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Pre-implantation Genetic Screening (PGS): What are we really talking about?

Pre-implantation Genetic Screening (PGS): What are we really talking about?

Pre-implantation Genetic Screening (PGS): What are we really talking about?

The tenant behind pre-implantation genetic screening (PGS) is to biopsy one or few cells from each embryo after creation, analyze the chromosomes for each embryo and transfer the ones that has normal chromosomes back into the uterus to boost IVF success and increase the live birth rate.

Central to this idea is that abnormal chromosomes in the embryos is the main reason why an embryo does not yield a newborn. It is logic then that PGS should allow the selection for the best embryo (preferably one only) for transfer into the uterus ending into one singleton newborn.

If this premise is accepted then the following assumptions should also be generally accepted

a. All or the majority of embryos reached the appropriate stage of development and expansion to allow biopsy.

b. Biopsy of the embryo does not harm its ability to implant

c. The cell or few cells obtained represent the rest of the embryo (has identical chromosomes to all the other cells in the embryo)

d. The platform used to analyze the embryo chromosomes is close to 100% accurate (otherwise some embryos will be wasted because they are abnormal according to the test, while they are actually normal). The platform reports only the chromosomes of the embryo and is not accountable for other elements of implantation i.e. the endometrium.

e. The delay (one or more days) needed to finish the testing does not affect embryo implantation

f. Freezing and then thawing of a biopsied embryo does not affect its implantation potential

g. Patients and physicians have agreed on how to calculate success: how many live births one would obtain from all embryos resulting from a single IVF cycle (all fresh and frozen embryos) i.e. total potential of one IVF cycle versus fresh embryo transfer only.

h. The added cost of biopsy and testing of embryos, potential increases the delivery rate and reduces the incidence of multiple pregnancy and miscarriage is cost-effective from the viewpoint of individual and a modern society.

The initial attempt to perform Preimplantation genetic diagnosis using an old technology called FISH that tested 7 to 9 chromosomes proved harmful few years ago and that its wide adoption at that time was a form of medical illiteracy : because it depends on logic not actual well conducted study. When the studies were conducted, they all showed that women universally achieved lower pregnancy rates after PGS.

New platforms are now available to test for all the chromosomes (array cGH and SNP array) and using cells (trophoectoderm) obtained from more advanced stages of the embryo (blastocyst). The question in hand is should we adopt these techniques, not as a research tool, but as the standard of care that should be offered to the majority of women undergoing IVF?

How Effective is PGS? The case for Logic

Applying logical thinking to modern pre-implantation genetic screening (PGS) methods indicates:

a. Not all embryos will reach the blastocyst stage (day 5) to be suitable for biopsy. Not all physicians and patients push their embryos to the blastocyst stage especially if few embryos exist in culture on day 3. Moreover, some normal embryos may not survive extended culture to blastocyst.

b. There are no conclusive evidence that biopsy of the trophoectoderm (the part that makes the placenta) of an embryo does not harm the embryo.

c. Mosaicism ; when one or few cells are different in chromosomes than the rest of the cells, is known to take place in embryos. The cells in the trophoectoderm maybe abnormal while the cells in the embryo maybe normal. Interestingly the embryo can later get rid of the abnormal cells in the trophoectoderm. This can lead in misdiagnosis of the embryo as abnormal while the embryo itself has the potential to implant and yield a healthy baby.

d. The platform used to analyze the embryo chromosomes is not 100% accurate either because of the accuracy of the test itself or because of mosaicism. The accuracy reported by labs administering the test is 97%. This means some normal embryos will be discarded and some abnormal embryos will be transferred. Actually the accuracy was not validated by many labs, only very few worldwide. Clinically some physicians have experienced much lower accuracy (80 or 90%). The platform reports only the chromosomes of the embryo and is not accountable for other elements of implantation i.e. the endometrium. So it is possible that the lower accuracy is due to other elements on embryo gentics (other than the number of chromosomes) or the lining of the uterus.

e. Currently the transfer of embryos into the uterus has to be delayed for one day (day 6) or several weeks (embryo has to be frozen then thawed back after results are obtained). This delay may reduce implantation of the embryo because it will not match the window of implantation in the lining of the uterus. This is a controversial point as some researchers found no difference in implantation between day 5 and day 6. This research, however, is not widely replicated.

f. After PGS some ‘normal’ embryos will be frozen. The survival of thawed and biopsied embryos is maybe reduced, potentially leading to loss of normal embryos. No large studies on survival of biopsied embryos after thaw exist.

g. Patients and physicians have agreed on how to calculate success: if success is calculated based on how many live births one would obtain from all embryos resulting from a single IVF cycle (fresh and frozen) i.e. total potential of one IVF cycle, then PGD has no value as it will not make an abnormal embryo normal or vice versa. If the success is based on what happens in the fresh cycle only with no regard to frozen embryos then PGS may improve the success rate of IVF. All assuming an excellent embryo freezing program.

For exampe If you are a young woman <38, with a good number of available embryo on day 5, say 4 blastocysts that are suitable for biopsy, you may elect to

i. transfer one embryo in the fresh cycle and freeze 3 embryos. If you are not pregnant, then transfer one embryo in each subsequent frozen cycle. If you are destined to get pregnant you will do that within a maximum of 3 months after your initial IVF and the risk for multiple pregnancy is minimized to 1% or less. If you were not destined to get pregnant no testing would have helped you or

ii. Alternatively, you may elect to test all your embryos in the fresh cycle, transfer one normal embryo, if any and freeze any normal embryo remaining. The potential benefit is getting pregnant in the fresh cycle instead of getting pregnant 1-3 months later. Also you will reduce the risk of miscarriage because abnormal embryos will likely be eliminated. The potential risks are misdiagnosis by PGS (not 100% accurate), loss of a thawed embryo (did not survive biopsy and freeze) and lower implantation potential of a normal embryo due to biopsy and delayed transfer.

h. A cost-effective analysis for PGS is not available at this time. The added costs are biopsy and testing of embryos. The potential benefits are increase in the delivery rate and reduction in multiple pregnancy and miscarriage. In the scenario above you either pay for i. frozen embryo transfer(s) if you do not get pregnant in the fresh cycle or ii. pay for ICSI (required for PGS by the majority of programs), biopsy and testing in the fresh cycle and frozen embryo transfer(s) if you do not get pregnant in the fresh cycle. In terms of multiple pregnancy, it can be minimized in either pathways if your physician is transfers one embryo anyway, tested or not. Things are not that simple, the payer will also make a difference: PGS is completely out of a patient pocket as it is not covered by any insurance while frozen embryo transfer may or may not be covered.

How Effective is PGS? The case for Published Studies

In general decision making in biological sciences is not amenable to logic, but determined by well designed ad well conducted studies. So far, three studies were published using the new platforms for embryo chromosome analysis, aiming at increasing IVF success. The studies were criticized because of

1. Restricted to young women (median age 31 to 32) so results cannot be generalized to the general IVF population: 2 studies

2. Did not account for frozen embryos: all studies

3. The studies did not demonstrate superiority of PGS to transfer best embryos based on morphology (shape): one study. Specifically a transfer of a tested embryo in the fresh cycle was not inferior to transfer of two untested embryos. Non inferiority does not mean superiority. Noninferiority study design is not suitable for a PGS study as patients and physicians are only interested in such an expensive treatment that can harm their embryos only if it promises superior results for their infertility treatment. Moreover, treatment could actually be inferior because a limit is placed that will make the outcome non inferior, in that study 20%. So if the difference is less than 20% PGS is considered not inferior.

4. End point should be live birth or ongoing pregnancy. Surrogate or intermediate endpoints as pregnancy, implantation (short of a baby in hand or at least pregnancy beyond 20 weeks) are not ideal outcomes.

Randomized studies related to pre-implantation genetic testing using newer platforms were independently analyzed. So far no study showed that PGS is superior to the strategy of transferring the best embryo based on morphology (the standard of care). Moreover due to factors related to the biology of reproduction and that the accuracy of the test is unlikely to reach 100% accuracy soon, it is unlikely that PGS will prove beneficial to women undergoing IVF for fertility treatment. PGS may only shorten the time to pregnancy but will not be able to improve the pregnancy rate and due to inaccuracies may even reduce it.

Alternatives to PGS are being studied. One alternative is time lapse photography of the embryos to observe the cell division of the embryo cells and select the best embryo for transfer. It is noninvasive but further studies are required before its ready for general use. Another alternative is polar body biopsy of oocytes but results of ongoing studies are not available yet.

It is possible that factors in this article could be interpreted differently in a specific situation by patients and their physicians, in conjunction with the number of mature eggs produced, but it does not appear that PGS is ready for generalized application in the majority of IVF population.

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Sex Selection

Sex Selection

Sex selection is considered for one of three reasons:

1. Avoiding sex related genetic disorders. These are genes mostly carried on the X chromosomes and affect boys more than girls since they have one X chromosome e.g hemophilia

2. Family balancing: couples that have children of one sex and desire a child of the opposite sex

1941-2011 Trend: Suppose you could only have one child. Would you prefer that it be a boy or a girl?

Gender Selection, Boy or a Girl

3. Preference: some prefer a child of certain sex due to social factors. Recent poll in The UK  indicates that when 2,129 recently married couples were surveyed, found that 47% admitted that they would prefer to have a son first, with the majority citing practical reasons like boys being “less hard work”. Only 21% of respondents said they would like to have a daughter as their firstborn, and 32% reported having no preference either way. Couples who wanted to have a daughter first see older girls as ‘better role models’ to their younger siblings. In the US a Gallup poll yielded similar answers by American parents, especially men, since 1940s. American women do not have a proportionate preference for girls. American women show essentially no preference either way: 31% say they would prefer a boy and 33% would prefer a girl. More recent trends indicates that American couples prefer girls.

In contrast couples on a waiting list for adoption prefer girls both in the US and India. There is also some evidence that sexual orientation may influence that preference. Gay men are more likely to have a gender preference for their adopted child whereas heterosexual men are the least likely. Couples in heterosexual relationships are more likely to prefer girls than people in same-gender relationships.

The preference is also influenced by geography and politics. The official family planning policy in China, applied to large portions of Chinese, allow only for one child and does not allow sex selection. In the US many couples desire to limit the number of children to 2. If the first child is of one sex they desire the second child to be of the opposite sex

 

How is the sex of the embryo determined?

Older methods of selecting sex through change in the position or timing of intercourse or sperm sorting are not accurate and are not suitable for sex selection in modern couples seeking a specific sex (the other sex maybe conceived in 30% or more of couples). Modern sex selection depends on genetics. After stimulation of the ovaries, egg retrieval and fertilization, one or few cells from the embryo is obtained. The cells are analysed for each embryo for the X and Y chromosome. Results are obtained and are accurate >99% of the time.

After identification of the X and Y chromosomes, the desired embryo is transferred into the uterus. The embryo that carries the correct chromosome, should survive and be of good quality. Sex selection is more likely to succeed in women with good ovarian reserve, producing a good number of eggs. The larger the number of embryos available for testing, the more likely a healthy embryo of the desired sex is available for transfer.

Learn more about gender selection.

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Should You Test Embryos Created after IVF for Chromosomal Abnormalities?

Should You Test Embryos Created after IVF for Chromosomal Abnormalities?

Many of the embryos created after IVF carry abnormal chromosomes. Normal embryo cells carry 46 chromosomes. The most common abnormalities are extra chromosome e. +21 (47 chromosomes) or missing a chromosome e.g -X (45 chromosomes). By far, abnormalities in the egg is the source of abnormal chromosome number.

Preimplantation genetic screening (PGS)

PGD: Testing of embryo chromosomes

Finding a ‘normal’ embryo is clearly advantages as it will theoretically lead to 1. The transfer of a single embryo instead of many  embryos and  2. can produce higher pregnancy rate than an embryo selected based on morphology (looks) alone. The process of embryo testing for the purpose of improving pregnancy rate is, however, not simple in relation to the accuracy of testing and many other issues

Preimplantation genetic screening for chromosomal abnormalites (PGS)

PGS require two steps: 1. Biopsy: obtaining a cell or a group of cells from the embryo and 2. genetic testing of the cells for chromosomes ideally in 1-2 days to obtain results and allow fresh transfer

Biopsy

Day 5 embryo biopsy

Biopsy of trohoectoderm cells of blastocyst

Obtained by removing a. a single cell of a day 3 embryo or  b. group of cells from the trophoectoderm (the outer part of the embryo that makes the placenta) of a day 5 embryo (blastocyst). Removal of cells nowadays uses a laser beam. Cells are fixed on a glass slide and sent for analysis.

Genetic Analysis of Embryos

In the past old technologies (FISH) was limited in its ability to test all chromosomes. Multiple studies in the past few years proved that PGD using FISH actually reduce the chance for pregnancy in many IVF populations and should not be used. Two newer technologies can test all the chromosomes in an embryo: cGH (comparative genomic hybridization) array and SNP (single nucleotide polymorphism) array. Some of these methods can report the results in 3 days allowing for delayed fresh transfer (day 6) and others require about a month for accurate testing, necessitating embryo freezing and transfer in frozen-thaw cycle. Labs offering these methods claim accuracy of 95 to 97%. There are more advanced methods e.g genome screening, that can test embryo chromosomes in as short as 6 hours. The ultimate method for testing is still evolving.

Should women test their embryos before transfer to the uterus?

My short answer is no, not routinely. The pros of testing embryos could be transferring less embryos , improving IVF outcomes (pregnancy rates) and avoiding pregnancy with a baby carrying chromosomal abnormalities. The cons are these aims are still not proven facts due to

1. The biopsy may hurt the embryo, reducing its ability to implant

2. The assumption that one cell represent the whole embryo may not be true (mosiacism); the cell may be abnormal while the rest of embryo is normal or vice versa

3. The methods of testing was not validated by independent large studies from multiple centers and maybe less accurate than claimed

4. Delay in transferring the embryo in the fresh cycle may reduce its implantation potential

5. Cost associated with biopsy and testing the embryo is approximately $5500 to $8000

6. Testing of an embryo will not improve the ‘pregnancy’ potential of that embryo. It will just tell you if the embryo is ‘normal’ or not. The potential from all the embryos obtained from IVF after an egg retrieval is not changed by testing. Assuming a very accurate test and an excellent freezing program,  tested embryo transfer should yield similar outcome as transferring untested embryo(s) in multiple cycles. That is the most important point to consider. If you are willing to be patient and transfer one or few embryos resulting from one ovarian stimulation successively in the fresh cycle then frozen cycles, the cumulative pregnancy and delivery rate should be the same at the end. For example in young women transferring one embryo, approximately 30- 40% of them will just achieve pregnancy in the fresh cycle. In the first frozen-thaw transfer another 30% or so will get pregnant. Frozen cycles are not as demanding as fresh IVF. Many women can have the embryo transferred in a natural cycle with no medications and minimal monitoring.

Embryo testing may help younger women, producing a large number of embryos and want to transfer only one. An alternative approach is to transfer one embryo at a time as their pregnancy rate is high even with a single untested embryo.

Testing of embryos from older women (40 or older) producing few embryos  (<6) is of little value as the alternative is to transfer 5 or so untested embryos in that age group because of the very high rate of chromosomal abnormalities in the embryos.

Testing may be helpful for older women (40 or older) producing a very large number of embryos (e.g >10 embryos) to eliminate the need for multiple transfers to get to the healthy embryo. This category (older women and very large number of eggs / embryos) is rare in IVF population.

Women contemplating testing of their embryos after IVF should consider many issues including age, number of embryos, history of unsuccessful fertility treatment if any, cost and sometimes tolerance for multiple pregnancy and fetal reduction. Moreover women should consider all these factors and be ready to modify their decision during the cycle depending on the number of available embryos.

All this does not apply to women testing the embryos for chromosome translocation, a specific genetic disease or sex.

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