Introduction and Background
Successful treatment of reproductive failure demands full prior identification and treatment of those factors that adversely influence both embryo “competence” (the ability of an embryo to propagate a normal pregnancy) and uterine “receptivity” (i.e., thickness of the uterine lining, immunologic modalities, anatomical integrity of the uterus, as well as infective and biochemical factors).

While advances both in methods and drugs used for ovarian stimulation as well as improvements in embryo culture techniques have undoubtedly had a positive influence, IVF success rates have lagged and even stagnated over the last 10 years. This is largely due to an inability to reliably identify and selectively transfer only “competent” embryos (those that are capable of producing a healthy baby) to the uterus. Even in young women, an embryo that “looks good” under a microscope is not necessarily competent. At best, it has a 25% chance of implanting. Furthermore, this statistic shrinks drastically with advancing age beyond 35 years.

Even the use of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) to identify chromosomes does not significantly improve this capability. As a result, many IVF specialists still transfer multiple embryos at a time to increase the odds that at least one competent embryo will reach the uterus and produce a pregnancy. The problem is that while this improves the chance of a pregnancy occurring, it also markedly increases the risk of multiple gestations/pregnancies.

A convenient and applicable parallel that we often draw to illustrate the relative importance of embryo competence versus uterine receptivity (in the IVF equation) is that of a “seed/soil relationship.” The ability of an embryo (the “seed”), upon reaching a receptive uterine environment (the “soil”) to successfully implant and develop into a healthy baby (the “plant”), is no different than what takes place in a regular agricultural setting. In simple terms, it is determined by establishing an ideal seed/soil relationship. It follows that it is no more possible to achieve a viable healthy pregnancy when a “competent” embryo is transferred to a “non-receptive” uterus than when an “incompetent” embryo is transferred to a receptive uterus.

In human reproduction, the establishment of an ideal seed/soil relationship is pivotal, since both embryo competence and uterine receptivity are indispensable to the development of a healthy baby. It is, however, an undeniable fact that reproductive failure (i.e. failed implantation, miscarriages and major birth anomalies) are far more likely to be due to embryo incompetence (70-75%) than to a lack of uterine receptivity (25-30%).

It is mostly (but not exclusively) the embryo’s chromosomal configuration that will determine its “competence”. The number of chromosomes in a cell is referred to as its ploidy. A cell with a normal number of chromosomes is referred to as euploid while one with an irregular chromosome number is aneuploid. It appears that it is the ploidy of the mature egg (rather than the sperm) that determines the post-fertilization chromosome configuration of the embryo. The embryo’s ploidy, in turn, determines its competence.

The relatively lax and unregulated IVF setting in the United States has provided a safety net for those wishing to transfer multiple embryos, and this in turn has led to a virtual explosion in the incidence of multiple births in this country. The enormous short and long term financial costs associated with IVF multiple births (many of which are related to prematurity) represents one of the main reasons why health insurance providers in this country are reluctant to cover the procedure.

There is a profound lack of correlation between the microscopic appearance (grading) of embryos and embryo “competence”. Moreover, Preimplantation Genetic Diagnosis/Screening (PGD/S) of human eggs and embryos for their chromosomal integrity, using traditional fluorescence in-situ hybridization (FISH) is only fractionally more reliable. The reason is that conventional FISH cannot fully access all the chromosomes – in fact, only about 12 of them. Thus, even when FISH reveals that all the accessed chromosomes are normal, there still remains more than a 40% chance of chromosomal aneuploidy involving those chromosomes not targeted by the test…and the incidence increases to above 50% by the time the woman reaches 40 years of age. This constitutes a serious drawback when it comes to attempting to select the most “competent” eggs or embryos for dispensation in ART.

We reported on studies involving the performance of CGH on a fragment of nuclear material (the first polar body or PB-1) that is routinely discharged from the egg during the chromosomal rearrangement that takes place following the “hCG trigger”. The PB-1 has a chromosomal makeup which is a mirror image of the chromosomes in the egg’s nucleus. Several hours following fertilization, the PB-1 divides and a second polar body (PB-2) appears alongside it. PB-1 biopsy involves removal of PB-1 from immediately below the outer envelopment (zona pellucida) of the pre-fertilized egg while a pb-2 biopsy refers to removal of the 2nd PB, soon after fertilization has occurred. Both PB-1 & PB-2 biopsy can be achieved without damaging the egg itself.

The study referred to above was performed on eggs extracted from women aged 25-42 years who underwent ovarian stimulation with fertility agents. We first performed PB-1 and PB-2 egg biopsies before and after fertilization. Thereupon, we biopsied the resulting day-3 embryos by removing a single cell (blastomere) from each. The PB-1, PB-2 and blastomere samples were sent for genetic testing using comparative genomic hybridization (CGH) to identify all of the chromosomes in the samples tested.

Subsequently we transferred up to 2 embryos derived from eggs that previously had tested chromosomally normal to the uteri of 35 women. The study revealed the following remarkable information:

• The chromosomal make-up of the egg, rather than the sperm, is the main determinant of an embryo’s chromosomal integrity and its ability to develop into a baby. (i.e., its “competence”).
• Even in young women, >60% of all mature eggs are likely to be aneuploid and thus incapable of producing “competent” embryos.
• The incidence of egg aneuploidy increases progressively with advancing age such that by the mid-forties it is probably above 90%.
• Eggs that have abnormal quotas of chromosomes (i.e. are aneuploid) will, upon fertilization, invariably propagate aneuploid, “incompetent” embryos. Such embryos will either fail to attach to the uterine lining or will attach and then subsequently miscarry early on in pregnancy.
• Approximately 85% of eggs that have a normal number of chromosomes (i.e. euploid) fertilized with normal sperm will subsequently develop into “competent” embryos.
• The transfer of 1-2 euploid (CGH-tested) embryos to a receptive uterine environment (free of immunologic and anatomical irregularities), has better than a 60% chance of resulting in a live birth.
• Embryos that fail to progress to the blastocyst stage will almost always develop into aneuploid, “incompetent” embryos. This finding all but dispels the erroneous contention that embryos might be better off being transferred to the uterus prior to reaching the blastocyst stage.
• Most IVF failures and early miscarriages are almost always attributable to embryo aneuploidy. It follows that by only transferring euploid, “competent” embryos, this risk will be significantly reduced.

Male Factor Contributions
Fertilization of an egg by a dysfunctional spermatozoon significantly increases sperm contribution to the development of aneuploid embryos. This is more likely to occur in cases of moderate to severe male factor infertility. Given that male infertility is responsible for more than 50% of infertility, it follows that it would be preferable to perform CGH analysis on the embryo (rather than the egg). This would improve the accuracy of CGH-diagnosis in diagnosing embryo competence. Accordingly when predicting embryo “competence” we shifted from egg to embryo CGH testing.

Our subsequent study reported in the prestigious medical journal, “Fertility and Sterility” (December, 2009), described the use of embryo (rather than egg-PB) CGH testing which confirmed the accuracy of this approach and showed that regardless of the age of the embryo recipient, the transfer of 1-2, CGH-normal embryos results in better than a 60% pregnancy rate and a marked reduction in miscarriage rate.

Staggered In Vitro Fertilization (St-IVF)

Staggered-IVF involves the use of CGH testing of day 3 embryos in order to identify those that are the most “competent” (i.e. chromosomally normal) embryos. St-IVF involves dividing the IVF cycle into 2 separate stages. The first stage involves ovarian stimulation, egg retrieval fertilization and embryo or blastocyst biopsy and ultra rapid freezing (vitrification) and storage of all blastocysts. The second stage which occurs, in a subsequent cycle, involves hormonal preparation of the recipients uterus followed by the transfer of one or more blastocysts. The reason for dividing the cycle into two parts is to allow for sufficient time to complete CGH analysis of DNA derived from the biopsied material removed in the second stage of St-IVF).
St-IVF improves the efficiency of the IVF process by:

• Markedly improving the birth rate per embryo transferred
• Avoiding the need to transfer several embryos at a time thereby reducing the likelihood of high order multiple pregnancies (triplets or greater)
• Reducing the incidence of chromosomal abnormalities in those embryos that are transferred

Egg/Embryo Competency Testing using CGH, while clearly a major breakthrough in the IVF arena is not a panacea. First, an embryo diagnosed to have all its chromosomes (euploid) through egg and/or embryo CGH testing will, in about 15% of cases, turn out to be “incompetent.” This is due to the fact that even chromosomally normal cells, upon further division at times can generate some aneuploid cells, leading to a condition known as Mosaicism (the presence of both chromosomally normal and abnormal cells) in the blastocyst. Depending on the percentage of aneuploid cells in the advanced embryo (blastocyst), mosaicism might not be lethal. Second, a competent embryo might fail to continue developing because of poor uterine receptivity rather than embryo aneuploidy. Third, Embryo transfer (ET), another rate-limiting factor in IVF, requires a great deal of technical expertise and there is a wide variation in such expertise.

The above serves to explain why the transfer of “competent” (CGH-normal) embryos will result in a live birth rate of 60-70%…not in 100% of cases.

Use of CGH technology represents a major step toward consistently achieving a pregnancy, but it has limitations. By understanding such limitations, we can work toward achieving even better results: First, an embryo, diagnosed to be euploid through single-blastomere CGH, is not always “competent”. Second, a competent embryo might not attach because of poorly understood uterine receptivity issues. Third, there is a wide variation in technical expertise when it comes to the performance of embryo transfer, a rate-limiting factor in IVF.

Using CGH to Select Eggs for Freezing (Banking)
In the past, egg freezing and banking has yielded dismal success (a 1-4% baby rate per frozen egg). After all, freezing a non-viable egg is a futile and fruitless exercise. The introduction of CGH to select only “competent” eggs for freezing has the potential to revolutionize this field. SIRM recently published a study that evaluated birth rates in women who underwent IVF using frozen/thawed eggs pre-selected by CGH. The study revealed that by combining CGH egg selection with a new freezing technique known as vitrification, birth rates from frozen eggs could be increased as much as 6-7 times over current averages!

About Array CGH (aCGH)
There are 2 types of CGH processes used in IVF: the first is metaphase CGH (mCGH) and the second is Array CGH (aCGH) (also called Microarray). To date we have used mCGH, a labor intensive and complex procedure that requires at least 5 days to perform once. Since a single run-through will yield inconclusive results in up to 20% of the DNA samples tested, we often need to repeat the process more than once in order to reduce the percentage of “inconclusive” results. This explains why it requires several weeks to obtain optimal results with mCGH and accordingly why it is necessary to defer embryo transfer to a subsequent cycle (Staggered IVF).

It is true that Array CGH is a much faster and less complex method for performing CGH. While aCGH can even be completed within several days, its accuracy is still in question, when used to evaluate a minute amount of DNA derived from a single PB or single blastomere. In fact, that is why aCGH (as currently applied to IVF) requires access to much more DNA derived from several cells at a time. This means that by and large, aCGH can only be reliably performed on blastocysts, where there are a large number of cells (>100), and several can be removed for testing without damaging the embryo. The problem is that most (if not all) blastocysts have some aneuploid cells (due to mosaicism). As such, when several cells are removed (biopsied) and CGH-tested at a time, there is presently no way of accurately knowing how to interpret the presence of one or more aneuploid cells and at what cut-off it would still be “safe” to transfer such blastocyst(s).

Also, the cost of performing aCGH is significantly higher per sample tested than for mCGH. Finally, as with mCGH done on day 3 embryos, blastocyst aCGH likewise mandates that the blastocyst(s) be frozen and then transferred in a subsequent cycle. But all this could change when/should it become possible to reliably conduct aCGH on single cells (as required for day 3 embryo testing). When this transpires, (provided that aCGH proves to be reliable when so applied and that the cost of aCGH decreases significantly) then it will become possible to perform fresh embryo transfers in the same cycle that the CGH testing was performed. We anticipate this will likely happen in the next few years. Until then it is our opinion that embryo-mCGH with St-IVF will remain the method of choice.

Conclusions

• CGH Embryo selection St-IVF does not improve embryo quality in a given cycle of ovarian stimulation. Rather, it allows for the identification and selection of high quality, “competent” embryos for transfer. As such, while it dramatically improves the live birth rate per embryo transferred and brings us much closer to a time where single embryo transfers will be come standard, it will not improve IVF outcome per stimulation cycle or per-egg retrieval.
• While CGH testing (as is also the case for FISH-PGD) markedly reduces the risk of numerical chromosomal birth defects such as Down’s syndrome, it does not absolutely preclude their occurrence. In fact, the anticipated error rate could be ≤5%. Thus all women undergoing CGH or FISH egg/embryo selection and who seek absolute confirmation that numerical chromosomal birth defects will not occur should still undergo prenatal genetic testing in the 1st or 2nd trimester.
• The potential application(s) of egg/embryo/blastocyst karyotyping through CGH should be regarded as a “work in progress.” Things are still very fluid, and are likely to change over time. Hopefully, with responsibility, honesty, and careful evaluation, this will ultimately evolve to the betterment of all.