Array CGH vs. Metaphase CGH in IVF: Which is Best & When to Test
The issue of whether to apply CGH technology to the testing of day-3 versus day-5 embryos and/or whether metaphase CGH (mCGH) or array CGH (aCGH) should be the preferred method used for testing eggs and embryos for their “competency” continues to rage. Central to this debate are several considerations.
- Fresh versus Frozen embryo transfers of CGH tested embryos:Is it better to transfer CGH-tested embryos “fresh”, i.e. during the same cycle in which the egg retrieval (ER) was performed, or to cryopreserve blastocysts to await the return of all CGH results, and then perform a frozen embryo transfer (FET) in a subsequent cycle (Staggered IVF)? There are many who, for reasons of convenience and/or for fear that freezing might harm their embryos, would prefer a fresh ET. However, I would like to set the record straight with regard to the latter concern. As recently as a few years ago, most embryo cryopreservation was done through “conventional” (slow) freezing, and ice tended to form in the embryo’s cells, thereby damaging them. This resulted in many such embryos failing to survive the freeze/thaw and a pregnancy rate of less than 25% following ET. However, with the advent of ultra-rapid cryopreservation (vitrification) a few years ago, the freezing process is now accomplished in a flash, so rapidly that no ice has time to form. Thus, about 90% of the embryos survive the freeze/thaw process and retain virtually the same potential to propagate a baby as do fresh embryos.I think that the issue of “convenience” in testing and transferring in the same cycle is also often over-stated, since the ability to delay ET for when you know that you have competent embryos for transfer has a very reassuring and calming effect. Using Staggered IVF, the pregnancy can be timed to the patients’ convenience, so I believe that there really is no medical downside to such an approach.
- When should aCGH versus mCGH be used?Array CGH could be problematic when it is performed on the very minute amount of DNA available from a single cell (e.g., egg polar body [PB] or a blastomere derived from a 5-9 cell, day-3 embryo). In contrast, metphase CGH can (for a variety of technical reasons) be performed far more accurately and reliably even on such a minute amount of DNA. That is why, when CGH is to be conducted on DNA derived from an egg-PB or on a single blastomere taken from a day-3 embryo, we maintain that mCGH is the best method.This having been said, it is an undeniable fact that mCGH analysis is a more manually intensive process than is aCGH, which in contrast, is largely automated. As such, mCGH takes much longer to perform and therefore it is not possible to have results available in time to select embryos for a fresh ET, two days later. This is why Staggered IVF is needed in such cases. Then again, regardless of whether aCGH or mCGH is used to test day 5-6 blastocysts, the results will also usually not be available in time available to allow for a fresh ET, so Staggered IVF will be required anyway.
- Embryo Mosaicism: What is it all about?Concern is often raised that some of the cells of an embryo, biopsied on day 3 might subsequently (over the next few days), undergo genetic (mitotic) alteration (i.e., mosaicism) and therefore, the later the biopsy for CGH is performed, the more reflective the results are likely to be of “embryo competence”. Such an assertion is, however, based on pure conjecture. You see, virtually all blastocysts have some degree of mosaicism. That is part of the natural order. So when one examines pooled DNA (as with aCGH) it is often difficult to interpret the significance of some DNA abnormalities and how/where to draw the line between abnormal (pathological) and benign “mosaicism”. It is this dilemma that has spawned speculation that some “CGH-abnormal” embryos might be capable of undergoing spontaneous “auto-correction” and subsequently develop into healthy, normal babies.Here, it is important to note that since chromosomal numerical abnormalities (aneuploidy) of the embryo almost always starts with the egg (rather than the sperm) a day or two prior to fertilization, it follows that the earlier one can pick up such irregularities, the more significant would be the implications. That is why , in our opinion, CGH chromosomal analysis of a 5-9 cell, day- 3 embryo is preferable to an assessment done on the hypercellular (>100 cell) blastocyst.
- Why is mCGH not more routinely offered in the U.S.A?To our knowledge, there are currently no reproductive genetic laboratories in this country that promote mCGH analyses on a routine basis. Those that have tried to do so locally (including ourselves) have found the analytical process to be so complex as well as time and technology-intensive that provision of such testing is prohibitive on multiple levels. That is why, when it comes to doing mCGH testing at SIRM centers, we have opted to have such analyses done in a highly seasoned and experienced genetics laboratory located in Europe (with whom we have an exclusive relationship).
- The Gender Selection IssueBoth mCGH and aCGH do provide information relating to embryo gender. However, there have been so many cases in this country where (regardless of the laboratory performing such testing) inaccuracies have occurred in gender identification that we now question the reliability of CGH testing for gender selection. We submit that it is probably preferable to use PGD with Fluorescence in-situ-hybridization (FISH) in these cases.
- Single Nucleotide Polymorphism Array (SNPa) genetic embryo testing.Another method of embryo chromosomal karyotyping, offered by a few genetics laboratories (e.g. Gene Security Network [GSN]), examines only a fraction of chromosomal DNA referred to as a single nucleotide polymorphism array (SNPa) rather than looking at the all genetic material on the embryo’s chromosomes, as is done in CGH testing.The SNPa process evokes a significant amount of “background noise” which makes interpretation of results difficult. This problem is quite creatively addressed by matching the embryo SNPa data with that derived from similar tests done on parental genetic material, usually derived from the mouth mucous membranes of both partners. While this approach has proven to be both powerful and precise, as with aCGH, SNPa is also prone to be somewhat unreliable when it comes to assessing the small amount of DNA available from a single cell (e.g., egg-PB or a day-3 embryo blastomere) and probably also lacks accuracy (as compared to PGD- FISH) when it comes to embryo gender determination.
In summary: There is no doubt that aCGH is faster, less cumbersome, and less expensive to perform than is mCGH. However, at present, aCGH still lacks reliability when it comes to testing single cell-DNA (i.e., eggs and day 3 embryos). This could well change with the evolution of technology. However, until then, when it comes to testing for aneuploidy in eggs and day 3 embryos (i.e., single cell analyses), mCGH with Staggered IVF is presently still the preferred approach. Both aCGH and mCGH can reliably be used to perform CGH on blastocysts.
So then… based on all this information, when is CGH embryo testing indicated? The following circumstances are in our opinion, best suited to egg/embryo CGH testing:
- Embryo Banking to slow the biological clock in older women and in those who have Diminished Ovarian Reserve (DOR)
- For Egg Banking to in cases of (FP) and for creating repositories of “competent” eggs for low cost and more efficient Egg Donation
- For the diagnosis of the causes for Repeated IVF Failure (to differentiate between embryo competency and implantation issues)
- For the development of Individualized Approaches to Ovarian Stimulation for IVF
And where will all this technology take us? In our view, it is inevitable that it will result in oocyte/embryo chromosomal screening becoming an ever more relevant part of the IVF experience. There is no turning back. Full embryo chromosomal karyotyping – whether performed through existing methods such as CGH, SNPa, or through newer/emerging approaches such as polymerase chain reaction (PCR) – is here to stay, and is destined to ultimately set a new standard of care in the field of Reproductive Medicine.