Array CGH vs. Metaphase CGH in IVF: Which is Best & When to Test

03 Apr
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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.

  1. 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.
  2. 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.
  3. 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.
  4. 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).
  5. 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.
  6. 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:

  1. Embryo Banking to slow the biological clock in older women and in those who have Diminished Ovarian Reserve (DOR)
  2. For Egg Banking to in cases of  (FP) and for creating repositories of “competent” eggs for low cost and more efficient Egg Donation
  3. For the diagnosis of the causes for Repeated IVF Failure (to differentiate between embryo competency and implantation issues)
  4. 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.

16 Comments

  • Subhadeep Sarkar says:

    Thanks a lot, Doctor. Much appreciated.

  • Subhadeep Sarkar says:

    We have been advised by our fertility clinic to conduct acgh-pgs on day-3 frozen embryos and then do a day-5 blastocyst transfer. Wouldn’t it be better to unfreeze and take the embryos to blastocyst stage and then do acgh-pgs and refreeze them for a staggered ivf?

    • Geoffrey Sher says:

      In my opinion, the day 3 embryos should be thawed, biopsied on day 3 and then taken to blastocyst and vitrified for Staggered IVF. I would also suggest doing next generation gene sequencing (NGS) rather than aCGH. I fully recognize that my strong position that day 3 biopsies for PGS is preferable to day 5 (blastocyst) will raise eyebrows because so many doctors have been brainwashed to believe that it is preferable to perform Preimplantation Genetic Sampling (PGS ) biopsies on blastocysts than on day 3 cleaved embryos. In my opinion this is incorrect and here is why:

      About 10 years ago, Levent Keskintepe PhD and I introduced Comparative Genomic Hybridization (CGH) into the clinical IVF arena, as a preimplantation genetic sampling (PGS) method that enables full karyotyping (numerically chromosomal analysis) of all 23 pairs of an embryo’s chromosomes so mas to determine its subsequent ability to propagate a viable pregnancy (i.e. its “competence”). Since then we have, over a period of a decade, authored many articles on the clinical utility and advantages associated with the selective performance of embryo PGS and with it have witnessed embryo karyotyping emerge as a valuable efficiency tool in the IVF arena. Several alternatives to CGH have since emerged and while none are perfect, they have all enhanced our ability to better select the most “competent embryos for transfer to the uterus, thereby improving the efficiency of IVF, and reducing the risk of chromosomal miscarriages and birth defects. One recently introduced method known as “Next Generation Gene Sequencing (NGS)” bears special mention since its improved accuracy and reliability over previously used methodologies, has established it as a method of choice when it comes to embryo karyotyping..
      Gene Sequencing is a method that determines the precise order of nucleotides within a DNA molecule. The method/ technology determines the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. A new generation of sequencing technologies known as NGS now provides unprecedented opportunities for high-throughput functional genomic research. NGS is currently being applied to identify the karyotype of the human embryo and in my opinion is more reliable than other available PGS methodologies.
      NGS can be conducted reliably on single blastomeres (derived from day 2-3, cleaved embryos) as well as on pooled cell samples biopsied from a blastocyst. When performed individually on several cells derived from a blastocyst NGS can help differentiate between meiotic and mitotic (mosaic) aneuploidy. Done selectively, this could have potential advantages because unlike meiotic aneuploidy which is permanent and often lethal to the embryo, mitotic aneuploidy (mosaicism) is often self-correcting with further embryo development.
      Optimal timing of embryo PGS biopsy is very important. You often hear told that embryo biopsy is more reliable if conducted on a blastocyst, rather than on an early (day 3) cleaved embryo. I disagree for 2 reasons. The first is that the earlier in embryo development that the biopsy is done for PGS, the more likely it is that a numerical chromosomal irregularity (aneuploidy) originated during egg (and rarely sperm) meiosis and accordingly is irreversible. In contrast, the later in embryonic development that the biopsy is done, the more likely it becomes that the aneuploidy occurred post-fertilization, affecting only some of the embryo’s cells during mitosis and is thus potentially autocorrectable over time. Thus, it is in my opinion preferable to perform embryo biopsies for PGS on day 3 rather than on day 5-6. The second reason that I prefer doing day 3 biopsies is to give the embryo time (over the ensuing 2-3 days that it develops into a blastocyst) to recover before being vitrified (ultrarapidly frozen). In my experience embryos that are biopsied earlier tend to survive the subsequent freeze/thaw in a much better condition than when they are biopsied as blastocysts whereupon thy are immediately frozen.
      Based upon available data, it is my opinion that the time has arrived to recommend that NGS be used as the preferred method for PGS in IVF.

      I urge you to access my new personal website at http://www.DrGeoffreySherIVF.com and from there, my new blog. When you get to the “home page” of the Blog, find the “search bar” and type in any of the articles below by title, “click” and you will immediately be taken to these. While on this blog, please take the opportunity to post any questions or comments with the full expectation that I will (as always) respond promptly.

      • Controlled Ovarian Stimulation (COS) for IVF: Selecting the ideal protocol
      • Ovarian Stimulation for IVF using GnRH Antagonists: Comparing the Agonist/Antagonist Conversion Protocol.(A/ACP) With the“Conventional” Antagonist Aproach
      • Ovarian Stimulation for IVF: Comparing “conventional” use of GnRH antagonists to the Agonist/Antagonist Conversion Protocol (A/ACP)
      • IVF: Factors Affecting Egg/Embryo “competency” during Controlled Ovarian Stimulation(COS)
      • The BCP: Does Launching a Cycle of Controlled Ovarian Stimulation (COS). Coming off the BCP Compromise Response?

      I invite you to call 702-699-7437 or 800-780-7437 and set up a one hour Skype consultation with me to discuss your case in detail.

      I also suggest that you access the 4th edition of my book ,”In Vitro Fertilization, the ART of Making Babies”. It is available as a down-load through http://www.Amazon.com or from most bookstores and public libraries.

      Geoff Sher

      For Preimplantation Genetic Sampling (PGS) using array CGH (aCGH) to be reliable and accurate, it requires access to more DNA than is available from a single cell biopsied from a day 3 embryo or from the 1st polar body of an egg. Presently, accurate PGS using single cell (blastomeres) is best performed using Next Generation Gene Sequencing (NGS). Biopsying a multicellular (>100cells) blastocysts by allowing aces to several cells at a time, provides much safe access to much more DNA than when a single cell (blastomere) is extracted from a 5-10 cell day 3 embryo. This serves to explain why those that champion PGS/ aCGH, favor blastocyst biopsies over day 3 biopsies, but does doing this enhance the reliability of PGS. I suggest not…

      Consider the fact most numerical chromosomal aberrations (aneuploidy) in day 3 embryos originate during maturational division (meiosis) in the egg and that many such meiotically aneuploid embryos never survive to the blastocyst stage. Embryos affected by meiotic aneuploidy are permanently “incompetent”. They cannot recover. In contrast, much aneuploidy detected in the hypercellular blastocyst, originates during post-fertilization cell replication (mitosis). In the latter situation, some blastocyst cells are aneuploid while others are chromosomally normal (i.e. mosaicism). While mitotic aneuploidy can reverse (autocorrect) with such embryos developing into chromosomally normal concepti. In contrast, as stated, meiotic aneuploidy is irreversible. Since it is presently not possible to reliably differentiate between mitotic and meiotic aneuploidy, it follows that in the process of discarding aneuploid blastocyst we could well (albeit unintentional) be destroying potentially normal concepti.

      Since events that occur during meiosis in the final maturation of the egg inevitably lead to “irreversible” egg/embryo “incompetence”, it is meiotic aneuploidy that needs to be identified. It follows that the closer to egg retrieval that we perform PGS/biopsies, the greater will be the likelihood that detected aneuploidy detected will be meiotic in origin. It is another e reason why I believe that day 3 PBS embryo biopsy is preferable to blastocyst biopsy.

      Finally, we recently observed that a blastocyst which is ultrarapidly frozen ( vitrified) immediately following biopsy for PGS , will have reduced viability as compared to cleaved embryos biopsied on day 3 and are then allowed to recover for a few days before being vitrified.

      Please go to my new personal website at http://www.DrGeoffreySherIVF.com and from there, my new blog. When you get to the “home page” of the Blog, find the “search bar” and type in any of the articles below by title, “click” and you will immediately be taken to these. While on this blog, please take the opportunity to post any questions or comments with the full expectation that I will (as always) respond promptly.

      • Controlled Ovarian Stimulation (COS) for IVF: Selecting the ideal protocol
      • Ovarian Stimulation for IVF using GnRH Antagonists: Comparing the Agonist/Antagonist Conversion Protocol.(A/ACP) With the“Conventional” Antagonist Aproach
      • Ovarian Stimulation For Women with Diminished Ovarian Reserve (DOR) and in Older Women undergoing IVF
      • Ovarian Stimulation for IVF: Comparing “conventional” use of GnRH antagonists to the Agonist/Antagonist Conversion Protocol (A/ACP)
      • IVF: Factors Affecting Egg/Embryo “competency” during Controlled Ovarian Stimulation(COS)
      • The BCP: Does Launching a Cycle of Controlled Ovarian Stimulation (COS). Coming off the BCP Compromise Response?
      • Measuring and Interpreting Blood hCG to Assess Pregnancy Viability Following ART Treatments.
      • Traveling for IVF from Out of State/Country–
      • A personalized, stepwise approach to IVF at SIRM”; Parts 1 & 2 (posted March, 2012)
      • The Role of Nutritional Supplements in Preparing for IVF
      • Frozen Embryo Transfer (FET): What Does it Involve?

      I invite you to call 702-699-7437 or 800-780-7437 and set up a one hour Skype consultation with me to discuss your case in detail.

      I also suggest that you access the 4th edition of my book ,”In Vitro Fertilization, the ART of Making Babies”. It is available as a down-load through http://www.Amazon.com or from most bookstores and public libraries.

      Geoff Sher

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