Fertilisation Flashcards
Steps in fertilisation
- Capacitation
- Acrosomal reaction
- Polyspermy block
- Completion of meiosis II (secondary oocyte to mature ovum)
- Zygote formation
Seminal fluid components
70% seminal vesicle fluid (fructose/prostaglandins/coagulase)
30% prostatic fluid (citrtate)
1% semen
Other - white cells, immature spematozoa, round cells, epithelial cells, micro-organsis.
Capacitation
Cellular and molecular changes that a sperm must go through to fertilise an egg.
Process which allows acrosome reaction, hyperactivated motility (much higher amplitude flagellar waves) and the ability to bind to the zona pellucida.
- removes molecules absorbed or incorporated into sperm plasma membrane (glycoproteins, cholesterol, sialic acid, antigens).
Cholesterol efflux and Ca2+ uptake crucial.
Decapacitation factors
Seminal plasma contains factor(s) that reversibly inhibit capacitation and the acrosome reaction in vitro. These decapacitation factors are the primary reason that spermatozoa must be isolated from seminal plasma before insemination in vitro.
Acrosome reaction
Modification and breakdown, followed by a merger, of the sperm cell membrane and the outer acrosomal membrane, allowing the release of enzymes and changes in the inner acrosomal membrane, necessary for fusion with the oocyte cell membrane.
Sperm head receptors bind to ZP3 ligand (via SPAM1 or PH-20 - membrane proteins on the sperm head), this induces an enzyme complex (tyrosine kinase and others) that induces the acrosome reaction. Calcium influx essential.
How does sperm penetrate ZP
Sperm head binds ZP3
Acrosome reaction
Release of acrosin and proteases break down ZP
Sperm moves into oocytes and binds to oocyte cell membrane
Polyspermy block - two steps and key proteins involved
- Fast block to polyspermy
Sperm head binds to vitteline memebrane (oocyte plasma membrane). Via PH-30/fertilin/vitronectin)
Rapid calcium and sodium transport intracellularly. Causee depolarisation of the oocyte memebrane and, this prevents penetration.
Only a transient block and further preventions requires corticol reaction.
Sperm cell releases pro-nucleus into the ooplasma. - Corticol reaction:
Ca2+ released from smooth endoplasmic reticulum within ooplasm> Phospholipase protein called sperm-specific phospholipase C zeta (PLCz) activates inositol and Ca+2 release. and activates lysosomes and these fuse with vitteline membrane, releases hydrolytic enzymes, these degrade the ZP and the ligands, hardens the ZP and prevents spermatozoa from binding.
What is the role of Phospholipase protein called sperm-specific phospholipase C zeta (PLCz)
Activates calcium release allowing for corticol reaction and is the key protein that initiates this calcium signalling once the pro-nucleus of the sperm cell has been released into the egg.
Completion of meiosis
Occurs after pro-nuleus of sperm has entered oolema.
Completed around 3 hours after fertilisation.
The second polar body is released leaving the egg with a haploid complenet of chromosomes.
Addition of the haploid chromosome complement from the sperm restores the diploid number to the fertilised egg (one maternal and one paternal set)
Formation of the zygote
The male and the female pronuclei migrate toward each other, and as they move into close proximity, the limiting membranes break down, and a spindle is formed on which the chromosomes become arranged. Thus, the stage is set for the first cell division.
Embryonic genome activity in the human begins early; DNA synthesis activity can be detected 9-10 hours after insemination.
Human gene expression (transcription) begins between the 4- and 8-cell stages of preimplantation cleavage, 2-3 days after fertilization.
Earlier embryonic signals may be derived from a store of maternal messenger RNAs, termed the “maternal legacy.”
In addition, proteomics have identified RNAs and transcription factors within sperm that suggest a mechanism for a paternal contribution to the early development of the embryo.
Clinical relevance: Embryo driven development usually from cleavage stage to blastocyst (sperm issues will therefore usually present with poor cleavage to blast development), earlier than this issues - from fertilisation to cleavage often maternal egg issue.
Oocyte activation as part of fertilisation
Competent fertilization requires:
Alleviation of meiotic arrest at the second metaphase of meiosis of unfertilised oocyte
Indications that this has occurred includes:
Second polar body extrusion
Formation of male and female pronuclei
Fertilization also involves:
Cortical granule exocytosis
Initiation of the first cell cycle
Oocyte activation is a distinct part of the fertilisation process
It can occur without sperm entry (and sperm entry can occur without oocyte activation)
Process:
Trigger for oocyte activation is an increase in cytosolic concentrations of free calcium (Ca2+)
Ca2+ oscillations occur
Direct consequence of inositol triphosphate (IP3-)-mediated Ca2+ release.
Initiated during or immediately after sperm-oocyte membrane fusion – suggesting that sperm introduces a factor into the oolemma to cause Ca2+ release.
Fertilisation in standard IVF
Each oocyte incubated with 50-100 thousand motile sperm for 12-18hrs at 37 degrees in 5-10% oxygen and 4-7% Co2
Conventional IVF achieves fertilisation rates between 60 (competency) -75% (benchmark)
Sperm-borne enzyme called phospholipase C enters the oocyte and activates it via the inositol-3-phosphate pathway.
Molecule binds to the corresponding receptor at the endoplasmic reticulum where it causes Ca2+ release in the form of oscillations
This Ca2+ response drives the extrusion of the second polar body and the formation of both pronuclei
Evaluated at 18hrs
Normally fertilised oocyte:
2 distinct pronuclei (1 from oocyte, 1 from sperm)
2 polar bodies in perivitelline space
Observe for >2 pronuclei (polyploid) -
Occurs in 5-10% embryos overall
-May result from polyspermy or digyny (fertilisation of a diploid oocyte) due to:
- Meiotic spindle errors
- Failure to extrude a polar body
- More commonly associated with immature, aging or postmature oocytes
Fertilisation process requires 24hrs and ends with first mitotic division (cleavage)
Indications for ICSI
Indications:
Previous failure of fertilisation
Severe male factor infertility
-Severe oligospermia <5milion/ml
-Asthenospermia <5% progressive motility
-Teratospermia <4% normal forms
-Surgically retrieved sperm
-PGT-M
- IVM
- Insemination of frozen oocyte
*May yield a higher fertilisation rate for oocytes matured in vivo and in cryopreserved oocytes which often exhibit a hardened zona (resistance to digestion by proteases)
ICSI process
Process
-Single selected sperm is immobilized by compressing the tail then drawn into pipette
-Oocyte stabilised with pipette at 9 o’clock position with polar body at 6 or 12 o’clock and entry at 3 o’clock
-Sperm injected directly into ooplasm
-ICSI does NOT require acrosome reaction or fuse with oocyte membrane
-Mechanical disruption of ooplasm and sperm membranes, triggers oocyte activation
-Achieves fertilisation rates comparable to those observed with conventional IVF in the absence of male factors
perm is microinjected directly into the oocyte cytosol
-With some male infertility, there is failure of some oocytes to activate, with embryos failing to either to protrude 2nd polar body OR successfully proceed past 1st cell division
Pitfalls:
-ICSI can damage the meiotic spindle even if the area adjacent to the 1st polar body is avoided because the 2nd meiotic spindle varies in position and is not always located immediately beneath the first polar body
Factors which impact on fertilisation rates:
Sperm motility
Patient age
ICSI operator
Vienna consensus KPIs for ART
ICSI damage rate - </= 10% (competency) </= 5% (benchmark)
ICSI normal fertilisation rate - >/= 65% (competency) >/= 80% (benchmark)
Fertilisation failure/low fertilisation
Oocyte activation deficiency assoc with PLC-z not producing enough Ca2+ oscillations reuqired for oocyte activation
A lot of low/failed fert is due to imparied semen characteristics or low oocyte number.
Some patient may have repeated failure despite normal semen parameters and good ovarian response.
