IVF Flashcards
1
Q
Intro
A
- Infertility affects 1 in 7 UK couples
- One method of assiting these couples = IVF
- IVF has prod ~5 million chldren since 1944
- Live birth rate = 32.6% but success rate decr w/ age
- Ethically controversial
- NICE rec. 3 free cycles per couple under 35yrs (funding varies with CCG)
- all IVF clinics require license from HFEA
- IVF bypasses uterine tubes
2
Q
- Shutting down HPG axis
Explain normal GnRH vs olfactory GnRH analogue (IVF)
A
- GnRh is normally secreted by AC of Hyp following kisspeptin stim acting to depolarise GnRH neurons
- binds to receptors in AP and causes subsequent LH surge which results in ovulation
- In IVF olfactory GnRH analogues are administered
- These downregulate the HPG axis, preventing the LH surge which would normally cause ovulation
- Continuous rel of gnRh causes cessation of LH/FSH, a key principle underlying IVF
3
Q
- Shutting down HPG axis
Explain the GnRH agonist used in IVF and how it differs to native GnRH
A
- A GnRH antagonist/agonist e.g. Lupron (agonist) is administered on day 21 of a womans cycle
- Endogenous GnRH = decapeptide consisting of 3 regions 1)Receptor binding region, 2) Receptor binding and activation region, 3) Activity determining region
- Lupron has an aa substitution od D-Leu (bacterial aa) for Gly at position 6, this stabilises conformation and prevents proteolytic cleavage
- native GnRH binds to GnRHRs on AP causing activation of signalling leading to g/trophin synthesis and secretion
- LH&FSH secretion causes dissociation of GnRH from receptor allowing receptor to become responsive to next GnRH pulse
- A GnRH agonist e.g. Lupron binds to the receptor and causes desensitisation of the receptor rather than dissociation. The receptor becomes unresponsive as it remains uncoupled
- Addition of Lupron or similar provides continuous slow dose of GnRHagonist which shuts down GnRH action and consequent LH/FSH release, preventing the LH surge that causes natural ovulation. Or in IVF would cause the premature ovulation of only one DF.
4
Q
2.Ovarian stim. & follicle monitoring
Explain what hormone is administered and different versions available
A
- FSH injection into SC fat of abdomen on Day2 of cycle for 12 days
- FSH starting dose decided according to woman’s age and clinical criteria
- AFC is a marker considered to be v. reliable in predicting ovarian response to FSH
- FSH administered is a purified form of FSH manufactured from human urine & purified to remove interfering proteins e.g. Bravelle
- Alternatively rFSH can be used –> Made by isolation of FSHB-subunit gene which is inserted into a vector then into CHO cell line
- Recombinant FSH e.g. Follistatim is identical to urinary/pituitary FSH in aa sequence, RBC, glycosylation site and in vitro biological activity
- Systematic review of 42 trials including 9606 couples found no sig diff in live birth rates resulting from urinary FSH/rFSH
- recommended to choose g/trophin depending on availability, cost & convenience
5
Q
- Ovarian Stim. & follicle monitoring
What does the FSH injection do?
A
- FSH causes hyperstimulation of ovaries by allowing selection of mx follicles for maturation into DFs, compared to natural selection process providing ‘window of opportunity’ for selection of a single DF
- prolonged FSH levels stim more follicles to mature and develop
- Becaus HPG axis is swtitched off, more than one follicle develops
- These all acquire hugh no.s of FSHR followed by LHR in the granulosa layer which heightens normal physiological E2 levels. LH causes androgen prod which is conv to E2 at granulosa by Aromatase
- High sustained E2 would usually prov pos FB to HPG axis and cause LH surge which would trigger ovulation
- Highlights important of first step in shutting down HPGto allow development & growth of multiple follicles, preventing premature ovulation of these follicles and maximising the no. of oocytes available for fertilisation
6
Q
- Ovarian Stim. & follicle monitoring
How are follicles monitored?
A
- Regular US to checkfollicle count every 1-3 days to ensure the pt does not develop OHSS
- OHSS would lead to VEGF release. (VEGF mRNA is expressed by granulosa & theca cells late in follicular development - provides permeability of theca blood vessels that occurs before ovulation) this results in oedema
- OHSS risk is incr in PCOS women and women with high ACF.
- Mild cases seen in ~35% normal women
7
Q
- Ovulation & Assessment of Oocytes
hCG administration?
A
- hCG is an LH analogue
- It is administered and binds to LHRs in theca/granulosa, allowing ovulation on demand (mimics LH surge)
- 34-38 hrs later, oocytes are surgically retrieved from the ovary via TVUS & laporoscopic surgery via the vaginal wall under local anaesthetic
- hCG injection confers risk of OHSS
- Using GnRH agonist is an option but live birth rate is 6% lower than using hCG
8
Q
- Ovulation & Assessment of Oocytes
How are oocytes assessed?
A
- A ‘good egg’ has completed meiosis I and extruded the 1st polar body
- Metaphase II oocytes show significantly higher fertilisation rates and more blastomeres per embryo than oocytes arrested in Metaphase I
- Morphological features of the oocyte can also be observed by light microscopy
- Techniques incl. ZP imaging > able to detect birefringence (optical activity and refractive index) between eggs which is a predictor of compaction, blastulation & pregnancy
9
Q
- Semen Preparation & insemination in vitro
Sperm collection & preparation?
A
- Semen is collected from the male via masturbation
- Wait for liquefaction to occur (prostatic enzymes e.g. prostatic acid phosphatase break down gelatinous proteins resulting in a thinner texture) ~15 mins
- Sperm washing is carried out
- involves density gradient centrifugation -> live sperm at the bottom of the tube, other components rise
- seminal fluid is removed > acts as capacitation i.e. maturation of sperm > destabilisation of acrosomal head allowing it to penetrate outer egg layer>Changes in tail allow incr sperm motility
- Sperm cells are conc in Hams F10 media and warmed to 37o
- A cryoprotectant is added if sperm are to be frozen
10
Q
- Semen Preparation & insemination in vitro
Insemination in vitro? incubation? ICSI?
A
-Healthy oocytes are inseminated in media (in vitro)
-very delicate process so lots of factors to control e.g. nutrients, acidity, gas comp of air, light exposure
-Sperm and egg are incubated at a ratio of 75,000:1
~4hrs after egg retreival
-A 2013 review found that 1-4hours incubation resulted in significantly higher pregnancy rates than coincubation for 16-24hrs.
-~65% eggs will fertilise (assessed after 28hrs)
-Intracytoplasmic sperm injection is used as an expansion of IVF mainly to overcome male infertility problems
-Sometimes used where eggs cannot easily be penetrated by sperm
-Can be used in teratozoospermia - when egg is fertilised abnormal sperm morphology does not appear to influence blastocyst development or morphology
11
Q
- Embryo culture and transfer
Cleavage stage: culture
A
- Most IVF labs culture embryos in droplets of media under oil overlay to provide the maximum osmolality range for embryonic development
- First sign of fertilisation = 2 pronuclei
- Fertilised egg is passed to medium and left for 2-4 days until egg consists of 6-8 cells
- Some die due to apoptosis
- Dividing, healthy embryos are selected for transfer
- Excess embryos can be cryopreserved
12
Q
- Embryo culture and transfer
Embryo grading
A
- Labs have developed grading methods to judge embryo quality
- Time lapse microscopy can be used to study embryo morphology
- Aneuploidy screening can check for common chromosomal abnormalities which are a major cause of implantation failure and miscarriage
- also check for Downs’ Syndrome
- Screening recommended in women >35yrs, family Hx, several unsuccessful IVF cycles & if sperm are known to be at risk of having chromosomal problems
- Involves embryologist removing one/two blastomeres from the embryo
13
Q
- Embryo culture and transfer
Blastocyst stage & transfer
A
- Embryos are graded by an embryologist based on amount of cells, evenness of growth and degree of fragmentation
- Healthy embryos chosen to be transfered to uterus
- In UK, canada, Aus & NZ a max of 2 embryos are transferred except in unusual circs
- In UK, HFEA regulations state that women >40yrs can have 3 embryos transferred as chance of successful preg. less
- Most clinics seek to reduce risk of multiple pregnancies
- At blastocyst stage (~100cells), healthy embryos are transferred to the uterus via the cervix using a catheter under US guidance
- Await pregnancy confirmation
14
Q
- Luteal support
Explain differences in luteal phase for non preg woman, preg woman and after IVF
Explain luteal support
A
- After pregnancy confirmation, support is provided through luteal phase via cyclogest (P4) to support implantation and early development of the embryo
-Non-Preg women: E2 & P peaks during 4 days after ovulation and declines a few days before menses
-Preg women: E2& P prod restored by hCG stim. of CL
> shift from ovarian to placental prod of gonadal steroids occurs ~7wks gestation
- After IVF: Luteal phase differs because of the prod of multiple CLs
- Causes incr P during luteal phase > a sharp and gradual P incr then a sharp decr in P prod
- In this way use of a GnRh agonist creates a short luteal phase
- P4 (Cyclogest is administered via a vaginal preparation to maintain the uterus >converts endometrium to secretory stage > to support embryo and maximise chance of live birth
15
Q
Recent IVF improvements?
A
- development of GnRH agonists/antag
- Purer FSH/LH preparations
- Recombinant g/trophins
- Better US monitorins = decr OHSS
- Decr in mx embryo tx
