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THE EVOLUTION OF CGH-EGG AND EMBRYO TESTING AT SIRM/REPROCURE Geoffrey Sher MD Three problems restricted the growth of IVF in the U.S: 1) Cost of service and lack of insurance reimbursement, 2) Ignorance of the fact that IVF is far more efficacious than other infertility treatment and, 3) OB-Gyn’s and/or Reproductive Endocrinologists (RE’s) that do not have the necessary expertise required to optimize ART outcome. Since most insurance providers do not cover IVF services, most IVF costs have to be shouldered by the consumer. The reason why insurance providers have been reluctant to cover IVF are: a) absence of a verifiable reporting system on IVF outcomes, b) poor IVF success rates per embryo transfer and, c) an alarming incidence of high-order multiple pregnancies. A “competent egg” is one that in most cases upon fertilization will propagate a chromosomally normal embryo and, a “competent embryo” is one that is karyotypically normal and which upon reaching a receptive uterus is most likely to spawn a viable pregnancy. Hitherto, the lack of reliably in identifying “competent eggs/embryos” has led to an inability to select the best embryo(s) for transfer. As a consequence implantation rates have been low (averaging at 15-25% per embryo in young women). In an attempt to improve I VF results many RE’s have tended to transfer several embryos at a time, which fact explains the virtual explosion in the IVF-induced high-order multiple birth (triplets or greater) rate in the U.S and reluctance on the part of most insurance companies to cover IVF services. . There is a profound lack of correlation between the microscopic appearance (grading) of embryos and embryo “competency”. Moreover Preimplantation Genetic Diagnosis/Sampling (PGD/S of human eggs and embryos for their chromosomal integrity, using Fluorescence in-situ Hybridization (FISH) is only fractionally more reliable. The reason is that FISH cannot fully access all the chromosomes….in fact only about 1/2 of them. Thus even when FISH reveals that all the accessed chromosomes are normal, there still remains more than a 45% chance of chromosomal aneuploidy involving those chromosomes not targeted by the test…and the incidence increases to about 60% by the time the woman reaches her forties. This constitutes a serious draw back when it comes to attempting to select the most “competent” eggs or embryos for dispensation in ART. In 2007 we reported in the journal, “Fertility and Sterility” on a study where we fully karyotyped mature eggs (M2 oocytes) using metaphase comparative genomic hybridization (mCGH) rather than FISH. Unlike FISH, CGH can identify all the chromosomes, thereby providing a reliable measure of “competency”. This study showed that the transfer of 1-2 embryos/blastocysts derived from CGH-normal eggs (mean=1.7 per woman), resulted in live births >70% of the time ( i.e. > double the national average), that in the process the risk of multiple pregnancies was markedly lower, and chromosomal miscarriages and birth defects were drastically traduced. Since CGH requires at least 5 days to be completed, it is not possible to perform the test on day 3 embryos and still be able to transfer them fresh to the uterus. It would, in such cases be necessary to cryobank and store the embryos until mCGH results become available. But, conventional (slow) freezing damages eggs/embryos because it results in ice forming in the blastomeres (embryo’s component cells), damaging the embryo and reducing viability. The solution arrived about 3 years ago through a major advance in freezing technology referred to as vitrification, an ultra-rapid freezing technique that avoids intracellular ice formation. As such vitrified embryos could be banked indefinitely while retaining virtually the same viability as their fresh counterparts. Thus it became possible to defer embryo transfer by vitrifying and cryobanking embryos until CGH results became available. Accordingly it is presently possible use CGH on the egg and/or the embryo in order to identify those that are most likely to be competent. . Since in the face of normal sperm function, 90% of the embryo’s chromosomal integrity is determined by the egg and NOT by the sperm, egg CGH can indeed be safely employed in cases where there is no perm dysfunction.. In cases of male infertility however, where sperm dysfunction increases the risk of embryo chromosomal aberrations (aneuploidy) we preferentially to biopsy the embryo so as to take the sperm’s contribution into account. The process of splitting the cycle in two ; i.e. performing CGH on the first polar body of the egg or on a blastomere taken from a day 3 embryo, allowing the embryos to go to blastocyst, cryobanking these and deferring the embryo transfer to another time, is referred to as Staggered –IVF (St-IVF). Our results using St-IVF have been very encouraging. The transfer of up to two “CGH- normal” embryos has consistently yielded about a 60% chance of a live birth, a 3- 4 fold reduction in the risk of miscarriages (which are usually due to aneuploidy), a minimal risk of chromosomal birth defects such as Down’s syndrome and a virtual elimination of high order multiple pregnancies (triplets or greater). It also provides an excellent (hitherto unavailable) tool by which to differentiate between embryo and implantation-related causes of IVF failure and pregnancy loss.. Egg freezing and banking is a much sought after technology. However, hitherto it has, (with good reason) been discouraged because of a dismal baby rate per frozen egg (about 3-4%). The introduction of CGH egg karyotyping combined with improved cryobanking using vitrification has changed all that. We recently reported in the prestigious journal, “RBMOnline”, on an 8 fold improvement in the baby rate per vitrified CGH-normal egg and a birth rate of >70% following the transfer up to 2 embryos derived from fertilization of such eggs . There were 16 live births using this approach. We believe that the selective vitrification of euploid (CGH-normal) eggs represents a “game changing”, major breakthrough, that finally promises to open the door to “commercial egg storage” for fertility preservation (FP) and for donor egg banking, thereby increasing reproductive choices for women. At SIRM/ReproCure we have transferred embryos derived from CGH-tested eggs and embryos, to more than 300 women resulting in >100 births and as many pregnancies still ongoing, thereby accounting for more CGH-derived pregnancies than the rest of the ART world combined. We are convinced believe that use of this technology will progressively grow and within the next 5-10 years will a “new standard of care” in the ART arena. Some IVF centers have recently started offering patients access to array-CGH (aCGH) The theoretical advantages in using aCGH for detection of aneuploidy in IVF is that it is easier to perform than mCGH, is less personnel intensive and can be performed much more rapidly. The latter would make it possible to perform the egg retrieval, CGH testing and embryo transfer in the same cycle (i.e. same cycle CGH) thus avoid having to defer embryo transfer to a subsequent cycle. However since even with aCGH the time required to get results is too long to allow for embryo biopsy and embryo transfer to be conducted in then same cycle egg rather embryo CGH would have to be done. This , as stated would virtually precluding its use in cases of male infertility (which represents about half the cases for which IVF is indicated). About Array CGH: In the past, aCGH has been used to evaluate pooled DNA derived from multiple \cells. When compared to mCGH, when it comes to evaluating DNA derived from a single cell (as in the case with egg and early embryo biopsies), aCGH (as presently available) lacks reliability. That is why currently, those who advocate using aCGH in the IVF setting recommend that its use be confined to the testing of multiple cells removed from the outer layer of advanced embryos (blastocysts). The problem that arises is that with growth and accelerated cell replication, even normal blastocysts can develop a few “reject”,(chromosomally abnormal)cells mixed in with a preponderance of normal cells (i.e. “mosaicism”), without this necessarily prejudicing its ability to subsequently propagate a normal baby.. The problem that arises when CGH is performed on pooled DNA derived from several cells is that the testing cannot reliably distinguish between benign (harmless) and “pathologic” chromosomal aneuploidy.. This explains why in 15-20% of cases where blastocyst biopsy diagnoses embryo “incompetence” the diagnosis is erroneous (“false positive”) and the embryos are in fact not compromised. Conversely, when CGH is performed on single cells derived from eggs or early embryos the detection of even a single chromosomally abnormal cell strongly suggests that the embryo will ultimately be pathologically compromised. The reason is that with continued embryo development, such a large percentage of its cells will be chromosomally abnormal as to ultimately prove lethal. Such embryos often would arrest (stop dividing) and not even develop into blastocysts. Those that did would often be microscopically readily identifiable as abnormal. In cases where abnormal blastocysts appeared to be normal they would often fail to survive the vitrification /warming (freeze thaw) process or upon being transferred to the uterus, would either fail to implant, or miscarry. Very rarely would such pathologically abnormal blastocysts propagate a chromosomally defective baby. The possibility (however small) of the latter occurrence is the reason why we strongly recommend that all pregnancies occurring following Preimplantation Genetic Diagnosis/Sampling (PGD/S) be thoroughly evaluated by prenatal genetic testing. Then there is the fact that in order to accomplish same-cycle CGH, it would be necessary to perform CGH on all mature eggs (MII’s) rather than only on the DNA of eggs or embryos that make it to blastocysts (as is the case of Staggered IVF). This could mean that many eggs (often well in excess of10might at a time). Since only the hard cost of each slide used to perform aCGH on every sample of DNA costs around $200.00 and this does not even take other hard and variable expenses inherent in performing the test into count, it is easy to see why same-cycle aCGH performed on eggs for Fertility Preservation (where each and every mature egg must be tested) and/or in cases of same- cycle CGH where all eggs and or 6-9 cell embryos likewise need to be tested, could be cost-prohibitive. Recent research in Europe has yielded a low cost, aCGH platform that would be at least as reliable as mCGH for single cell aneuploidy detection. Since aCGH is much easier (than mCGH) to perform and interpret, expected demand will likely drive down its cost to appoint that it will likely supplant mCGH altogether. In the interim however,, low-cost mCGH remains a method of choice for fully karyotyping eggs and embryos.
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