Human infertility – what can the lab do to
overcome conception failure?
Nicola Winston, PhD, HCLD
IVF Lab Director
Department of Obstetrics and Gynecology
Cell types within the testes
•Spermatogonium
Undifferentiated
primordial germ cell
(diploid)
•Sertoli cells
Provide crucial
support for
spermatogenesis
• Leydig cells
Interstitial cells
between sertoli
cells Î produce
testosterone
SPERMATOGENESIS
• Spermatozoa develop within the
Seminiferous tubules in close association
with the Sertoli cells which provide
structure, signals and nutrients.
• Has 3 main phases;
i) mitotic proliferation
ii) meiotic division
iii) cytodifferentiation
• Spermatogonia (diploid)
Spermatozoon (haploid)
• takes 64 days
• several hundred million sperm reach
maturity daily
• Temperature sensitive < 36oC
Sperm cell differentiation
(Packaging)
• post-meiotic ‘haploid’ cell
changes shape from round
spermatid to elongated ‘mature’
sperm
• Head: consists primarily of the nuclear material.
• Acrosome: contains ~20 different enzymes essential
for ova penetration during fertilization.
• Neck / Midpiece: essential components include the
centriole required for chromosome organization in the
fertilized egg and the mitochondria which provide energy
for the movement of the tail.
• Tail: essential for forward progression.
Hormones controlling
testes function
Hypothalamus
GnRH +
Pituitary
Inhibin
Testosterone
-
LH +
FSH +
-
Activin
+
Leydig cells:
Sertoli Cells:
steroidogenesis spermatogenesis
Testis
World Health Organization Reference Values
Volume
pH
Sperm concentration
Total sperm number
Motility
> 2.0 ml
> 7.2
> 20 x 106 spermatozoa / ml
> 40 x 106 spermatozoa per ejaculate
> 50% motile within 60 mins of ejaculation
WHO NOMENCLATURE FOR SOME SEMEN VARIABLES
Normospermia
Normal ejaculate as defined by the reference values
Oligospermia
Sperm concentration less than reference value
Asthenospermia
Less than the reference value for motility
Teratospermia
Less than the reference value for morphology
Oligoastheno-
Signifies disturbance of all three variables
Teratospermia
Azoospermia
No spermatozoa in the ejaculate
Aspermia
No ejaculate
Teratospermia - “Abnormal Forms”
• Most indicate testicular disorder due to genetic, hormonal, vascular, stress,
infection, radiation, drugs, toxic exposure.
Examples:
Tapered Heads = Varicocele
Amorphous Heads = Allergic Reactions
Immature Spermatozoa (presence of cytoplasmic
droplets) = Recent ejaculation (< 24 hrs) or epididymal
dysfunction – male tract involved in sperm maturation and
storage.
Male infertility due to Infection
• Scaring of tract obstruction or gland secretions
Example: obstructed prostrate semen liquifaction disorder
• Bacterial and Leukocyte products impaired sperm motility
• sperm agglutination
• incidence of antisperm antibodies
• female tract infections Infertility
• sperm count
Oligospermia solutions:
• IUI / IUD: intrauterine insemination with partner or donor sperm – increases
the sperm reservoir in the uterine cavity – low success rate but often a
requirement of insurance companies before IVF coverage considered.
AZOOSPERMIA
Obstructive or non-obstructive azoospermia can
be overcome using in vitro fertilization. Sperm
can be collected directly either from the
epididymis or the testis.
Microscopic epididymal sperm aspiration (MESA)
was the first method to be introduced in 1985.
Percutaneous epididymal sperm aspiration (PESA)
method followed in 1995.
Testicular sperm aspiration (TESA) used when no
motile spermatozoa are retrieved from the
epididymis. The testis is punctured once with a
needle through the stretched skin of the
scrotum.
Testicular Sperm Extraction (TESE) is mandatory in
all cases of non-obstructive azoospermia with
a residual, often focally developed
spermatogenesis. Achieved by open testicular
biopsy or by microdissection TESE with limited
excision of testicular tissue.
OOGENESIS
CONTROLLED OVARIAN HYPERSTIMULATION (COH)
• Aims: 1) increase the number of oocytes and hence embryos
available for transfer to the uterus.
2) recruitment of a cohort of ovarian follicles that develop
synchronously
• Clomiphene citrate (anti-estrogen) and human menopausal gonadotrophin
(hMG: Pergonal) Î risk of premature LH surge
• Gonadotrophin releasing-hormone agonists (GnRH-a); use for IVF first described in 1984
(Porter et al.) – Shown to prevent premature luteinization and cycle cancellation,
increase the number of follicles stimulated to develop and to improve pregnancy rates
(Droesch et al., Hughes et al.) Cause an initial rise in the circulating levels of endogenous
gonadotropins. Longer administration leads to pituitary desensitization and ovarian quiescence.
• GnRH antagonists: most recently introduced. Administered late in the follicular phase Îavoids
ovarian suppression during follicular recruitment. Free of side effects associated with long
administration of GnRH-a. Bind competitively to and block GnRH receptors, immediately
inhibiting endogenous gonadotrophin release and a premature LH surge.
• Induction of ovulation using human chorionic gonadotrophin (hCG) which binds to LH
receptors
• Recombinant (vs) urinary hormones
OOCYTE MATURATION
First Polar
Body (PB1)
Ovulation
Germinal
Metaphase I
Vesicle (GV)
46 chrs (Diploid)
46 chrs* (Diploid)
Secondary
oocyte
Metaphase II
23 chrs (Diploid)
Metaphase I (A) and mature metaphase II (B) oocytes
Immature germinal
Vesicle (GV) oocyte
-arrested in prophase of
Meiosis I
Maturing metaphase
I (MI) oocyte
Mature metaphase
II (MII) oocyte
Sperm function:
To fertilize an oocyte, the sperm must first undergo the process of capacitation which
brings about changes necessary for sperm to penetrate the egg.
Female secretions strip layers of protein off the heads of sperm. Normally occurs in the
female reproductive tract but can be induced in vitro by washing the sperm out of the
seminal fluid.
= The Acrosome Reaction
Primary Sperm Receptor on egg =
ZP3/ZP4
Primary egg binding protein on sperm
= Sperm Membrane Protein
Secondary Sperm Receptor on egg = ZP2
Secondary egg-binding protein on inner
acrosomal membrane of sperm = Proacrosin
Sperm Interaction with the egg =Fusion
• Sperm fuses with the egg plasma membrane (oolemma)
• Entire head and tail of the spermatozoa are incorporated into the egg
Intracytoplasmic Sperm Injection (ICSI)
Microinjection of an intact spermatozoon directly into oocyte cytoplasm
Î Bypasses: Zona penetration, Fusion, Incorporation
Benefits: dramatic rise in the successful treatment of idiopathic infertility in humans
(+preservation of endangered animals species and rare breeds).
Controversy: Male infertility is known to be associated with elevated levels of chromosomal
and other genetic anomalies; may unwittingly assist the transmission of genetic disease or
reproductive dysfunction in the offspring.
• The best characterized of these so far is infertility associated with Cystic Fibrosis (CF)
carrier status. Such men can present with obstructive azoospermia through congenital
absence of the vas deferens and are thus obvious candidates for ICSI.
Extrusion of the second
polar body /
completion of Meiosis II
Formation of pronuclei
- one male, one female
Oocyte Function - Block to polyspermy
The cortical granules to fuse with the egg plasma
membrane and release proteases clip off the ZP binding
receptors, removing ZP sperm-binding properties.
Failure can be associated with immature eggs.
Polyploid (Multi-pronucleate) Fertilized Eggs
Two cell embryo
(Day 1: ~18-24
hours postinsemination
Abnormal
two cell embryo
Four cell embryo
(Day 2: ~40-50 hours post-insemination)
Eight cell embryo
(Day 3: ~68-72 hours post insemination)
• On average, 15% of embryos such as this will implant after
being transferred – female age dependent.
Embryo Quality
•Embryos for transfer to the uterus are evaluated for 'quality' based on;
• the number of cells present
• size and regularity of the cells
• degree of fragmentation observed
Level 1: 8 cell embryo, no fragmentation
Level 2: 8 cell embryo with equal-sized cells but 5-25% fragmentation
Level 3: ~8 cell embryo with >25% fragmentation and irregular sized cells
• The origin and cause of fragmentation is unknown
• Level 1 & 2 embryos are more likely to implant
• No evidence of increased abnormalities or birth defects associated
with poorer embryo morphology.
Implantation-stage embryo = Blastocyst (Day 5-6)
•Has a fluid filled cavity, the blastoceol (C).
Comprised of 2 Cell Types;
• outer layer of cells = trophoblast (T)
Î become the placenta after implantation.
• dense mass of cells = inner cell mass (ICM)
Î become the fetus.
• Prior to implantation, the embryo must hatch
out of the zona pellucida.
• Hatching is achieved by the actions of
hydrostatic pressure and uterine secretions.
Assisted Hatching
A small hole is “drilled” into the zona pellucida using an acidic
solution in cases where hardening of the protein coating
might impede the embryo’s ability to hatch out.
Indications for Assisted Zona Hatching (AZH): problems of
hatching has been associated with the length of time in
culture, female age, high FSH levels.
Preimplantation Genetic Diagnosis (PGD)
• An early alternative to prenatal diagnosis.
• Suitable for patients at substantial risk of conceiving a pregnancy affected by a
known genetic defect.
• Has been applied to;
i) the analysis of numerical and structural chromosomal abnormalities that can result
in handicap or recurrent miscarriage
ii) the identification of sex to prevent transmission of X linked disease
iii) the detection of specific serious monogenic disorders
Embryo Biopsy
• A single blastomere is removed from each 8-cell human embryo for the
purposes of preimplantation genetic diagnosis.
• Embryos found to be unaffected by the genetic disorder are transferred to
the uterus and / or cryopreserved.
• For single gene disorders, such as cystic fibrosis and spinal muscular atrophy, the polymerase
chain reaction is used to amplify the region of the DNA containing the genetic lesion to levels
where a diagnostic test can be carried out.
• PGD of sex linked diseases for which the specific genetic
defect is unknown or not amenable to molecular diagnosis at
the single cell level can be done using fluorescence in situ
hybridization (FISH).
• Specific probes, which bind to either X (green) or Y (red)
chromosomes in the interphase nucleus fluoresce under
ultraviolet illumination are used to determine the sex of the
embryo.
• Probes that bind to specific chromosomal telomeres can
be used to identify balanced or unbalanced products in
Robertsonian and reciprocal translocations.
Screening for Genetic Abnormalities
Uses fluorescent probes to up to a maximum
of 9 chromosomes at one time.
Normal embryos are diploid and have two
spots for each colour (A).
Many different abnormalities have been seen;
•A complete extra set of chromosomes (one)
triploid (B) and (two) tetraploid (C) – generally
demise shortly after implantation.
•A set of chromosomes is missing in haploid
embryos (D).
•Abnormalities of individual chromosomes are
also seen (E – Aneuploidy Screening), such as
monosomy, trisomy, and double trisomy (F).