NEW DIRECTIONS IN THE EVALUATION OF
RECURRENT MISCARRIAGE
William H. Kutteh, M.D., Ph.D., H.C.L.D.
Professor of Obstetrics and Gynecology
Vanderbilt University Medical Center
DISCLOSURES
• None
LEARNING OBJECTIVES
At the conclusion of this presentation,
participants should be able to:
1. Discuss the current trends in the diagnosis and
treatment of RPL.
2. Describe the different society definitions of
“pregnancy” and “RPL”.
3. Appreciate the role of genetic testing in
developing a strategy for the evaluation of RPL
4. Understand the effect of maternal age and
number of prior losses on predicting future live
births
CONTROVERSIES in RPL
• How many losses diagnose RPL?
• What counts as a pregnancy loss?
• Should we get karyotypes on parents?
• Should we get karyotypes on POC?
• What is the prognosis for a live birth?
Natural miscarriage history
Reference
Alberman, 1988
(study of female MD)
Wilcox et al., 1988
(preclinical + clinical)
Risk of 1
loss
10.4%
Risk of 2 Risk of 3
Losses Losses
2.3%
0.34%
63/198
31.3%
Kutteh, 1995*
(unselected women)
766/59,035
1.3%
*Considered a minimum estimate as many lost to follow up.
Population 1/3 hispanic, 1/3 White, 1/3 African-American
Kutteh, WH. Williams Obstetrics. Supp 15:1-4, 1995
Theoretical Incidence of RPL
Based on Number of Miscarriages Used to Define
# Miscarriages to Define RPL
Incidence of RPL
Two
1/45
Three
1/300
Four
1/2000
Five
1/13,000
Six
1/90,000
Seven
1/600,000
Eight
1/4,000,000
Incidence based on mean sporadic miscarriage rate of 15% (=μ).
Incidence=μnumber of miscarriages (μ = sporadic miscarriage rate of 15%).
Saravelos SH, Regan LR. Obstet Gynecol Clinics N Am. 2014.
Spontaneous Pregnancy Loss:
Role of Maternal Age
American Society for Reproductive Medicine: Patient’s Fact Sheet: RPL. 2005.
Hassold T et al 1980 Ann Hum Genet 44:151-178
7
Theoretical Incidence of RPL
Based on Maternal Age
Maternal Age
Incidence of RPL
20
1/85
25
1/70
30
1/45
35
1/16
40
1/4
45
1/2
Incidence based on mean sporadic miscarriage rate according to age.
Incidence=μ2 (μ = sporadic miscarriage rate for age).
Saravelos SH, Regan LR. Obstet Gynecol Clinics N Am. 2014.
Maternal Age is Related to Aneuploidy
in oocytes
Aneuploidy Risk
10%
30%
50%
100%
Maternal Age
<35 years
40 years
43 years
> 45 years
Pellester F, Andreo B,Arnal F, Humeau C, Demaille J
Maternal aging and chromosomal anormalities:new data drawn from in
vitro unfertilized human oocytes, Hum Genet 112 : 195, 2003
How many losses? -Traditional
• Three or more
spontaneous,
consecutive pregnancy
losses (fathered by the
same partner)
Williams OB 21st Edition “most generally accepted definition”
How many losses? - ACOG
• “RPL is typically defined as two
or three or more consecutive
pregnancy losses”
• “Patients with two or more
consecutive, spontaneous losses
are candidates for an evaluation
to determine the etiology”
ACOG Practice Bulletin No. 24, February 2001 (withdrawn)
How many losses? - ASRM
• “RPL is a disease distinct from
infertility defined by two or more
failed consecutive failed
pregnancies”
• “Clinical evaluation may proceed
following two first trimester
pregnancy losses”
ASRM Committee Opinion Fertil Steril. 99:63, 2012
ASRM Practice Committee Fertil Steril 98:1103-1101, 2012
How many losses? - Insurance
• After two consecutive
losses, most insurance
companies will pay for a
complete evaluation of
recurrent pregnancy
loss
Kutteh experience over last 20 years of clinical practice.
Does the Number of losses affect the frequency of
abnormal findings in women with RPL?
Frequency of abnormal tests in 1020 women with RPL
EVIDENCE BASED TESTS
INVESTIGATIVE TESTS
Karyotpe parents
Prolactin
Evaluate uterine anatomy
Antiphosphatidyl serine
Lupus anticoagulant
Midluteal progesterone
Anticardiolipin antibodies
Mycoplasma/ureaplasma
Thyroid stimulating hormone
Factor II (prothrombin) DNA
Factor V Leiden DNA
MTHFR/Homocysteine
Christiansen et al. Semin Reprod Med 24;5-16,2006.
Jauniaux etal. Hum Reprod 21:2216-2222, 2006.
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010
Theoretical Incidence of RPL occurring by Chance
for Women with one, two and three miscarriages
AGES (years)
1 miscarriage
By chance
2 miscarriages
By chance
3 miscarriages
By chance
20-24
11%
1.21%
0.13%
25-29
12%
1.44%
0.17%
30-34
15%
2.25%
0.34%
35-39
25%
6.25%
1.56%
Saravelos SH and LiTC. Human Reprod. 27:1882-1886, 2012
Possible RPL Etiologies based on Number of Losses
Frequency of abnormal tests in 1020 women with RPL
# of prior
losses
2
(n=447)
3
4 or more P value
(n=343) (n=230) 2,3,or 4
Evidence based
test results
41%
40%
42%
NS
Investigative test
results
20%
22%
21%
NS
Total abnormal
test results
61%
62%
63%
NS
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.
Spectrum of Pregnancy Loss
•
•
•
•
•
•
Pregnancy of Unknown
Location (PUL)
Early embryonic (< 6 wks)
Embryonic (> 6 to 9 wks)
Fetal loss (> 9 to 20 wks)
Miscarriage (< 20 wks)
Stillbirth (> 20 wks)
Silver et al. Obstet Gynecol 118: 1402-1408, 2011.
What counts as a Loss? -Traditional
• Miscarriage is the
loss of a pregnancy
before 20 weeks of
gestation or less
than 500g
Williams OB 21st Edition “most generally accepted definition”
What counts as a loss? - ACOG
• “Loss of a recognized pregnancy
in the first or early second
trimester <15 wks)”
• “Most are evident by the 12th
week and the demise precedes
clinical features of pregnancy
loss by one or more weeks”
ACOG Practice Bulletin No. 24, February 2001 (withdrawn)
What counts as a loss? - ASRM
• “Pregnancy is defined
as a clinical pregnancy
documented by
ultrasonography or
histopathologic
examination”
ASRM Committee Opinion Fertil Steril. 99:63, 2012
ASRM Practice Committee Fertil Steril 98:1103-1101, 2012
What counts as a loss? - Patient
• A positive pregnancy
test from home (or
their doctors office)
that does not result in
a baby
Kutteh experience over last 20 years of clinical practice
What counts as a loss?- My Opinion
• A pregnancy that is documented by
an appropriately rising quantitative
hCG that fails
• Using this definition there is
< 7% chance of being an ectopic
Kutteh experience over last 20 years of practice
Barnhart KT. Obstet Gynecol 104:50-55, 2004
What is a complete workup? - ACOG
• Karyotypes on both partners
• Uterine cavity evaluation
• Glucose level
• LAC, aCL, β2-glycoprotein
(No inherited thrombophilias)
ACOG Bulletin No. 24, Feb 2001 (withdrawn)
ACOG Bulletin No. 124, September 2011
What is evidence-based?- Genetics
Frequency of abnormal tests in 1020 women with RPL
Control # of prior
losses
0.4% Parental
2
3
>4
P value
n=447 n=343 n=230 2,3,or 4
2.8% 5.4% 5.2%
NS
genetics
7.5%
0.5%
6.7%
3.9%
6.8%
Anatomy
18.7% 18.2% 16.7%
5.0% 2.9% 1.9%
NS
NS
4.2%
NS
NS
NS
Lupus
anticoagulant
Anticardiolipin 15.6% 13.1% 17.1%
TSH
8.1% 6.5% 6.2%
Factor V
8.1%
10.3%
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.
What is a complete workup? - ASRM
• Karyotypes on both partners
• Uterine cavity evaluation
• Prog, PCOS, HgbA1c
• LAC, aCL, antiβ2 GP1
(No inherited thrombophilias)
ASRM Committee Opinion Fertil Steril. 99:63, 2012
ASRM Practice Committee Fertil Steril 98:1103-1101, 2012
Parental Genetic Abnormalities
(found in 3-5% of couples with RPL)
•
•
•
•
•
Reciprocal translocation 59%
Robertsonian translocation 27%
Inversions 9%
Sex chromosome aneuploidy 4%
Supernumerary chromosome 1%
Balanced translocation
Prognosis based on parental karyotypes
The karyotype results from the parents provides
prognostic information for subsequent pregnancies
Parents Karyotype
Subsequent Miscarriage
Reciprocal translocation
Robertsonian translocation
50-70%
30-50%
(Exception is translocation to same chromosome)
Inversions
Normal
30%
30%
Brigham, Hum Reprod. 1999 Nov;14(11):2868-71
Engels, Am J Med Genet A. 2008 Oct 15;146A(20):2611-6
Neri, Am J Med Genet. 1983 Dec;16(4):535-61
Stephenson, Hum Reprod. 2006 Apr;21(4):1076-82. Epub 2006 Jan 5
Sugiura-Ogasawara, Fertil Steril. 2004 Feb;81(2):367-73.
Carp, Fertil Steril. 2006 Feb;85(2):446-50
27
Karyotype of POC provides prognosis
for subsequent pregnancy
•If the POC of the first
miscarriage are normal,
the second miscarriage will
be aneuploid in 35%
•If the POC of the first
miscarriage are aneuploid,
the second miscarriage will
be aneuploid in 65%
70
% Aneuploid in 2nd Miscarriage
60
50
40
30
20
10
0
Euploid
Miscarriage
Ogasawara, Fertil Steril. 2000 Feb;73(2):300-4
Carp, Fertil Steril. 2001 Apr;75(4):678-82
Hassold TJ Am J Hum Genet 1980; 32: 723-730
Aneuploid
Miscarriage
Risk of Aneuploidy based on Maternal Age
Sporadic (Control) vs. Recurrent miscarriage
Risk of cytogenetic abnormality in miscarriage
80%
70%
Control
60%
50%
RPL
40%
30%
20%
10%
0%
18-29*
30-35*
36-39
Maternal Age in years
>40
* P<0.05
Stephenson et al., Hum Reprod. 2002 Feb;17(2):446-51
Aneuploidy in Products of Conception
Possibility exists that
aneuploidies on these
chromosomes survived
longer and thus allowed
a karyotype to be
obtained from POC
•
•
Chromosome Number
% of All Trisomies
16
22
21
15
13
18
14
7
2
8
9
4
20
10
12
6
3
17
11
5
19
1
24.7 %
13.9 %
12.3 %
8.3 %
6.8 %
4.8 %
4.4 %
3.4 %
3.2 %
3.0 %
2.9 %
2.8 %
2.7 %
1.5 %
1.2 %
1.0 %
0.9 %
0.9 %
0.5 %
0.4 %
0.2 %
0%
59.2%
8% of all SAB are
45, X
6.6%
Monni G, Ibba RM, Zoppi MA. Prenatal Genetic Diagnosis through Chorionic Villus Sampling. In: Milunsky A, Milunsky JM, eds. Genetic Disorders and the Fetus. 6th ed. Oxford, UK: Wiley-Blackwell. 2010.
Kearns WG, er.al Preimplantation genetic diagnosis and screening. Semin Reprod Med. 2005 Nov;23(4):336-47. Review.
Maternal Cell Contamination:
Parental Support Technology vs. Traditional Karyotype
46 XX
MCC
13%
ANEUPLOID
46 XY
46 XX
ANEUPLOID
46 XY
14%
15%
37%
25%
49%
47%
600 POC cases analyzed
using Parental Support
1920 POC cases analyzed using
cytogenetic karyotyping
GSN data. First 600 sequential cases (448
fetal results)
Menasha et al. Genetics in Medicine 2005; 7(4): 251-264
31
Aneuploidy Exists in all Chromosomes
• Aneuploidy in the developing
embryo exists at significant rates in
all 23 pairs of chromosomes at both
the cleavage and blastocyst stage as
identified by SNP microarray PGS
preformed on couples with > 2
previous pregnancy losses
Brezina, Kearns, Kutteh. J Assist Reprod Genetics. In Press, 2014.
Results: % Aneuploidy by Chromosome in RPL
(After PGS on 1702 embryos from RPL patients)
1702 SNP microarrays obtained
1404 (82%) Cleavage Stage
298 (18%) Blastocyst Stage
759 (45%) Euploid Embryos
943 (55%) Aneuploid Embryo
Significant levels of
aneuploidy
occurs in all chromosomes
during early human
Embryogenesis
Range of aneuploidy was
From 3.1% to 5.8%
Ch21
4.8%
Ch22
4.7%
X/Y
3.1%
Ch1
4.9%
Ch2
5.2%
Ch3
3.9%
Ch20
4.5%
Ch4
3.7%
Ch19
3.5%
Ch5
4.1%
Ch18
4.2%
Ch6
3.9%
Ch17
4.8%
Ch7
4.0%
Ch8
4.6%
Ch16
5.8%
Ch9
4.9%
Ch15
4.6%
Ch14
4.1%
Ch13
4.2%
Ch12
4.0%
Ch11
4.4%
Ch10
4.0%
Brezina, Kearns, Kutteh. J Assist Reprod Genetics. In Press, 2014.
What is evidence-based?- Anatomy
Frequency of abnormal tests in 1020 women with RPL
Control # of prior
losses
0.4% Parental
2
3
>4
P value
n=447 n=343 n=230 2,3,or 4
2.8% 5.4% 5.2%
NS
genetics
7.5%
0.5%
6.7%
3.9%
6.8%
Anatomy
18.7% 18.2% 16.7%
5.0% 2.9% 1.9%
NS
NS
4.2%
NS
NS
NS
Lupus
anticoagulant
Anticardiolipin 15.6% 13.1% 17.1%
TSH
8.1% 6.5% 6.2%
Factor V
8.1%
10.3%
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.
Congenital Uterine Anomalies
3-D Sonohysterograpy for the Evaluation of the Uterine Cavity
Prevalence of uterine anomalies among 904
consecutive patients with RM.
Total frequency of anomaliesa
Congenital anomalies
Bicornuate uterus
Didelphic uterus
Septate uterus
T-shaped uterus
Unicornuate uterus
Acquired anomalies
Adhesions
Fibroid(s)
Polyp(s)
% occurrence (n)
19.5(176)
6.7 (61)
0.8 (7)
0.2 (2)
4.8 (43)
0.3 (3)
0.7 (6)
13.3(120)
4.1 (37)
6.4 (58)
3.2 (29)
Jaslow and Kutteh. Fertil Steril 99: 1916-22, 2013.
aFive
patients (0.6%) had both congenital and acquired anomalies. The combinations were septum and adhesions,
septum and fibroid(s), septum and polyp(s), bicornuate uterus and fibroid(s), and unicornuate uterus and polyp(s).
Comparison of Uterine Anomalies
Primary RM compared with Secondary RM
Primary RM
(n = 479)
Secondary RM
(n = 425)
P
22.8 (109)
15.8 (67)
0.009
8.8 (42)
4.5 (19)
0.011
Bicornuate uterus
1.0
(5)
0.5
(2)
ns
Didelphic uterus
0.2
(1)
0.2
(1)
ns
Septate uterus
6.3 (30)
3.1 (13)
T-shaped uterus
0.4
(2)
0.2
(1)
ns
Unicornuate uterus
0.8
(4)
0.5
(2)
ns
14.6 (70)
11.8 (50)
ns
Adhesions
4.0 (19)
4.2 (18)
ns
Fibroid(s)
7.3 (35)
5.4 (23)
ns
Polyp(s)
4.0 (19)
2.4 (10)
ns
All uterine anomalies
Congenital anomalies
Acquired anomalies
Values are % occurrence (n).
0.028
Jaslow and Kutteh. 99:1916-22, 2013.
What is evidence-based?- Autoimmune
Frequency of abnormal tests in 1020 women with RPL
Control # of prior
losses
0.4% Parental
2
3
>4
P value
n=447 n=343 n=230 2,3,or 4
2.8% 5.4% 5.2%
NS
genetics
7.5%
0.5%
6.7%
3.9%
6.8%
Anatomy
18.7% 18.2% 16.7%
5.0% 2.9% 1.9%
NS
NS
4.2%
NS
NS
NS
Lupus
anticoagulant
Anticardiolipin 15.6% 13.1% 17.1%
TSH
8.1% 6.5% 6.2%
Factor V
8.1%
10.3%
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.
Pathophysiology of aPL
IT’S NOT JUST ANTICOAGULATION !
• Inhibit hCG release from placental explants
• Block of in vitro trophoblast migration &invasion
• Inhibit formation of giant, multinucleated cell
• Inhibit of trophoblast cell adhesion molecules
(alpha 1 and 5 integrins, E and VE cadherins)
• Activate complement on the trophoblast surface
inducing an inflammatory response
Girardi,Redecha,Salmon. Nature Med 10:1222-1226, 2005
What is evidence-based?- Endocrine
Frequency of abnormal tests in 1020 women with RPL
Control # of prior
losses
0.4% Parental
2
3
>4
P value
n=447 n=343 n=230 2,3,or 4
2.8% 5.4% 5.2%
NS
genetics
7.5%
0.5%
6.7%
3.9%
6.9%
Anatomy
18.7% 18.2% 16.7%
5.0% 2.9% 1.9%
NS
NS
14.1% 16.3% 17.4%
NS
NS
NS
Lupus
anticoagulant
Anticardiolipin 15.6% 13.1% 17.1%
TSH
8.1% 6.5% 6.2%
HgbA1c
Jaslow & Kutteh. Fertil Steril 93:1234-43, 2010.
Thyroid Function and RPL
• Increased pregnancy loss rate in thyroid
antibody negative women with TSH levels
between 2.5 and 5.0 in the first trimester
• 4,123 women prospectively evaluated
• Miscarriage rate doubled in group with TSH
2.5 to 5.0 compared to below 2.5 group
• Endocrine Society Guidelines advise TSH
between 1.0 and 2.5 for pregnancy
Schwartz et al. J Clin Endocrinol Metab 95: 44-48, 2010
Metformin Improves Pregnancy Outcomes
Meta analysis of metformin use in pregnant women
• All women with PCO by Rotterdam Criteria
• Total of 8 studies and 1,106 women
•
OUTCOME
Odds Ratio (95% CI)
Miscarriage
0.32 (0.19-0.55)
Gestational DM
0.37 (0.25-0.56)
Preeclampsia
0.53 (0.30-0.95)
Preterm delivery
0.30 (0.13-0.68)
Zheng and Shan. J Endocrinol Invest 36:797-802, 2013
What about Lifestyle Factors?
Risks of miscarriage increase 1.5 -2 fold
• Tobacco (>15/day)
• Ethanol (> 5/week)
• Obesity (BMI > 30)
• Caffeine (> 2-3 cups/day)
Initial Evaluation for Early RPL
Miscarriage #1
(No action unless clinically indicated
2nd Miscarriage
Aneuploid karyotype
Obtain Miscarriage
Karyotype
No further evaluation
Euploid karyotype
RPL Workup
Brezina and Kutteh. Clin Reprod Med Surg. 2nd Ed.pp197-208,2013.
Modified from Bernardi et al. Fertil Steril 98:156-161,2012
Unbalanced chromosomal
translocation or inversion
Perform parental
karyotypes and offer
preimplantation genetic
diagnosis for future
pregnancy attempts
Workup for Early RPL
Euploid POC After ≥2 Pregnancy Losses
OR
At Least 2 Consecutive Miscarriages With No POC Diagnosis
Or
At Least 3 Nonconsecutive Miscarriages With No POC Diagnosis
Anatomic
Evaluation
(Ex: HSG, SHG)
Endocrinologic
Evaluation
(EX: TSH, Prolactin,
Hyperglycemia)
Targeted
Surgical
Correction
Targeted
Medical or
Surgical
Correction
Add
Progesterone
Support to
Future
Pregnancies
Until 10 Weeks
Gestation
Autoimmune
Factors:
aPL
LAC
β2GP 1
Evaluation of
Lifestyle/Environment
(Ex: Caffeine, Tobacco,
Alcohol, Environmental
Exposures, Obesity)
Genetics:
Karyotype of
Parents if no
POC
karyotype
Obtained
Start ASA, SQ
heparin, Calcium,
& Vitamin D
preconceptually
and continue
until delivery
(Follow CBC)
Appropriate
Alterations to
Lifestyle,
Nutrition, or
Environment
Preimplantation
Genetic Testing if
Appropriate and
Desired: PGS/PGD
Brezina and Kutteh. Clin Reprod Med Surg.
2nd Ed.pp197-208,2013
.
What about True Unexplained RPL?
•
•
•
•
•
•
Current evaluation completed
Test results all return as normal
Chromosomes on POC are normal
Subsequent live birth is 40% to 80%
Depends on maternal age
Depends on number of prior losses
48
Chance of Live Birth based on # Prior Losses
Current Diagnostic and Treatment Strategies
(n-665)
Lund et al. Obstet Gynecol 119: 37-43, 2012
Chance of Live Birth based on Maternal Age
Current Diagnostic and Treatment Strategies
(n=665)
Lund et al. Obstet Gynecol 119: 37-43, 2012