Fertilization, Implantation and Assisted Reproductive Technologies

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Fertilization, Implantation and Assisted
Reproductive Technologies

• Sanjay K. Agarwal, MD
• Associate Professor of Reproductive Medicine,
  UCSD
• San Diego, CA

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Relationship between capacitation, acrosome
reaction and fertilization
CAPACITATION
Changes in tail
Changes in head
Potential to undergo acrosome reaction
Hyperactivation
Spermatozoon Binding to ZP3 ACROSOME REACTION
Zona penetration
Fertilization
G. Erickson
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1. Capacitation
changes that enable the spermatozoa to
undergo the acrosome reaction and
hyperactivation. Normally, this occurs when
semen interacts with the vaginal fluids.
G. Erickson
4
Human Cervical mucus plays a role in regulating
sperm entry into the uterus.
Periovulatory mucus is watery facilitating
sperm movement through the open External os.
Luteal mucus is thick and resistant to sperm
motion, and the External os is closed.
External os
Cervix
External os
Thin cervical mucus
G. Erickson
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As a result of capacitation, spermatozoa acquire
the capacity to bind to the zona pellucida. This
event is believed to result from the unmasking
of a species specific ZP-3 receptor in the sperm
membrane.
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2. ACROSOME REACTION
Acrosome Reaction Discharge of proteases
acrosome
Spermatozoon Head
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Zona penetration
Tail hyperactivity
As a consequence of the acrosome reaction, the
zona pellucida is locally hydrolyzed. This
together with hyperactation, allows the
spematozoon to penetrate the zona pellucida.
Zona pellucida
proteases
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The steps in fertilization
Metaphase II oocyte
G. Erickson
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Acrosome reacted sperm penetrating zona pellucida
egg
ZP
Sperm
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Sperm/oocyte contact triggers the cortical
granule reaction that sweeps around the egg.
Cortical Granules
Met II oocyte
Sperm-egg contact
Cortical granule reaction
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The Mechanisms of the Cortical Granule Reaction
Sperm/egg binding
Sperm/egg membrane fusion
Cortical reaction
Ca2 release
G. Erickson
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Diagram showing the course of cortical granule
reaction.
Oolemma
Cortical Granule/oolemma fusion
EXOCYTOSIS Cortical granule discharge of protein
material
Cortical granule
G. Erickson
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Cortical Granules intact
After release, the contents of the cortical
granules polymerize into a proteinaceous
matrix that alters the structure of the zona
pellucida.
Cortical Granules discharged
G. Erickson
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Cortical reaction blocks sperm binding to
fertilized egg.
Capacitated sperm do not bind to fertilized egg
Capacitated sperm bind to unfertilized egg
G. Erickson
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The key events that occur during fertilization

• 1. Capacitated sperm binds to zona pellucida
• protein-3 (ZP-3) induces the acrosome
  reaction.
• 2. The acrosomal process facilitates ZP
  penetration.
• 3. Sperm-egg contact induces the cortical
  granule
• reaction and the block to polyspermy

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The oviduct and uterus are critical components of
the female reproductive tract.
Ampulla is the site of fertilization
oviduct
endometrium
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Time course of the development of the human
embryo from fertilization to implantation.
myometrium
endometrium
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Physiological functions of the uterus

• 1. Endometrium - to prepare for and participate
  in implantation, and to form the maternal portion
  of the placenta.
• 2. Myometrium - to participate in the sloughing
  of the endometrium during menstruation and
  expulsion of the fetus, placenta, and fetal
  membranes during parturition or child birth.

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Histology of the Endometrium
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The Endometrial Cycle
progesterone
estrogen
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During the preimplantation period, the
fertilized egg develops into a blastocyst while
remaining encapsulated by the zona pellicudia.
2 cell
4 cell
morula
8 cell
Inner cell mass
blastocyst
trophoblast
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Zygote with Fusion of pronuclei at 24 hrs
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Two-cell human embryo at 30 hr
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Four cell human embryo at 2 days
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Human blastocyst at 5 days
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Time course of the development of the human
embryo from fertilization to implantation.
myometrium
endometrium
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Implantation phases

• A. Hatching phase
• B. Adhesion phase
• C. Invasive phase

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Hatching phase
Site of zona rupture
Blastocyst escaping from the zona pellucida
G. Erickson
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Invasive phase After attachment, the trophoblast
cells begin to penetrate the uterine epithelium.
Trophoblast Cells invading endometrium
uterine epithelium
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Human embryo completely embedded in the
endometrium
embryo
endometrium
Secretory glands
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Early Placental Development
Sinusoids filled with maternal blood
syncytiotrophoblast
Compact layer venous islands
cytotrophoblast
Endometrial epithelium
G. Erickson
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Human chorionic gonadotropin (hCG) secreted by
the syncytiotrophoblasts during implantation
inhibits luteolysis, which in turn transforms
the corpus luteum of the cycle into the corpus
luteum of pregnancy.
Corpus luteum
hCG
progesterone
Inhibits myometrial contraction
G. Erickson
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Corpus luteum-placental shiftat 7th weeks of
pregnancy, the placenta produces the progesterone
necessary for maintaining pregnancy.
Progesterone ng/ml
G. Erickson
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Assisted Reproductive Technologies
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Infertility

• Defined as the inability to conceive after one
  year of unprotected intercourse
• 6 months for women over 35?

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Statistics

• Overall, about 85 of couples will conceive
  within 1 year of unprotected intercourse
• Therefore, about 15 of US couples are infertile
  by definition (3 million couples)

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Etiologies

• Sperm disorders 30
• Anovulation/oligoovulation 30
• Tubal disease 16
• Unexplained 14
• Peritoneal factors 10

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Overview of Infertility Evaluation
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1. Normal Semen Analysis (WHO 1992)
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2. Detection of ovulation

• Regular menses
• Midluteal serum progesterone
• Urinary LH (ovulation) Kits
• BBT charts
• Ultrasound
• Endometrial biopsy

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3. Tubal patency 4. Uterine cavity
Hysterosalpingography
Hysteroscopy
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5. Age related decline in ovarian
reserveRecruitment (reflected in the decline
of primordial follicle number) is bi-exponential
during aging. At 37 years, i.e. when the human
ovaries have 25,000 primordial follicles, the
rate of loss of primordial follicles increases
2-fold.
Number of primordial follicles
Accelerated loss of oocytes
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G Erickson
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Female fecundity declines with age.
FSH Antral follicle count
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Peritoneum (Endometriosis)

• Requires surgery for diagnosis
• Relationship between minimal endometriosis and
  fertility controversial

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Common treatment options

• Male factor
• Anovulation
• Tubal occlusion
• Endometriosis
• Decreased ovarian reserve

• - Insemination, ICSI or donor
• - Ovulation induction
• - In-vitro fertilization vs surgery
• - Ovulation induction or IVF
• - Oocyte donation

Increasingly complex and expensive treatments
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Ovulation induction
Clomiphene citrate Blocks central estrogen
feedback - Increases endogenous FSH
Aromatase inhibitors
Gonadotropins Direct ovarian effect
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Intrauterine Insemination
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In-Vitro FertilizationOvarian stimulation
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Oocyte retrieval
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IntraCytoplasmic Sperm Injection (ICSI)
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Zygote with Fusion of pronuclei at 24 hrs
G. Erickson
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Human blastocyst at 5 days
G. Erickson
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Embryo Transfer
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Many controversies, and ethical issues- just
because we can, doesnt mean we should

• Maternal age limit 50y, 60y, any age?
• Single parent male or female?
• Single sex parents male or female?
• For parent with terminal disease?
• Legally limit number of embryos transferred?
• Preimplatation genetic testing
• Sex selection?
• Physical characteristics?