Assisted Reproductive Technologies

Question 1

History of ART

Answer 1

• 18th-20th century → artificial insemination in domestic animals
• IVF of rabbit oocyte and successful embryo transfer giving live birth (Pincus and Enzmann, 1934)
• 1960/70s → Attempts to first fertilise human oocytes in vitro (Edwards)
• 1978 → First test tube baby, Louise Brown born
• 1982/84 → Warnock committee of enquiry in the UK and Warnock report publication
− Established to enquire into the technologies of IVF
− In response to the concern at the speed of which they are developing, and also the birth of Louise Brown
− Aim to develop princiles for the regulation of IVF and embryology
− Concluded that the embryo should be protected, but research and IVF is permissible given the appropriate safeguard
− Propose the establishement of a regulatory authority – the HFEA.
• 1989 → Fertilisation and Human Embryology Bill UK
• 1990 → HFE Act
• 2010 → Edwards wins Nobel prize

Question 2

Regulation of IVF in the UK

Answer 2

• HEF Act 1990 → under this act, the HFEA regulates IVF and similar ARTs
• The major clinical remit is:
− Consideration of the clinical welfare of the couple and upholding of clinical standards
− Major consideration in decision for treatment is the welfare of the child

Question 3

When are a couple considered to have a fertility problem?

Answer 3

If they do not concieve after 1 year of unprotected intercourse

Question 4

What are potential female causes of infertility? (30%)

Answer 4

Failure to ovulate → failure to respond to gonadotrophins, or failure of the normal pattern of gonadotrophins
Blocked/fluid filled fallopian tubes
Endometriosis - where the endometrium grows outside the uterus
Low ovarian reserve → could be due to elevated FSH
Unexplained, or multifactorial

Question 5

What are potential male causes of infertility? (30%)

Answer 5

Low sperm number
Poor sperm motility
Suboptimal number of normal looking sperm

Question 6

Possible ARTs

Answer 6

• Ovulation induction
− Clomiphene: inhibits the estrogen receptor in the hypothalamus, preventing the negative feedback on gonadotrophin production
− Gonadotrophins: overcomes insufficient production
• Superovulation (ovulation induction) + intrauterine insemination
• Egg, sperm or embryo donation
• Donor sperm insemination
• Gamete intrafallopian transfer – historically
• IVF
• ICSI – used when sperm is suboptimal
• In vitro oocyte maturation followed by IVF → oocyte needs to be in arrested metaphase II

Question 7

IVF statistics:

Answer 7

• Over 15,000 babies born through IVF in the UK in 2008 – rise 10% year on year
• Currently 2% of all babies in the UK are born via IVF
• The live birth rate/treatment is about 20%
• Failure of IVF is related to poor embryo development and implantation/pregnancy failure
• The major factor in success is womans age due to loss of ovarian reserve and poor oocyte quality → one study indicates that 12% of the pre-birth ovarian reserve remains at 30, only 3% by 40.

• It isn’t surprising there are so many failures with IVF – it is being performed on a population with inherent fertility problems.

Question 8

Overview of the IVF Cycle

Answer 8

• The duration of each treatment cycle is 6-8 weeks
− Normal menstrual cycle is 28 days, but the first part of treatment involves switching off the womens own menstrual cycle, so the clinican can impose an artificial cycle.
1. Natural menstrual cycle is downregulated (the natural cycle may actually be aberrant)
− Daily Buserelin injections from day 18-25 of cycle → suppresses gonadotrophins, and gives menstrual bleed
− Buserelin is a GnRH agonist, causes continuous stimulation of the pituitary and therefore decrease pituitary secretions of LH and FSH
2. The woman is given GnRH + gonadotrophins + buserelin injections to stimulate oocyte growth and control the timing of ovulation.
− These injections are a harsh treatment, thought it may be better to give kisspeptin to naturally stimulate gonadotrophin production.
3. Monitor oocyte growth and graffian follicle formation (monitor by ultrasound scans and blood tests)
4. When ready for oocyte collection, give late night hCG injection, triggering meiotic maturation
− hCG would normally trigger ovulation, but you don’t want them to be ovulated. So you go in early, and pick the oocytes from antral follicles of ideally up to 12mm.
5. Oocyte collection 36 hours later
6. Fertilise
• The oocyte and sperm are mixed, and then 16-18 hours later we look for the presence of an oocyte with two pronuclei.
• With IVF/ICSI – 60 to 70% of the eggs fertilise and form embryos.
• Then, in an incubator at 37C, we get cleavage stages
7. Day 5 embryo transfer - allow the embryo to develop in vitro, so you can see which have successfully gone through preimplantation development.
8. Pregnancy test in 2 weeks

Question 9

When do ultrasounds occur during the IVF cycle?

Answer 9

• Occurs around 4-5 tmes

1. During downregulation, to confirm thinning of the uterine wall and the presence of quiescent ovaries with no follicles
2. 2-3 scans during the stimulation phase to confirm response to gonadotrophin injections

Question 10

How are eggs collected for IVF?

Answer 10

• Vaginal ultrasound guided procedure to aspirate oocytes from suitable follicles
• Usually >12mm diameter
• If they are too big, may be becoming atretic.
• Generally, 70-75% of follicles seen on scans give oocytes (much more than natural cycle)
• Harvesting eggs is tricky and invasive

Question 11

How is sperm collected for IVF?

Answer 11

• Partner provides fresh semen sample
• Sperm separated on a gel gradient to select for most normal sperm
• If sperm are suboptimal, clinician considers ICSI.

Question 12

How does the preimplantation embryo culture change?

Answer 12

• Single stage mediate (day 2/3 replacement)
• Single or 2 stage media (day 5 replacemnt)
• Reflects the different metabolic requirement (Early embryo has low energy requirement, releies on anaerobic metabolism of pyruvate and lactate, blastocyst on the other hand has a full glycolytic cycle).

Question 13

What are the pros/cons of early/late embryo replacement?

Answer 13

• Early transfer avoids potentially damaging culture conditions but:
i. No chance to assess which embryos are likely to be viable and healthy
ii. If you put a cleavage stage embryo back in the uterus, this is technically the worng environment, as in vivo, blastomeres undergo cleavage in the fallopian tube. In other animals, the embryo wouldn’t survive in the wrong environment. May be okay in humasn as tube is wider, allowing mixing of follicular and uterine fluid
• Day 5 transfer allows morphological assessment and choice of best blastocyst but:
i. Culture environment may be damaging
ii. If the embryos don’t survive, none can be replaced

Question 14

How are oocytes selected for quality?

Answer 14

Morphology -

Question 15

How is sperm selected for quality?

Answer 15

Morphology, motility, DNA fragmentation

Question 16

How are embryos selected for quality?

Answer 16

• Appearance of 2 pro-nuclei within 18 hours
• Non-invasive amino acid profiling of culture medium
• Prenatal genetic screening
• Pyruvate, glucose and oxygen consumption
• Time-lapse videoing for morphology and analysis of length of first cleavage (recent) :
Timing is everything! → Kiessling 2010
− A new human life depends upon robust steadily increasing signals from the fertlised egg to the mother
− How the early human embryo sends the robust, steadily increasing signals is poorly understood, byt one mechanism could be rapid duplication of embryonic DNA with a concomitant increase in signaling output to the mother → this suggests a direct correlation between the speed of chromosome duplication, cell divison and successful pregnancy.
− Fertilised on day 1, an egg that has duplicated its chomosomes twice and reached the 4 cell stage by day 2, and reached the 8 cell stage by day 3, has a higher likelihood of giving rise to an offspring than an egg that duplicated its chromosomes only once, and reached the 2 cell stage on day 2, and 4 cell stage on day 3.
− mouse embryo model has taught us that the rapid cleavage rate that occurs in vivo between the 4 cell and 16 cell stage is not reproduced in vitro under existing culture conditons. Mouse embryos developed in vivo have more than twice as many cells at blastocysts at the blastocyst stage than embryos developed in culture.
− Should the situation be the same for human embryos, extended culture would lead to blastocysts with fewer cells available to form a fetus → possible explanation for the low birth weight reported for some IVF.
− Time-lapse videoing by Wong et al, discovered 3 characteristics that could predict progression to the blastocyst stage:
i. First cleavage furor should be only 14 mins from the beginning to the appearance of 2 cells → may not hold, not used clinically
ii. The 2 cell stage should last only 11 hours → best indicator
iii. The cleavage of each of the 2 cells into 4 daughters should occur within 1-2 hours of each other

Question 17

How is timing also crucial in terms of the mother?

Answer 17

• Think 15% of implantation failures are due to poor timing
• In most women, there is a 2 to 4 day stretch where the endometrium sends out crucial chemical signals that allow the embryo to attach. For some women, the fertile window is shifted earlier or later in the cycle, or is unsually brief.
• In the new test, a biopsy of the endometrium is taken, and the gene activity is analysed. As the cells enter the receptive phase, a series of genes switch on or off in a reliable sequence that can be ready.
• 33% of women treated with the test had successful implantation, significantly more than would be typically seen.

Question 18

How have microRNAs recently been found to play a role?

Answer 18

• Women who repeatedly suffer IVF failure shown to have altered microRNA expression in the endometrium
• Study suggests the presence of IGF1R is required for embryo attachment – IGF1R is a target or miR-145
• miR-145 overexpression inhibits the growth of IGF1R
• Developing treatments to suppress miR-145 could lead to improved rates of attachment

Question 19

Is aneuploidy common?

Answer 19

• 5-20% of oocytes carry errors from one or both meiotic divisions
• Incidence of aneuploidy similar in normal and ART population but possible causes of differences include ovarian stimulation or culture conditions, or errors in early cleavages.
• Infertile parents may carry chromosomal abnormalities

Question 20

What are the risks of IVF?

Answer 20

• Ovarian hyperstimulation syndrome (pain and bloating, but short term)
• Maternal cancer
• Increase in perinatal death, pre-term delivery and small for dates babies → low birth weight is main concern, 2.6 fold increase in risk
• Accepted small increase in congenital abnormalities (not known if this reflects the infertility status of the parents) → the majority do not have birth defects. However, some studies suggest increase risk – 6.2% of IVF children had birth defects compared with 4.4% naturally conceived children.
• Controversial association between ART and imprinting disorders (normal switching off of an allele during imprinting may not occur)
− Beckwith Wiederman syndrome → Excess IGF-II driven somatic overgrowth and other features found in 4% of children. Aberrant imprinting and DNA methylation
− Angelman syndrome → severe mental retardation, sporadic defects in imprinting with loss of methylation
− Prada Willis syndrome
− These errors not really considered to be a risk , corrected for maternal age and other factors.

Incidence of multiple births:
• Multiple pregnancy is the single biggest risk → correlated with small for dates babies, subject to miscarriage and diseases such as cerebral palsy
• Although children from triplet and higher order multiple gestations are greatest risk, study showed that IVF twins were more likely than IVF singletons to be admitted to a neonatal ICU, require surgical intervention and to have special needs → the mothers of these twins gave lower ratings for their general health.
• Women who carry multiple fetus have greater need for bed rest, higher risk of premature labour, hypertension, postpartum hemorrhage, C-section and although rare, death.
• Excess hospital costs for multiple births resulting from IVF estimated at $640 million per year in the US

→ Because of this, there is a maximum 2 embryo transfer policy in the UK, recommened by NICe