lecture 13: ART and IVF

Question 1

What is the ‘receptive period’?

Answer 1

• time of endometrial receptivity
• the ‘window of implantation’
• the endometrium has to be sufficiently primed for the embryo
• soil for the seed
• over the course of your cycle the endometrium gets thicker - increased vasculature
• only receptive for a couple of days
• potentially a screening method for early embryos - maybe something is wrong with it if it arrives too early or too late

Question 2

What is the silent sickness in society?

Answer 2

• infertility
• people don’t like to talk about it
• men don’t talk about while women might share more
• massive impact
• very stressful

Question 3

What is the definition of infertility?

Answer 3

• unprotected intercourse for 1 year without conception

Question 4

How frequent is assisted human conception?

Answer 4

• 1 in 6 couples will require medical intervention to conceive
• around 3-4% of all children born in Australia are so-called ‘test-tube’ babies
• there are over 5 million test-tube babies world-wide

Question 5

What is the age-related decline in human female fertility?

Answer 5

• fertility starts to decline in the late 20s
• chances of conceiving are very low in late 30s to 40s

Question 6

Who was the world’s first test-tube baby?

Answer 6

• Louise
• delivered by Robert Edwards, Jean Purdy, and Patrick Steptoe
• born in 1978 in north england
• IVF is the biggest team sport ever played

Question 7

Is IVF a cure? What is the paradox of infertility and multiple gestations?

Answer 7

• percentages of ART cycles using fresh nondonor eggs or embryos that resulted in live births, by woman’s age and number of previous live births
• not a miracle cure
• older women still have low chances of having a successful pregnancy with IVF
• risks of having multiple-foetus pregnancy and multiple-infant live birth from ART cycles fresh nondonor eggs or embryos
• 1/3 of IVF births are twins or more
• 25% of multiple births in Australia come from IVF
• the goal of human assisted conception should be the birth of a healthy singleton conceived through the transfer of a single embryo → becoming a reality over the last few years

Question 8

What is the significance of single embryo transfer?

Answer 8

• medical
• financial
• social

Question 9

What are risks to the mother of having twins +?

Answer 9

• hypertension
• thrombo-embolism
• urinary tract infection
• anaemia
• placental abruption
• emergency caesarean section
• increased maternal mortality rate

Question 10

What is the incidence of cerebral palsy compared to singletons/100 pregnancies?

Answer 10

• affects the child/family for life
• incidence in twins is 8 fold higher
• triplets: 20
• quads/+: 50

Question 11

What is the rate of infant mortality?

Answer 11

• deaths less than 1 year per 1000 lives births
• 0.3% of singletons die in the first year of life
• twins: 4%
• triplets: 8%
• quads: 10%

Question 12

What are the costs of prematurity at delivery?

Answer 12

• twins: $30,000
• triplets: $100,000
• quads: $400,000

Question 13

How much are ART-associated multiple pregnancies estimated to cost US health care annually?

Answer 13

• around $890 million ($1 bil australian)

Question 14

What is required in order to attain single embryo transfer?

Answer 14

• optimise embryo development in culture
• ability to select the most viable/normal embryo
• optimise cryopreservation procedures

Question 15

What is the process of IVF?

Answer 15

• Stage 1: follicles are stimulated using gonadotrophins
  
  • patients are given exogenous gonadotrophins to recruit more than one oocyte and hCG to induce the maturation process
• Stage 2: mature eggs are removed from ovaries
  
  • typically 10 oocytes are collected from each patient
  • oocytes are collected (before ovulation) by inserting a needle into each follicle and aspirating the contents
  • this is performed using a local anaesthetic and transvaginal ultrasound
• Stage 3: eggs are fertlised with sperm
  
  • at the same time as the eggs are being retrieved they will do a sperm collection
  • if the sperm are motile they will add the live sperm and let them attack the egg like normal
  • sometimes there are no motile sperm or very few
  • can use ICSI
  • about half the worlds test tube babies are born following this
• Stage 4: fertilised embryos are grown in the laboratory and subsequently replaced into the uterus
  
  • grow embryos on little drops of culture medium
  • layer of oil to prevent dehydration
  • use all kinds of incubation chambers
  • analysis of uterine and fallopian fluid showed that there were concentration gradients
  • generated a culture system that was mirrored on what was in the female reproductive tract

Question 16

What is the social impact of multiple births?

Answer 16

• the divorce rate is 8% higher in parents with twins

Question 17

What is the embryo culture?

Answer 17

• pronucleate oocytes are cultured in groups of 4 in 50µl drops of phase I medium under paraffin oil for 48h until Day 3 in 6% CO₂, 5% O₂, @ 37°C (up to 8 cell stage)
• blastocyst culture:
  
  • on Day 3 embryos are moved to 50µl drops of Phase II medium under oil for 48h to the blastocyst stage (Day 5) in 6% CO_2,5%O₂, @ 37°C

Question 18

What day should we perform embryo transfer?

Answer 18

• Day 1 at the pronuclear stage?
• Day 2 or 3 at the cleavage stage?
• Day 5 at the blastocyst stage?
• putting the embryo back prematurely significantly compromises embryo development and implantation

Question 19

Transfer of Embryos at the Blastocyst Stage: a means of increasing implantation rate?

Answer 19

• identification of those embryos with little, if any, developmental potential
• synchronisation of the embryo with the female reproductive tract
• minimise exposure of embryo to uterine environment
• reduced uterine contractions on day 5
• assessment of true embryo viability (post genome activation)

Question 20

What was seen in a prospective randomised trial of Day 3 vs Day 5 embryo culture and transfer?

Answer 20

• implantation rates were much much higher on day 5

Question 21

How many live births per transfer?

Answer 21

• goes down with age and use of own eggs
• if you use an egg donor the chances of getting pregnant stay exactly the same regardless of age (average age of an egg donor is 27)
• therefore it is not the endometrium it is the egg

Question 22

What is genetic analysis?

Answer 22

• when anyone in your family has any kind of genetic disorder you should go and see a counsellor
• can find out risks
• used to have to either not take the risk or get pregnant and wait for amniocentesis
• terrible position to be in - elected termination or baby born with potentially awful disease
• this provides an alternative

Question 23

How is aneuploidy affected by maternal age?

Answer 23

• increases
• human aneuploidy: mechanisms and new insights into an age-old problem, Pat Hunt
• very cruel increase in the incidence of downs syndrome past 30s

Question 24

What can you do to avoid implanting embryos with aneuploidy?

Answer 24

• extracting polar bodies (will have the opposite number of chromosomes as in the nucleus of the oocyte)
• take a blastomere: human embryo biopsy, single cell genetic analysis
  
  • have taken one eighth if the
• trophectoderm biopsy, taking placental tissue
• FISH: can only do very few chromosomes and had quite a high error rate
• more recently: multiple genetic analysis, array CGH (comparative genomic hybridisation on a chip, can screen with confidence for every chromosome, can do on another chip specific genetic disorders

Question 25

What is analysis of embryo physiology?

Answer 25

• genomics
• proteomics
• metabolomics
• embryo is smaller than a full stop on a printed page so have very little material to work with
• have had to work out ways of scaling down experiments to be able to work on single cells

Question 26

What is glucose uptake on day 4 of development and pregnancy outcome?

Answer 26

• embryos that consume more glucose are actually more able to produce pregnancies
• relation to embryo sex: females tend to use more glucose than males

Question 27

Can viable human blastocysts be sorted from thos which fail to produce a pregnancy?

Answer 27

• viable human blastocysts have a different metabolic footprint to those which fail to produce a pregnancy

Question 28

What is lab on a chip?

Answer 28

• metabolic analysis of embryos
• allows us to work in small volumes - more reflective of the biochemistry of the body
• nano litres, pico litres
• fluids behave different
• more analagous to physiology
• able to control volumes for analysis
• can get down to single cell analysis
• phenomenal sensitivity

Question 29

What is the importance of cryopreservation for IVF?

Answer 29

• with the move toward reducing the number of embryos transferred there is an increased need to optimise the cryopreservation of supernumerary embryos
• dehydration:
  
  • need to take the water out of a cell if you want to cryopreserve it
  • if you don’t the water in the cell will crystallise
  • ice crystals will sheer everything inside, rip membranes apart
• can slow freeze by gradual dehydration or vitrify (liquid to glass instantaneously)
• if you slow freeze you need a freezing machine
• vitrification only needs a very elegant little device
• cryoprotectant + base medium went to a glass after freezing with liquid nitrogen, while base medium alone went to ice crystals

Question 30

What are methods of cryopreservation of oocytes and embryos?

Answer 30

• slow-freezing
  
  • low levels of cryoprotectants
  • slow controlled rates of cooling (0.3°C/min)
  • slow dehydration to minimise ice-crystal formation
  • takes hours
• vitrification
  
  • high levels of cryoprotectants
  • very fast cooling rates
  • ~20,000°C/min
  • fast cooling rates result in solidification of solution into glass-like structure (no crystallisation)
  • takes seconds
• can store them for decades

Question 31

What is the cryoloop?

Answer 31

• film of cryoprotectant on the loop
• high viscosity
• nylon loop (20µm wide; 0.5-0.7 mm in diameter)
• thin film of cryoprotectant solution by surface tension
• embryos are placed by pipette
• dipped directly into liquid nitrogen

Question 32

summary

Answer 32

• human infertility is very common, and one mode of treatment is the so-called “test-tube baby” method, or IVF
• patients are induced to develop several oocytes at one time
• fertilisation is by insemination or direct injection of a sperm into the oocyte
• embryos are culture up to day 5 before embryo transfer
• diagnosis of the embryo (both genetic and physiology) can be performed prior to implantation
• genetic analysis is invasive requiring embryo biopsy
• embryos not transferred are readily cryopreserved through vitrification
• lab-on-a-chip technologies may have a role in the future of assisted conception