L8 RDA Fertilisation and Implantation

Overview

This lecture follows the embryo from fertilisation in the uterine tube through to early implantation in the uterus. It covers the four sequential steps of fertilisation and the polyspermy block, the resulting zygote, cleavage and compaction into a morula and then a blastocyst, hatching and implantation timing, ectopic pregnancy, and how the blastocyst differentiates into trophoblast and inner cell mass, which in turn establish the placenta, amniotic cavity, yolk sac and early nutrition of the embryo.

Gametes and site of fertilisation

• Fertilisation is the fusion of the pronuclei of two gametes: the spermatozoon and the secondary oocyte.
• Occurs in the ampulla of the uterine tube.
• Spermatozoon: survives ~48 hours; motile and active; has an acrosomal cap containing acrosin.
• Secondary oocyte: survives ~24 hours; surrounded by granulosa cells (corona radiata); the zona pellucida (ZP) lies between the granulosa cells and the oocyte plasma membrane, bridged by granulosa microvilli.

Stages of fertilisation and the polyspermy block

Four sequential stages:

1. Capacitation - secretions of the female genital tract remove the sperm’s glycoprotein coat and seminal proteins, so the sperm can contact the zona pellucida and bind ZP3 glycoprotein receptors on it.
2. Acrosomal reaction - the acrosomal membrane is perforated and acrosin is released, digesting the zona pellucida around the sperm head; the plasma membrane fuses with the outer acrosomal membrane, vesiculates, and the sperm (now acrosome-reacted) enters the perivitelline space and then the oocyte surface.
3. Cortical reaction - a weak membrane potential change triggers a calcium wave in the oocyte, starting at the point of sperm entry. This (a) signals resumption of cell division, completing meiosis II, and (b) triggers release of cortical granules containing lysosomal enzymes.
4. Zonal reaction - the released lysosomal enzymes act on the zona pellucida in two ways, together forming the polyspermy block: hydrolysis of the ZP3 receptor prevents further sperm binding/acrosomal reaction, and modification of the oocyte plasma membrane makes it less susceptible to further sperm-oocyte fusion. Correspondingly, the zona pellucida surface changes from an open honeycomb/mesh structure before fertilisation to a smoother, collapsed mesh after fertilisation.

Outcome of fertilisation: the zygote

Fertilisation produces a zygote, which achieves:

• Restoration of the diploid (2n) chromosome number
• Completion of meiosis II
• Sex determination (occurs at fertilisation)
• Initiation of cleavage

Cleavage, morula and blastocyst formation

• Cleavage: the zygote’s first cell divisions, distributing cytoplasm equally between daughter cells.
• Up to the 8-cell stage: division is continuous (non-stop); cells are spherical, loosely touching, each with a central nucleus, and there are no special cell junctions.
• After the 8-cell stage: cells flatten to maximise contact area and form tight junctions, a process called compaction.
• Morula = 16-cell embryo.
• Blastocyst forms at the 32-64 cell stage, when a fluid-filled cavity (blastocoele) is generated.
• All of these changes occur inside the zona pellucida.
• Sequence along the uterine tube to the uterus: oocyte penetrated by sperm -> zygote -> two-cell -> four-cell -> eight-cell -> morula -> blastocyst (forming near the posterior uterine wall).

Hatching and implantation

• Hatching (day 6-7 post-ovulation): dissolution of the zona pellucida, penetration of trophoblast into the endometrium, and migration of the blastocyst into the endometrium.
• Implantation: occurs 7-12 days post-ovulation, at the endometrium of the posterior wall of the uterus, nearer the fundus.

Ectopic pregnancy

Implantation site frequencies from a 10-year population-based study of 1800 ectopic pregnancies:

• Tubal: 95% overall, comprising Ampullary 70%, Isthmic 12%, Fimbrial 11%, Interstitial 2-3%
• Ovarian: 3%
• Intraligamentous or abdominal: 1%
• Cervical: <1%
• Caesarean scar: <1%

The great majority of ectopic pregnancies are tubal, and within the tube the ampulla (also the normal site of fertilisation) is by far the commonest location.

Advanced abdominal ectopic pregnancy can result in a fetus situated within the abdominal cavity, outside the uterus.

Blastocyst differentiation during implantation

• The blastocyst has two cell types: the inner cell mass (Embryoblast), which develops into the embryo, and the outer cell mass (Trophoblast), which eventually gives rise to the placenta.
• Trophoblast differentiates into two layers:
  1. Cytotrophoblast: mononuclear layer, remains germinative (proliferative) throughout pregnancy.
  2. Syncytiotrophoblast: multinucleated, ill-defined cell mass (a syncytium); has endocrine function and secretes hCG, the hormone detected by early pregnancy tests.
• Inner cell mass: surface cells have microvilli that transfer fluid into the inner cell mass; the amniotic cavity forms; by week 2 of development the inner cell mass becomes a bilaminar germ disc, in which the epiblast floors the amniotic cavity and the hypoblast roofs the yolk sac.

Progression of structures seen across the implantation sequence:

• Day 8: outer to inner - blood vessels, syncytiotrophoblast, cytotrophoblast, amnioblasts, endometrium (stroma/epithelium), amniotic cavity, epiblast, hypoblast, blastocoele.
• Day 9: maternal vascular sinusoids, trophoblastic lacunae, exocoelomic (Heuser) membrane, coagulation plug, primitive yolk sac.
• Later: trophoblastic lacunae fill with maternal blood; extraembryonic mesoderm and extraembryonic coelom appear, formed by the hypoblast. [slide does not elaborate on the mechanism further]
• Further stage: the extraembryonic coelom expands into the chorionic cavity; the yolk sac becomes the secondary yolk sac; an exocoelomic cyst is present; a connecting stalk links the embryonic disc to the trophoblast; primary villi project from the trophoblast into the maternal blood-filled lacunae.
• Later stages: dilated maternal blood vessels surround an expanding chorionic cavity containing the amniotic cavity and yolk sac; the embryo develops within the amniotic cavity, surrounded by the chorionic cavity and chorion (villi). By later pregnancy, the arrangement is: outer decidua, a discrete placenta, an inner chorionic sac containing the amniotic cavity (amnion as its inner membrane) housing the fetus.

Several of the later implantation diagrams (the day-8/day-9 sequence onward) carried no slide title in the source, and some diagram elements (e.g. a yellow-circled region of the chorion, an unlabelled blue/yellow ellipsoid shape) were shown without any explanatory text, so their specific meaning is not established by the transcript.

Nutrition of the early embryo

• Early on, surface microvilli of the inner cell mass transfer fluid into the inner cell mass.
• As implantation progresses, trophoblastic lacunae fill with maternal blood and primary villi project from the trophoblast into these maternal blood-filled lacunae, bringing the embryo into contact with the maternal circulation. [slide does not elaborate further on the nutritional mechanism]

Self-test

1. Define fertilisation and state where in the female reproductive tract it normally occurs.
2. Compare the lifespan and key surface features of the spermatozoon and the secondary oocyte.
3. List the four stages of fertilisation in order.
4. Describe the steps of capacitation, from removal of the sperm’s glycoprotein coat to ZP3 binding.
5. Describe the acrosomal reaction, from perforation of the acrosomal membrane to the sperm entering the oocyte surface.
6. Explain how the calcium wave of the cortical reaction produces its two downstream effects.
7. Explain the two mechanisms of the zonal reaction and how together they form the polyspermy block.
8. List the four outcomes of fertilisation that define the zygote.
9. Distinguish cell behaviour before and after the 8-cell stage of cleavage, and name the process that occurs after 8 cells.
10. Distinguish the morula from the blastocyst by cell number and by the key structural feature that appears in the blastocyst.
11. Describe the events and timing of hatching, and give the timing and site of implantation.
12. A patient has a positive pregnancy test and is found to have an ectopic pregnancy. Which implantation site is most likely, and what proportion of ectopic pregnancies does it represent?
13. Distinguish the inner cell mass from the trophoblast in terms of fate.
14. Distinguish the cytotrophoblast from the syncytiotrophoblast, and explain why the syncytiotrophoblast is clinically relevant to a home pregnancy test.
15. Describe how the bilaminar germ disc is organised in week 2 of development.
16. Describe two ways the early embryo obtains nutrition as implantation proceeds.
17. Trace the sequence of extraembryonic structures from the primitive yolk sac (day 9) to the appearance of primary villi, and explain what change in trophoblast-maternal contact this sequence represents.

Answers

Reveal answers

1. Fertilisation is the fusion of the pronuclei of a spermatozoon and a secondary oocyte; it normally occurs in the ampulla of the uterine tube.
2. The spermatozoon survives ~48 hours, is motile/active, and has an acrosomal cap containing acrosin. The secondary oocyte survives ~24 hours and is surrounded by granulosa cells (corona radiata), with the zona pellucida lying between the granulosa cells and the oocyte plasma membrane.
3. Capacitation, acrosomal reaction, cortical reaction, zonal reaction.
4. Secretions of the female genital tract strip the glycoprotein coat and seminal proteins from the sperm surface, allowing the sperm to contact the zona pellucida; the sperm’s glycoprotein receptors then bind the ZP3 protein on the zona pellucida.
5. The acrosomal membrane is perforated and acrosin is released, digesting the zona pellucida around the sperm head; the sperm plasma membrane fuses with the outer acrosomal membrane and vesiculates; the acrosome-reacted sperm enters the perivitelline space and then the oocyte surface.
6. The calcium wave (starting at the sperm entry point) signals resumption of cell division, completing meiosis II, and also triggers release of cortical granules containing lysosomal enzymes.
7. The lysosomal enzymes released in the cortical reaction hydrolyse the ZP3 receptor on the zona pellucida (preventing further sperm binding/acrosomal reaction) and modify the oocyte plasma membrane (making it less susceptible to further sperm fusion); together these block further sperm entry, i.e. the polyspermy block.
8. Restoration of the 2n chromosome number, completion of meiosis II, sex determination, and initiation of cleavage.
9. Up to the 8-cell stage, division is continuous, cells are spherical, loosely touching, with a central nucleus and no special junctions. After the 8-cell stage, cells flatten to maximise contact and form tight junctions, a process called compaction.
10. The morula is a 16-cell embryo with no internal cavity; the blastocyst forms at the 32-64 cell stage, when a fluid-filled cavity (blastocoele) develops.
11. Hatching (day 6-7 post-ovulation) involves dissolution of the zona pellucida, penetration of trophoblast into the endometrium, and migration of the blastocyst into the endometrium. Implantation occurs 7-12 days post-ovulation, at the endometrium of the posterior uterine wall near the fundus.
12. Tubal, most likely in the ampulla; tubal sites account for 95% of ectopic pregnancies overall, and the ampulla alone accounts for 70%.
13. The inner cell mass (embryoblast) develops into the embryo; the trophoblast (outer cell mass) eventually gives rise to the placenta.
14. The cytotrophoblast is a mononuclear layer that remains germinative throughout pregnancy; the syncytiotrophoblast is a multinucleated syncytium with endocrine function that secretes hCG, which is the hormone detected by early home pregnancy tests.
15. In week 2, the inner cell mass becomes a bilaminar germ disc: the epiblast floors the amniotic cavity and the hypoblast roofs the yolk sac.
16. Initially, surface microvilli of the inner cell mass transfer fluid into the inner cell mass; later, as trophoblastic lacunae fill with maternal blood, primary villi project from the trophoblast into these blood-filled lacunae, bringing the embryo into contact with the maternal circulation.
17. From the primitive yolk sac (day 9), trophoblastic lacunae fill with maternal blood, extraembryonic mesoderm and coelom form, the coelom expands into the chorionic cavity, the yolk sac becomes the secondary yolk sac, a connecting stalk links the embryonic disc to the trophoblast, and primary villi project from the trophoblast into the maternal blood-filled lacunae. This sequence represents progressively closer and more organised contact between the trophoblast and the maternal circulation, laying the foundation for the placenta.