Assisted Reproductive Technology Flashcards
Define infertility and list potential causes.
- The WHO define infertility as the failure to conceive after 1 year of regular, unprotected intercourse. Have to define what is meant by regular, unprotected intercourse; need to check whether the patients are doing it properly, e.g. how it is being carried out, what times of the month, how often etc. Then it can be investigated.
- Around 15% of couples have a problem conceiving
- The chance of getting pregnant each month is the same, but it is unlikely not to get pregnant after a year if not infertile and done properly.
1) Mechanical blockage to egg and sperm meeting =
a. Infection/occlusion of vas deferens or uterine tubes. One of the main reasons for occlusion of uterine tubes in women is endometriosis.
b. Previous ligation for sterilisation, e.g. vasectomy reversal issues like scarring or antibodies to sperm. Usually, there is a blood testes barrier (tight junctions between the Sertoli cells), so the immune system does not usually see the contents of the testes. During a vasectomy, this may happen, and anti-sperm antibodies can form.
c. Endometriosis = Endometriosis = cells from the lining of the uterus (endometrial tissue) implant in other areas of the pelvis and continue normal function as if they were in the uterus. During the first half of the menstrual cycle, when oestrogen levels are very high and the follicles are developing (the follicular phase), these cells (wherever they are) continue to respond to the high oestrogen and proliferate just as if they were in the uterus. At the end of the cycle, they differentiate during the second half of the cycle under the action of progesterone. At the end of the cycle, when the corpus luteum dies, progesterone levels fall, and they shed/bleed wherever they are (just as if they were in the uterus). Cells from the endometrium can only enter the pelvis through one route (through the uterine tubes); endometriosis in uterine tubes will completely block them, endometriosis on the outside of the tubes may cause inflammation and hydrosalpinx (fluid accumulation in the uterine tubes that will again block them). Any kind of inflammation in the pelvis might cause the uterine tubes to be blocked, e.g. chlamydia (most common infection that causes inflammation in the pelvis that may cause infertility in a women due to blockage of the uterine tubes), pelvic inflammatory disease. The uterine tube is where fertilisation occurs. Also, the first 5/6 days of embryo development occurs in the uterine tubes. It is not just a passive tube connecting the ovaries with the uterus; it is a highly specialised environment where fertilisation and early embryo development occurs. Anything that disturbs this will lead to infertility. This can be bypassed completely when fertilisation (egg and sperm meeting) occurs in vitro. One of the main reasons for occlusion of uterine tubes in women.
d. Congenital defects. Congenital defects include absence of the vas deferens mainly. Blockage or congenital absence of vas deferens means sperm will not be expelled.
2) Failure of gamete production or release.
a. Anovulation, maternal age, PCOS. Ovarian reserve decreases with maternal age (as menopause approaches). Decreased ovarian reserve = irregular cycles, lower quality eggs. As women reach their mid 30s, they start panicking and reach the the IVF clinic to find out their ovarian reserve and how much time they have. Maternal age is quite common.
PCOS is another common one; can cause anovulation/ problems with ovulation.
b. Azoospermia (no sperm), asthenozoospermia (sperm don’t swim well), teratozoospermia (increased abnormal morphology), oligozoospermia (low count of sperm). In terms of men, it is about sperm quality. Azoospermia is no sperm at all, which is quite difficult to overcome, unless it is caused by one of these other sperm conditions, e.g. asthenozoospermia or tetrazoospermia. Abnormal sperm can make up over 90% and this would still be considered normal. Oligozoospermia is a low count of sperm (not necessarily infertile). These conditions don’t necessarily mean that a man is infertile, but it decreases the chances of getting pregnant each month and so patients may choose to seek help.
3) Failure of fertilisation/implantation & miscarriage.
a. Genetic factors. Failure of fertilisation/implantation or early miscarriage can sometimes be due to genetic factors, but often the embryo is not quite right. The embryo doesn’t divide properly at first; the first mitosis isn’t quite right, doesn’t have enough chromosomes or has too many chromosomes. A lot of these embryos will automatically undergo apoptosis; they may not implant or there is early pregnancy loss (if they do implant). It can often be due to a genetic factor, sometimes it is that the egg quality was not sufficient or the sperm genetic quality.
b. Endometrial receptivity, maternal age. Endometrial receptivity can be affected by PCOS or hormonal abnormalities in the cycle. This again can come back to maternal age; poor egg quality, irregular cycle, not as receptive endometrium.
4) Unknown/unexplained = About 40% of all cases are idiopathic.
- This list highlights major causes of infertility, then medical interventions can be decided.
What are possible methods of Assisted Reproductive Technology (ART)?
1) Inducing ovulation with exogenous hormones. Begin with something as mild as possible; ovulation may be able to be induced by administering exogenous hormones if the problem is just anovulation (irregular cycle/failure to ovulate). If this doesn’t work, the uterine tube can be bypassed entirely; the egg and sperm can meet in vitro.
2) By-passing the uterine tube (IVF); the egg and sperm can meet in vitro.
3) Direct collection of sperm from the testis/epididymis.
4) Direct insertion of the sperm into the egg (ICSI). The sperm can be directly injected into the egg; a fine needle can be injected into the testis and a few sperm can be aspirated. ICSI is useful when the sperm are dysfunctional, e.g. don’t swim properly. The sperm that are aspirated, in an azoospermic patient for example, are not swimming in the testes (they start swimming on the way out).
5) Donor gametes. If all else fails, donor eggs/sperm can be used.
6) Combination of the above. With all of these tools at our disposal, with something as mild as possible, the real treatment might be a combination of all.
When are gonadotrophins used to induce ovulation?
- Exogenous gonadotrophin used to treat women who are anovulatory or who have oligo (irregular)/amenorrhoea (no menstrual cycles; no period, not ovulating).
- The aim is to induce single dominant follicle.
- Can be daily injections and monitored by ultrasound during the cycle.
- A few oocytes are recruited into the cycle and start to grow by an intercycle rise in FSH. As they all start to grow, about a dozen of them in both ovaries, they produce some oestrogen. This oestrogen thickens the lining of the uterus in the first half of the cycle, but it also feeds back negatively on the cycle. It acts on the hypothalamus and pituitary, reducing the FSH. That reduction in FSH causes most of them to undergo apoptosis, because they are FSH-dependant. The most advanced one, which has the most FSH receptors and has also started to induce some LH receptors, survives and goes on to become the dominant follicle.
- In a situation where this hasn’t quite happened, there may be a few follicles which are not quite producing enough oestrogen to cause ovulation. When oestrogen reaches a certain threshold for a couple of days, the feedback turns positive and there is a massive surge in LH which causes ovulation. There isn’t enough oestrogen for this, but there isn’t enough FSH to get over the line and further develop the follicles. If this has not happened the normal way, exogenous gonadotrophins injected, particularly FSH, should promote follicle development again. The oocytes will work again, the follicles will be stimulated, and this will allow the threshold to be reached for selection of a dominant follicle.
- Exogenous gonadotrophins can be injected. Can either be daily injections or a single injection sometimes. When doing this, it is important to be really careful and monitor by ultrasound. Too much FSH can prevent dominant follicle selection. This is because FSH stays high, since it is being injected, and lots of follicles reach maturity. When it began in the ‘60s and ‘70s, FSH used to keep being injected and it would result in stories of women having octuplets etc. This is very dangerous for the mother and the children. The aim is to stimulate the follicles and obtain one dominant follicle; if a lot of follicles start to be stimulated, the drug can be stopped.
- Fostimon is an example of a drug that can be given intramuscularly but is usually given subcutaneously (patients can inject it into themselves). This one is called Urofollitropin. There are two sources of this = the modern source is recombinant technology (a cell line has been transfected with the gene for FSH, grown in vitro, purified), the older method is to collect it from post-menopausal women’s pee and purify it (still used today, Fostimon is an example). Post menopausal women have a lot of FSH in their urine, because they have low oestrogen and progesterone levels. This means that there are no brakes on the hypothalamus and pituitary, so they have higher levels of FSH and LH which are metabolised and excreted into the urine.
Aside from using exogenous gonadotrophins, what’s another way of manipulating the HPG axis to induce ovulation?
- Gonadotrophin levels may be normal, but are not cyclical. Exogenous gonadotrophins is just adding FSH. A more sophisticated way of doing it could be to remove the negative feedback.
- At the end of a cycle, there is death of the corpus luteum and progesterone levels fall. That fall in progesterone levels takes the brake off and there is a rise in FSH naturally that starts the next cycle. Inter-cycle rise in FSH relies on death of the corpus luteum, ie. fall in levels of progesterone and estradiol.
- There is no corpus luteum in the absence of ovulation. This can’t be done here, as ovulation hasn’t been reached; this is anovulatory. We are in the first half of the cycle, with a little bit of oestrogen and a bit of FSH, but not going anywhere.
Removing the feedback caused by oestrogen would cause a natural rise in FSH, so it would not need to be administrated. - Cannot reduce progesterone as there has not been a corpus luteum to make any.
- There are follicles in the ovary making estradiol so we can remove the negative feedback of this.
- Removing the feedback caused by oestrogen would cause a natural rise in FSH, so it would not need to be administrated.
What are the two ways in which negative feedback is removed to induce ovulation?
- There is a bit of FSH, but not enough to stimulate the gonads. There are some follicles producing oestrogen, but not enough to cause ovulation. Stuck!
- Removing the feedback from the oestrogen will release the negative feedback on the hypothalamus and pituitary, so endogenous FSH will rise and get the follicles moving again.
- 2 ways to remove estradiol feedback:
1) The first way to do it is to block the oestrogen (E2) receptors on the hypothalamus and gonadotroph cells of the hypothalamus using a selective oestrogen receptor modulator (SERM) = Clomid/Clomiphene is a common one. It blocks these without stimulating them. Although there is oestrogen present, the hypothalamus and pituitary do not respond to it and remove the brakes on feedback, so FSH is produced.
2) Stop E2 being made in the gonads by using an aromatase inhibitor . To go from androgens to oestrogens, there is an enzyme that removes a carbon atom (found in the granulosa cells). This enzyme can be stopped using a specific enzyme inhibitor. A lot of enzyme inhibitors end in ‘zole’. Letrozole blocks the aromatase and oestrogen can’t be made. Oestrogen levels fall, the brakes have been taken off and there is increased endogenous FSH.
Outline the process of IVF.
- Exogenous gonadotrophins is probably the first line of treatment with an ovulation problem (it may not be an ovulation problem, e.g. may be a sperm problem). If it is an ovulation problem and that doesn’t cure it, in vitro fertilisation will likely be the next choice.
1) Hypothalamic-pituitary down regulation (GnRH). Normally, GnRH is produced in a pulsatile manner; fast pulses (~every 60 minutes) causes the pituitary to favour LH. IVF begins with downregulation of the HP axis. Exogenous GnRH is administered, which shuts down the axis. This prevents ovulation, so follicles are not lost in the LH surge. If many follicles are growing and all producing oestrogen, the oestrogen threshold will be released reached very early (within two to three days); the HPG axis is turned off to prevent premature ovulation.
2) Ovarian stimulation (monitoring follicles). The next step is stimulating the follicles and trying to get as many as possible. Daily FSH is given for the first 12 to 13 days with ultrasound monitoring. There will be no dominant follicle selection as FSH will remain high (administered everyday). There will be a lot of follicles and they will be triggered for ovulation.
3) hCG trigger
4) Oocyte retrieval
5) Fertilisation in vitro. Can collect the eggs and bypass the cycle. The eggs can be mixed with the sperm in a lab and an embryo can be grown, then implanted back into the woman. The oocytes will be collected and then they will be mixed with sperm in a dish. The embryos will be cultured for a few days and then transferred back into the mother. Hopefully, a pregnancy test will come out positive after this. Usually, only one embryo is transferred, but the rest will be frozen if there are more.
6) Embryo culture 3 – 5 days
7) Embryo or Blastocyst transfer
8) Pregnancy confirmation
9) Luteal phase support - Cyclogest (progesterone). Luteal phase support (progesterone) may be given early in the pregnancy to help maintain a good endometrium. Failure will occur at every stage; not every egg is mature, not every egg gets fertilised and some embryos are genetically wrong from the beginning and undergo apoptosis. Naturally, one egg is released every month. We want to do this once, so lots of eggs are collected to produce as many embryos as possible. Losses will occur at each stage, but embryos that aren’t used can be frozen for additional attempts later. - As failure will occur at each stage, we require as many eggs as possible and so hyper-stimulate the ovaries to increase follicle numbers.
How are multiple follicles selected in IVF?
- Multiple follicle selection with exogenous gonadotrophin
- Tweaking the menstrual cycle to favour the desired outcome.
- Typically, when the CL dies and progesterone levels decrease, the brake is taken off the cycle. No more negative feedback means there is an intercycle rise in FSH. Normally, there are dozens of small antral follicles in the ovary. These are follicles that left the primordial pool months ago and have been growing due to growth factors that we don’t yet understand (gonadotropin-independent). There is a constant conveyor belt of them and they develop through the stages of folliculogenesis. When they get to the small antral stage (start to have a fluid-filled space inside), they start to express FSH receptors. At that stage, as soon as the next menstrual cycle starts and FSH rises, they all get recruited into the current cycle. They’ll start growing and produce oestrogen. That oestrogen starts to reduce FSH (which they require to grow). It is nature’s way of selecting one out of the whole cohort. The lack of FSH will kill most of them, but the most advanced one will have the most FSH receptors and it will also have induced some LH receptors so it will escape this. It will progress as the dominant follicle, continue to produce oestrogen, the oestrogen will have a negative effect on the FSH and this will cause apoptosis (atresia when it comes to the ovary).
- When carrying out IVF, it is different. Exogenous FSH is administered everyday and endogenous FSH is switched off. Pituitary/hypothalamus action has been blocked off with GnRH. Rather than pulses, a constant level is administered to the patient which shuts down the pituitary and hypothalamus completely (pulsatile delivery is required); does not allow the receptors to get recycled, just shuts it all down. Either the agonist or antagonist will do it. This prevents ovulation so follicles are not lost in the LH surge.
- Exogenous FSH is being administered and no matter how much oestrogen is being produced, it has no effect. If many follicles are growing and all producing oestrogen, the oestrogen threshold will be released reached very early (within two to three days); the HPG axis is turned off to prevent premature ovulation.
All the follicles are selected, so hopefully there about 10 to 15. Too many can be dangerous, so it is monitored by ultrasound; if we start to get too many, the dose of FSH can be reduced or may just stop entirely.
How is the ovary stimulated in a controlled manner?
- Downregulate Hypothamo-pituitary gonadal axis using GnRH antagonist or agonist. The antagonist or the agonist can be used to downregulate the hypothalamus and pituitary. She has multiple follicles growing and all producing oestrogen. She will reach the high oestrogen threshold for ovulation very quickly. If the HPG axis was not downregulated, oestrogen levels would go so high that she would get an LH spike suddenly after only a few days and this would cause ovulation (all the follicles will be lost). This is the reason why it is turned off before starting.
- As failure will occur at each stage, we require as many eggs as possible and so hyper-stimulate the ovaries to increase follicle numbers.
- Give FSH by subcutaneous injection. Growth of multiple follicles. Sometimes it is a mixture of FSH and LH, but mainly FSH. It is a subcutaneous injection; the patient will inject it at the same time everyday from the beginning of her cycle after downregulation with the GnRH. She will be monitored by ultrasound every few days. Multiple follicles grow under close monitoring. When the follicles are nearly at the right size, they need to get ready for ovulation.
- Monitor follicle growth with ultrasound until most follicles 12–19mm. At this point hCG trigger given (GnRH agonist or Kisspeptin may be used).
- 36 hours allowed for completion of meiosis I and initiation of meiosis II before egg collection. At ovulation, the LH spike causes two main events. Firstly, it causes resumption of meiosis I. Meiosis I begins in the ovary of the baby still in the womb (the follicles are very primitive = granulosa cells with an oocyte in the middle; meiosis I begins in this oocyte) and then the primordial follicles arrest. As the girl grows up, the primordial follicles in her ovaries are all arrested in meiosis I. When she becomes adolescent and starts cycling (during puberty), every egg that grows is in the middle of meiosis I. The LH surge causes resumption of meiosis I when the egg ovulates, it is completed, meiosis II begins and then it arrests during meiosis II (until fertilisation). Eggs are now in a position to be fertilized. Meiosis I needs to resume, but we don’t want her to ovulate (the eggs will be lost). An injection of LH could be used; there will not be an endogenous LH spike as GnRH has been used to shutdown the system. An artificial LH spike would cause resumption of meiosis I, completion of it and initiation of meiosis II. LH is not given though, because it is expensive and has a short half-life. Therefore, hCG is used; it binds to the LH receptor and does the same thing. There are some more modern methods, but they amount to the same thing. Classically, hCG is given because it is cheap, it is found in massive abundance (in pregnancy and there also recombinant ones), it binds to the LH receptor, and it does the same thing (causes resumption of meiosis I and then ovulation not long after that). The patient is told they are being given an injection of hCG and that they will ovulate around 40 hours later. By 36 hours, meiosis I will have resumed, completed and meiosis II will have begun. There is this window of opportunity where the eggs will be fertile (hCG trigger), but the eggs won’t have been lost so they can be collected.
- Important to note that we want to complete meiosis I, begin meiosis II and then collect the eggs.
How are oocytes retrieved in IVF?
- 34-38 hours post hCG trigger
- This is how the eggs are collected. The transvaginal needle goes through the wall of the vagina. The patient is mildly sedated. Under ultrasound guidance (transvaginal ultrasound probe), the needle is taken to the follicle and the follicular fluid is aspirated (the egg comes out with it). The embryologist is looking at each aspiration and ensuring there is an egg, the ultrasonographer is looking at the ultrasound picture and the doctor is doing the collecting.
- Can see how inflamed it is; the ovary is ready to release the egg in an inflammatory reaction caused by hCG (LH in a normal cycle). It is pierced, collapses and comes out with gentle aspiration under ultrasound guidance.
How is sperm prepared for IUI of IVF?
- The sperm has to be prepared. There’s a density gradient in the test tube, where it is denser at the bottom compared to the top (gets less concentrated). The semen is layered on top of the density gradient.
- After the semen is floated on top, it is centrifuged. Centrifugal force goes down and sperm that are not fully mature or the cellular fragments are less dense (sperm start off big and get smaller by shedding their cytoplasm so they become more dense). As it is centrifuged, they stop at whatever level of density they have. Only the most mature sperm get to the bottom of the tube. These can then be used in IVF; a petri dish contains droplets of media underneath mineral oil.
- The sperm and the egg are incubated together in the petri dish at a ratio of about 75,000:1. Duration of this co-incubation traditionally 16 – 18 hours. Approximately 65% of the eggs will fertilize. Incubated together overnight with a large ration of sperm to egg.
- Control factors such as nutrients, acidity, humidity, temperature, gas composition of air, and exposure to light.
What are the different stages the embryo can be seen in during IVF?
1) The fertilized egg has 2 pronuclei.This is the first sign of fertilization. Giving the hCG leads to resumption and completion of meiosis I. Meiosis I is cell division. When the egg divides, it divides asymmetrically. The egg contains all the cellular machinery for the early embryo (largest cell in the body). It contains all the ribosomes, the endoplasmic reticulum, the nutrients, the machinery for cell division etc., while the sperm just brings its DNA. Baby’s mitochondria is identical to mother’s mitochondria; it all comes from the egg. The sperm has its own mitochondria, but it does not contribute to the mitochondria in the embryo. When it divides, it keeps one half and then produces a polar body. After meiosis I, one polar body is visible. This means it is an egg that can be fertilised. After meiosis II, which happens at fertilisation, there is a second polar body. These two polar bodies mean two meiotic divisions have occurred. One pronuclei has the male DNA while the other has the female DNA = two pronuclei. They move together and fuse. The cell becomes diploid and mitosis occurs after this. Can see the unsuccessful sperm around the edge. Two pronuclei shows fertilisation and this is an embryo. Without the two pronuclei, especially with one polar body, the egg hasn’t been fertilised.
2) The developing embryo contains 6-8 cells 3 days after fertilization. After about three days, a human embryo having undergone a few cell divisions is seen. This used to be the limit to which an embryo could be grown in the lab; growing them for longer than three days would make them die. A few would be put into the mother with the idea that they would be better off in the uterus than the lab, but they shouldn’t be in the uterus at this point (supposed to be in the uterine tube). Therefore, a lot of them would not implant, but there were some pregnancies.
3) Blastocyst 5 days old approximately 100 cells. With better techniques, they can be grown to the blastocyst stage (around 5 days). The first thing a blastocyst does is differentiate the inner cell mass from the trophoblast. The trophoblast cells become the embryos’ part of the placenta and the inner cell mass of the embryo becomes the baby (this is where stem cells come from). When it has been grown to 5 days, the blastocyst has a better implantation rate (this is more like what is expected to emerge from the uterine tube ready for the uterus). Usually, only one is transferred.
How is the embryo transferred into the uterus?
- Embryo transferred to the patient’s uterus through catheter, which goes through the vagina and cervix, usually under ultrasound guidance. This is not as traumatic as collecting the egg, because there is a route this time to where you want to go. There is a similar fine catheter that goes through the vagina and cervix, straight into the uterus where it drops the embryo with hope it will implant into the uterine wall. Ultrasound guidance is used; there is some debate about where is the best place to drop the embryo in the uterus (no indicative evidence in support of this).
- Single embryo transfer is the norm in order to avoid multiple pregnancies, though 2 – 3 may be transferred in women over 40 or who have had repeated implantation failure. Two embryos may be placed in certain situations after being discussed with the patient, e.g. advanced maternal age, a few failed attempts etc. Three embryos is very rare; one is most common nowadays.
What are the success rates of IVF (considering maternal age)?
- Around 6.5 million children born worldwide to date.
- Approximately 60,000 to 70,000 cycles per year in the UK.
- Over the age of 44, the chances of a live birth are less than 1%.
- Success rates are very slightly better now. With increased maternal age, IVF is less likely to be successful.
- In under 35-year-olds, around 30% of the cycles will be successful. Can try again, especially if there are frozen embryos still remaining (don’t have to go through the whole process again).
What is intracytoplasmic sperm injection (ICSI)?
- Used in low sperm count, low motility or repeated fertilisation failure.
- Single sperm used so can collect sperm by needle aspiration from epididymis or testis.
- Inject sperm into the egg.
- The microscope has two arms that go onto the sample. On the ends of them, there are two ultrafine glass pipettes (around 10 microns in width). These pipettes are connected to these machines which are controlled by these two controllers. These two controllers allow movement of the two pipettes, as well as a sucking and blowing action (allows manipulation).
- The embryologist will be in a microscopic world, controlling these two pipettes. The egg can be held in one hand. The egg has one polar body, meaning it has undergone meiosis I. It is probably halfway through meiosis II, so it is ready to be fertilised. In one hand, the embryologist holds the egg with a holding pipette. The other hand controls the second pipette; one sperm can be injected into the egg (the sperm may not even be swimming).
- The fertilisation rate for this technique is 90%.
Is ICSI safe?
- Natural means of sperm selection is bypassed.
- Some evidence of increased genetic damage, but equivocal.
- Other defects 9.9% compared with 5% of non-ICSI.
- Patients may be being pushed to ICSI as higher fertilisation rates. In 2013 there were more ICSI cycles than IVF for the first time.
- Biggest risk with infertility treatment is still multiple pregnancy.
- The fertilisation rate for this one is around 90%. Having said this, nature has arranged it so that the sperm have to race to the egg. In females, there is a low number but high quality system and in males, there is a high number but low quality system. Nature arranges it so that the sperm that reaches the egg is expected to be ready. If a random sperm is picked, this system is bypassed. Are we causing genetic abnormalities with this? Most embryos that have something wrong chromosomally or genetically will undergo apoptosis in the first few divisions. The first generation of children born this way are around 22 years old. In terms of the children born, a lot of studies have been carried out and do not show much evidence to suggest that there are higher rates of genetic abnormalities. There is a slight increase in genetic abnormalities but not significant. The rates are so low that it is hard to tell anyway.
