosphatemic rickets, and McCune-Albright syndrome (267).
All display hypophosphatemia, relatively high FGF23, inappropriately normal or low calcitriol, normal to low PTH,
normal calcium, myopathy, and rickets or osteomalacia
(19, 161, 267). X-linked hypophosphatemic rickets is the
seminal example of excess FGF23 action. It can be diagnosed during the first year when suspected by family history
and early onset of hypophosphatemia (452), but in the absence of family history it more often presents at 2 or 3 years
of age with bowing of the long bones or growth failure, or
at later ages with short stature (102, 172, 588). In four cases
in which affected parents prompted an early diagnosis, serum phosphorus was low in all four between 2 and 6 wk of
age, tubular reabsorption of phosphorus was low at 9 days
of age in one but normal in the others until 6 mo, and
radiographic findings of rickets did not develop until 3– 6
mo (452).
explain why loss of the estrogen receptor has no obvious
effect during fetal development. Deletion of RANKL results
in osteopetrotic changes in mice by 2 days after birth,
whereas growth and serum calcium, phosphorus, and alkaline phosphatase remain normal until weaning (322). Deletion of RANK similarly leads to a normal appearance at
birth. However, the mice are stunted and osteopetrotic by 3
wk of age, tooth eruption fails to occur, and they also develop hypocalcemia, hypophosphatemia, and secondary
hyperparathyroidism (370). An opposing phenotype of severe osteoporosis with arterial calcifications occurs in mice
lacking OPG (85, 447). Although grossly normal until a
month of age, slight trabecular and cortical thinning are
evident in femurs and vertebral bodies within 7 days after
birth, and fractures of spine and long bones can occur before weaning (85). No studies have been done in fetuses.
F. Summary of Key Points
The only human data come from case reports, such as a
man with an inactivating mutation of the estrogen receptor who had normal birth weight, length, and early development (630). He later presented with a severe form of
osteoporosis.
FGF23 does not contribute importantly to fetal regulation
of serum phosphorus, placental phosphorus transport, or
skeletal development and mineralization. Its effects may be
curtailed by the limited role that the fetal kidneys play in
mineral homeostasis. FGF23 begins to have significant effects within days after birth such that deficiency or excess of
FGF23 alter serum phosphorus and calcitriol, although
clinical manifestations may not appear until years later.
XI. SEX STEROIDS
A. Animal Data
Mice lacking estrogen receptor alpha or beta, or the aromatase, have normal skeletal lengths at birth and do not develop altered skeletal metabolism until several weeks after
birth (125, 391, 462, 711, 738). At 3 wk of age (the first
postnatal measurement) in male estrogen receptor knockout mice, there was no difference in total weight, crownrump length, femur length, and bone density at multiple
skeletal sites (711). Therefore, it is likely that no difference
is present at the end of fetal development and that mineral
metabolism is undisturbed through at least 3 wk of age.
However, no fetal studies have been done.
Estradiol exerts its effects on osteoblasts in part by downregulating the expression of receptor activator of nuclear
factor kappa-B ligand (RANKL) and upregulating the expression of its decoy receptor osteoprotegerin (OPG). The
RANKL-OPG-RANK system plays a critical role in regulating the formation, recruitment, and function of osteoclasts within the adult. However, it may be relatively unimportant from the perspective of the fetal skeleton and
regulation of mineral homeostasis, and in turn, this may
1196
B. Human Data
C. Summary of Key Points
Overall, although estradiol and testosterone play important
roles in regulating calcium and bone metabolism in the
adult, in part through regulating RANKL and OPG, the
available evidence is compatible with these hormones (and
the RANKL-OPG system) being unimportant for mineral
and bone metabolism in the fetus and neonate. Detailed
study of bone and mineral metabolism in these models during fetal development is needed.
XII. FETAL AND NEONATAL RESPONSES
TO MATERNAL MINERAL
DISTURBANCES
A. Primary Hyperparathyroidism in the
Mother
1. Animal data
Maternal hypercalcemia adversely affects fetal parathyroid
function. An acute infusion of calcium raised the serum
calcium of pregnant ewes from 2.25 to 3.75 mM, and this in
turn caused an increase in the fetal serum calcium from 3.00
to 3.5 mM and a ⬎50% fall in the fetal PTH level (295). In
contrast, exogenous PTH administration to the mother
failed to raise the fetal serum calcium level (295), consistent
with the failure of PTH to be transported across the placenta. These results suggest that maternal hypercalcemia
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FETAL AND NEONATAL MINERAL METABOLISM
will increase the fetal blood calcium and, in turn, suppress
the fetal parathyroids. Furthermore, hypercalcemia within
the fetal circulation did not suppress active transport of
calcium across the perfused guinea pig placenta (433). The
lack of a compensatory decrease in placental calcium transport means that the effect of maternal hypercalcemia can be
magnified to cause fetal hypercalcemia and suppression of
fetal parathyroid function.
2. Human data
Maternal hypercalcemia likely increases the flow of calcium
across the placenta, and in turn it may lead to a higher than
normal cord blood calcium (610). Importantly, maternal
hypercalcemia suppresses the fetal parathyroids and can
cause significant fetal morbidity and mortality in up to 80%
of cases (597). These outcomes include stillbirth, miscarriage, and neonatal tetany. Comparison of older to more
recent case series suggest that these outcomes have improved (609); however, a recent sobering report found that
fetal death occurred during the second or third trimester in
30 of 62 medically managed cases, representing a 3.5-fold
increased risk of fetal mortality compared with surgically
treated cases (477).
After experiencing higher than normal calcium levels in
utero, the neonatal calcium level may decline more slowly
after birth (610). Failure of the neonate’s parathyroids to
upregulate promptly after birth may precipitate hypocalcemia and tetany. In several case series 50% of neonates had
tetany or other complications of maternal hypercalcemia,
while 25–30% of them died (149, 304, 393, 718). Hypoparathyroidism normally resolves by 3–5 mo after birth
(609), but permanent hypoparathyroidism has also occurred (79, 393, 609). Some cases have had delayed onset or
recognition until several weeks or months after birth (282,
469, 688).
A common clinical impression is that mild maternal hypercalcemia during pregnancy may be unlikely to cause fetal or
neonatal problems. However, neonatal hypocalcemia and
tetany have occurred after seemingly mild cases of primary
hyperparathyroidism (522). A twin pregnancy complicated
by maternal hypercalcemia provides a seminal example of
the variability: one neonate remained normocalcemic while
the other had hypocalcemic seizures (431).
B. Familial Hypocalciuric Hypercalcemia in
the Mother
1. Animal data
Study of Casr⫹/⫺ mice, the murine equivalent of FHH, has
shown that fetal PTH is suppressed compared with the PTH
values obtained in fetuses from WT mothers (338).
2. Human data
Despite the seemingly benign nature of hypercalcemia in
adults with FHH, maternal hypercalcemia during pregnancy has caused suppression of the fetal parathyroids, and
subsequent neonatal hypocalcemia and tetany (529, 673,
674). This includes the heterozygous neonate who may still
develop symptomatic hypocalcemia and tetany, before
eventually becoming hypercalcemic when the parathyroids
gain full function and achieve the higher calcium level set by
the mutated CaSR (672).
C. Hypoparathyroidism and Activating
Mutations of the Calcium-Sensing
Receptor in the Mother
1. Animal data
Maternal hypocalcemia compromises the supply of mineral
available to the fetus. Thyroparathyroidectomy has been
used in rats and ewes to mimic the effects of maternal hypocalcemia due to hypoparathyroidism during pregnancy.
These fetal rats (67, 276) and lambs (564) had normal serum calcium levels despite severe maternal hypocalcemia,
although the serum calcium declined during the last several
days of gestation in other studies of parathyroidectomized
rats (106, 208, 220). A normal serum calcium likely requires upregulation of placental calcium transfer to maintain it, and this is supported by the observation that an
acute calcium infusion caused a marked rise in serum calcium of fetuses from thyroparathyroidectomized rats, but
not in fetuses from thyroidectomized rats (106). The fetal
skeleton may also be compromised by resorbing itself to
maintain the normal high level of calcium in fetal blood, as
seen by enlargement of the fetal parathyroids (206, 625),
increased resorption in fetal rat bones that requires intact
fetal parathyroids for it to occur (542), and reduced femur
length and mineral ash content (104). But even with overt
hypocalcemia throughout pregnancy, the neonate can have
a perfectly normal skeleton (217).
2. Human data
When maternal hypocalcemia during pregnancy has been
prolonged and severe, fetal hyperparathyroidism has developed, in turn causing a severely demineralized skeleton, and fractures in utero or during birth (11, 76, 389,
586, 652). Spontaneous abortion, stillbirth, and neonatal
death have also occurred (25, 170, 302). The cord blood
calcium is variably normal, low, or even increased,
whereas the fetal parathyroids may be enlarged and hyperplastic. Although this is secondary hyperparathyroidism in the fetus, the function remains autonomous for a
time, such that even if the fetus is normal at birth, hypercalcemia and skeletal demineralization can develop in the
neonate before the parathyroid function subsides to nor-
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CHRISTOPHER S. KOVACS
mal (672). Avoiding the magnitude of hypocalcemia that
can occur with hypoparathyroidism should prevent these
outcomes during pregnancy.
The same issues apply to activating mutations of the
CaSR in the mother. In two pregnancies of an affected
woman whose serum calcium was below normal despite
supplemental calcium and calcitriol, her babies (who did
not inherit the mutation) had low-normal serum calcium
and elevated PTH at birth (492). This suggests that some
secondary hyperparathyroidism had developed in utero.
But in another report of three pregnancies complicated
by maternal hypocalcemia, the babies were described as
normal at birth, although no cord blood calcium or PTH
values were provided (13). Maintaining the maternal calcium concentration in or near the normal range prevents
the development of fetal hyperparathyroidism (217).
D. Pseudohypoparathyroidism in the Mother
1. Animal data
No animal models have been studied during pregnancy.
2. Human data
In women, hypocalcemia due to maternal pseudohypoparathyroidism during pregnancy can lead to the same
fetal outcomes that have been associated with maternal
hypoparathyroidism, including parathyroid hyperplasia,
skeletal demineralization, and fractures (226, 710). Postnatally, the relatively autonomous parathyroid function
can cause neonatal hypercalcemia before the parathyroids involute. Maintaining normocalcemia in the
mother during pregnancy should prevent these complications.
E. Vitamin D-Related Disorders in the
Mother
1. Animal data
Vitamin D deficiency in the mother will cause low levels of
25-hydroxvitamin D in the fetus, and the consequences of
this have already been addressed in section VIII. The fetuses
show normal serum minerals, PTH, and skeletal lengths
and mineral content.
Despite marked hypocalcemia in VdrBos null mice during
pregnancy, the only abnormality that their fetuses have
shown is a somewhat smaller size compared with their
counterparts from VdrBos⫹/⫺ mothers, with a normal ash
weight and mineral content after adjustment for their
smaller size (342). Serum calcium, phosphorus, and PTH
are all normal (342). Similarly hypocalcemic 1␣-hydroxy-
1198
lase null pigs bore fetuses that had normal serum mineral
concentrations (352, 353). These results indicate that the
fetus will be able to maintain normal serum mineral concentrations and skeletal development despite maternal hypocalcemia. The benign outcome differs from the fetal hyperparathyroidism that can develop in response to maternal
hypoparathyroidism, and this is likely because maternal
hypocalcemia is more modest with disorders of vitamin D
physiology because secondary hyperparathyroidism offsets
the fall in serum calcium. With a more modest decline in
maternal serum calcium, the fetus is less likely to be adversely affected.
2. Human data
The potential effect of maternal vitamin D deficiency during
pregnancy has been addressed in detail in section VIII.
Pregnancies have been uneventful in women who lack 1␣hydroxylase (VDDR-I) (229). Treatment with calcium and
physiological doses of calcitriol or 1␣-cholecalciferol have
been adjusted as needed to maintain a near-normal ionized
or albumin-corrected serum calcium.
Pregnancy has also been uneventful in women who lack
functional vitamin D receptors (VDDR-II) (408). Depending on the nature of the mutation, there may be a partial
response to calcitriol or cholecalciferol or complete loss of
calcitriol-induced effects. The calcium content of the diet
may need to be increased to maintain a near-normal ionized
or albumin-corrected serum calcium.
If maternal hypercalcemia occurs from excess consumption of calcitriol, 1␣-cholecalciferol, or vitamin D (hypervitaminosis D), then fetal parathyroid suppression and
neonatal hypocalcemia and hypoparathyroidism can result. This effect is mediated by the high maternal calcium
concentration, and not directly by the vitamin D metabolites. If women consume modestly high doses of vitamin
D during pregnancy but remain normocalcemic, the neonate is unlikely to be adversely affected based on available clinical data. This was shown by a randomized clinical trial that administered 400, 2,000 or 4,000 IU of
vitamin D daily to women during the second half of
pregnancy (262). No maternal or fetal hypercalcemia
was reported during the study. Maternal 25-hydroxyvitamin D at the end of pregnancy ranged from 79 nM in
the low-dose group to 111 nM in the highest dose group,
while fetal 25-hydroxyvitamin D values in the corresponding offspring were 46 and 66 nM (262). Intense
placental activity of 24-hydroxylase causes preferential
catabolism of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D (as described earlier in sect. IIB2), and
likely explains why the fetal rise in 25-hydroxyvitamin D
was more modest than in the mothers.
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FETAL AND NEONATAL MINERAL METABOLISM
F. Maternal Magnesium Infusions (Tocolytic
Therapy)
Intravenous magnesium is used in pregnant women to inhibit uterine contractions of premature labor and to prevent
seizures from eclampsia. The infusion causes fetal hypermagnesemia, suppressed PTH and parathyroid responsiveness,
and variable effects on the total and ionized calcium of
neonates (130, 159). Prolonged exposure has also caused
defective ossification of bone and enamel in the teeth (358),
and abnormal mineralization within the metaphyses of long
bones (132, 402, 587). Severe hypermagnesemia (⬎7 mg/
dl) is more likely to cause hypotonia, respiratory depression, and bone abnormalities (159, 379, 402, 591).
G. Hypercalcemia of Malignancy in the
Mother
Maternal hypercalcemia in these cases is more extreme and
likely to suppress fetal parathyroid function, thereby provoking neonatal hypocalcemia, tetany, and possibly permanent hypoparathyroidism. Hypercalcemia may be present
in cord blood and the first few postnatal samples (131, 278,
700), but the calcium will fall and may reach severely hypocalcemic values with attendant risk of respiratory distress, tetany, and seizures (9, 454). One of four babies died
where the outcome was reported (454).
H. Pseudohyperparathyroidism in the Mother
Excess maternal production of PTHrP causes pseudohyperparathyroidism, a less common cause of maternal hypercalcemia during pregnancy. It can occur as a result of excess
mammary production of PTHrP and present with hypercalcemia during late pregnancy or in the puerperium (283,
307, 590); it has also occurred in nonpregnant women with
large breasts (418). Placental PTHrP can also cause pseudohyperparathyroidism, as shown by a pregnant woman
who presented with severe hypercalcemia (5.25 mM), undetectable PTH, and a serum PTHrP of 21 pM (173). Six
hours after an urgent C-section, she was profoundly hypocalcemic with undetectable PTHrP and elevated PTH.
Her breasts were of unremarkable size and she did not
breast feed. The rapid change in her status after delivery is
most consistent with the placenta being the source of
PTHrP (173). In all cases of pseudohyperparathyroidism,
hypercalcemia may be present in cord blood (590), and in
turn maternal hypercalcemia increases the risk of neonatal
hypocalcemia and hypoparathyroidism.
I. Maternal Diabetes Causing Fetal and
Neonatal Hypoparathyroidism
Poorly controlled maternal diabetes during pregnancy can
cause hypocalcemia, seizures, and tetany within the first
FIGURE 12. Relative roles of PTH, PTHrP, and calcitriol during fetal and neonatal life. The placenta is the
main source of mineral during fetal life. PTH and PTHrP are expressed within the placenta but may also act on
it from systemic sources to stimulate calcium transfer. The intestines are a trivial source of mineral in the fetus
but are the main source for the neonate. Intestinal calcium absorption is initially passive but later becomes
active, saturable, and calcitriol-dependent in the infant. Within the endochondral skeleton, PTHrP is produced
by proliferating chondrocytes and perichondrial cells (arrowheads) and delays terminal differentiation of
prehypertrophic chondrocytes. PTHrP is also produced within preosteoblasts and osteoblasts and stimulates
bone formation (semicircular arrows). During fetal life PTHrP and PTH act together to maintain high blood
calcium and phosphorus levels to facilitate mineralization; loss of either PTHrP or PTH causes hypocalcemia
and hyperphosphatemia. Calcitriol is not required to regulate blood calcium, endochondral bone formation, or
skeletal mineralization in the fetus. [From Kovacs (329), with permission from Elsevier.]
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CHRISTOPHER S. KOVACS
24 –72 h after birth. Hyperphosphatemia is commonly present, indicating that transient neonatal hypoparathyroidism
is contributing to the hypocalcemia. The mechanism for this
is unknown. One case series found high ionized and total
serum calcium levels in cord blood of infants of diabetic
mothers (684), which could account for suppression of the
fetal parathyroids. But why maternal diabetes should cause
fetal hypercalcemia is unclear, unless some aspect of the
maternal diabetic state leads to increased maternal-fetal
flow of calcium. Another possibility is that glucosuria
caused by uncontrolled diabetes during pregnancy may
cause renal wasting of magnesium and maternal hypomagnesemia, which in turn could cause fetal hypomagnesemia
and parathyroid suppression. However, a small clinical trial
found no effect of postnatal magnesium supplementation
on the incidence of neonatal hypocalcemia in infants of
diabetic mothers (434). Other factors that contribute to
hypocalcemia in babies born of diabetic mothers are associated increased risks of preterm birth, lung immaturity,
and asphyxia.
XIII. CONCLUSIONS
The fetal and neonatal skeletons require adequate mineral delivery to develop and mineralize normally (FIGURE
12). Placental mineral transfer requires PTHrP and possibly PTH, but not calcitriol, calcitonin, or FGF23. PTH
and PTHrP work together to maintain serum calcium and
phosphorus at high levels in utero for optimal bone mineralization, while calcitriol, calcitonin, and FGF23 are
not required. PTH and PTHrP are required to regulate
endochondral bone development during gestation,
whereas calcitriol, calcitonin, FGF23, and the sex steroids are not. During the neonatal period, intestinal calcium absorption becomes dependent on vitamin D/calcitriol. This means that skeletal development and mineralization in turn become dependent on vitamin
D/calcitriol as well. However, this is not an absolute
requirement because the role of calcitriol can be bypassed
by increasing the calcium content of the diet or by administering parenteral calcium.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: C.
Kovacs, Health Sciences Centre, 300 Prince Philip Dr., St.
John’s, NL A1B 3V6, Canada (e-mail: ckovacs@mun.ca).
GRANTS
This work was supported by Canadian Institutes of Health
Research Grants 133413, 126469, and 84253 as well as by
Research & Development Corporation of Newfoundland
and Labrador Grant 5404.1145.102.
1200
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the author.
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