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 “CGHnormal” 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 samecycle 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.