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Pgd - Fish Vs Cgh

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#1 christiyen

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Posted 01 October 2007 - 08:42 AM

Hi Dr. Sher,

Can you walk me through the CGH process?  
Is CGH now a standard test or still in experimental phase?  Is it offered in Dallas?
At age 38/39 category, what's the % of normal embryo with CGH vs FISH?
What's CGH's success rate (live birth) / transfer compared to FISH?
How does it thaw w/vitrification vs slow cryo?  
FET success rate / transfer w/ CGH vs FISH?
What's the cost differential CGH vs FISH?

#2 Geoffrey Sher, MD

Geoffrey Sher, MD
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Posted 01 October 2007 - 12:15 PM

It is in the developmental stages and it is available in Dalas. Talk to Dr Saleh about it.

Geoff Sher


Staggered In Vitro Fertilization (St-IVF):  Rational Expectations

Our reported IVF results following egg biopsy, our unique experience in the performance of CGH, and our preliminary and growing experience with St-IVF, suggest a 60-70% chance of a live birth following the transfer of up to two “competent” blastocysts to a “receptive” uterus.
         GS & LK
A. Introduction
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 (one that upon reaching a “receptive” is 60%-70% likely to develop into a viable pregnancy) is  transferred to a “non-receptive” uterus that when an “incompetent” embryo is transferred to a “receptive uterus”.
Throughout human reproduction, the establishment of an ideal “seed-soil” relationship is pivotal, since both embryo “competency” as well as uterine receptivity is 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%).
Successful treatment of reproductive failure  demands full prior identification and treatment of those factors  that adversely influence both  embryo “competence” as well as 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 to the uterus. Even in young women, an embryo that “looks good” under a microscope is not necessarily “competent”. At best it has a 30% 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, in the hope that this will 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 pregnancies. The relatively 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 financial cost associated with the short and long term prematurity related consequences of IVF multiple births  represents one of  the main reasons why health insurance providers in this country are reluctant to cover the procedure.

B. What methods are currently in use to differentiate between “competent” and “incompetent” embryos.
The following methods currently employed in IVF clinics across the world to evaluate embryo quality all, to a lesser or greater degree, fail to differentiate reliably between “competent” and “incompetent” embryos”.
1. Microscopic Embryo Grading Systems:  These evaluate and grade embryos based upon structural appearance (morphology). They are all unreliable. Even those embryos that appear to be morphologically normal are much more likely to be “incompetent” than “competent”. Furthermore, the older the woman who produces the eggs, the less reliable morphologic embryo grading becomes. For example, a morphologically normal embryo derived through fertilization of an egg taken from the ovary of a 30 year old woman might, on average  have about a  25% chance of propagating a pregnancy, while a morphologically comparable embryo derived from the egg of a 45 year old, is 7-10 times less likely to do so.
2. Prolonged embryo culture to the Blastocyst: This approach relies on the fact that by extending the embryo culture process, many poorer quality embryos will be culled out. Thus embryos that survive to the blastocysts stage are more likely to be “competent”. This approach, while helpful, is far from perfect because many “incompetent” embryos in fact will progress to the blastocyst stage.  
3. Preimplantation genetic diagnosis (PGD): Here, one or two cells (blastomeres) is/are  microsurgically removed from the embryo on day 3 post-fertilization. The blastomere(s) are thereupon stained to reveal specific chromosomes, using a method known as fluorescence in situ hybridization (FISH). However since PGD/FISH only targets some (i.e. 8-12) of 23 chromosome pairs, it is unable to reliably differentiate between “competent” and “incompetent embryos. In fact an embryo that tests normal with FISH still has at least a 47% chance of having chromosome irregularities that involve those chromosome pairs that could not be evaluated by FISH. Accordingly PGD/FISH is of questionable value in selecting the best embryos for transfer.  
4. Embryo Competency testing (ECT):  ECT is a new method by which DNA derived from blastomeres removed from day 3 embryos is tested using comparative genomic hybridization (“CGH”). CGH accurately identifies all the chromosomes in a cell and is thus >80% accurate in evaluating embryo “competence”. The reason that CGH is not 100% accurate in assessing embryo “competence” is because only one (sometimes two) blastomeres are tested with ECT, leaving the remaining blastomeres untested.  (see “mosaicism”  below)

C. What makes one embryo “competent” and another, “incompetent?
It is mostly (but not exclusively) the chromosomal configuration of the embryo that determines its “competence”. The number of chromosomes in a cell is referred to as its ploidy. A cell with a normal number of chromosomes is 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”.
We recently reported (Fertility and Sterility, May, 2007) on a two phase  study involving the performance of CGH on a fragment of nuclear material (the first polar body or PB-1) discharged from the egg during the chromosomal rearrangement that takes place following the hCG trigger. The PB-1 has a chromosomal make up which is a mirror image of the chromosomes in the egg’s nucleus. Several hours following fertilization, PB-1 divides and a second polar body (i.e. PB-2) appears alongside it. Polar body biopsy involves removal of PB-1 and/or PB-2 from immediately below the outer envelopment (zona pellucida) of the pre or post-fertilized egg. This is achieved without damaging the egg itself.  
Our study (referred to above) was performed on eggs extracted from women aged 25-42 years who underwent ovarian stimulation with fertility agents.  Phase 1 involved performance of PB-1 and PB-2 egg biopsies before and after fertilization, and subsequently, on blastomeres removed from the corresponding day 3 embryos. Phase 2 involved a second study involving 35 women where we selectively transferred up to 2 embryos derived from eggs that previously had tested chromosomally normal following PB-1, CGH. The study produced the following, hitherto unknown information:
1. It is definitively the chromosomal make-up of the egg, rather than the sperm, that is the main determinant of an embryo’s “competence”.
2. Even in young women, >60% of all mature eggs are likely to be aneuploid and thus incapable of propagating “competent” embryos.
3. The incidence of egg aneuploidy increases progressively with advancing age such that by the mid-forties it is probably near or above 90%.
4. Eggs that have an abnormal number of chromosomes (i.e. aneuploid)  will , upon fertilization always propagate aneuploid, “incompetent” embryos. Such embryos will either fail to attach to the uterine lining, attach and then subsequently miscarry, or produce chromosomal birth defects such as Trisomy 21 (Down’s syndrome).
5. Approximately 85% of eggs that have a normal number of chromosomes (ie euploid) fertilized with normal sperm subsequently  result in euploid, “competent” embryos.
6. In the absence of male infertility and untreated immunologic implantation dysfunction, the transfer of 1-2 euploid, (CGH-tested)  embryos to a receptive uterine environment that is free of immunologic, anatomical and biochemical impediments, has better than a 70% chance of resulting in a live birth.  
7. Appropriately cultured embryos that fail to progress to the blastocyst stage are in fact almost always aneuploid and thus “incompetent”. 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.
8. Most early miscarriages and many birth defects (e.g Down’s syndrome) are attributable to embryo aneuploidy. It follows that by only transferring euploid, “competent” embryos, will this risk be significantly reduced.

D. What is Staggered- In Vitro Fertilization (St-IVF)?
St-IVF is a new and improved IVF process that is capable of improving IVF success, reducing the risk of miscarriages, minimizing the risk of chromosomal birth defects such as Down’s syndrome, and reducing the incidence of multiple births. St-IVF involves dividing the IVF cycle up into 2 segments. The first segment involves ovarian stimulation and egg retrieval while the second comprises the embryo transfer (that can be performed weeks, months or even years later). . The purpose of dividing up the cycle into two parts is to allow the genetics laboratory  sufficient time to complete CGH on one or more of the embryo’s  cells (i.e. blastomeres) that were  surgically removed from the embryo, 3 days following fertilization by intracytoplasmic sperm injection (ICSI).
First Segment:  
• Ovarian stimulation, ultrasound and hormonal monitoring, egg retrieval and ICSI are performed. The patient returns home following the egg retrieval..  
• Three days later, one (rarely two) blastomere(s) is/are removed (blastomere biopsy) from embryo(s) that have reached a 6-9 cell stage. Blastomere  DNA is then processed in preparation for CGH analysis which is a highly complex process that takes a week or longer to complete.
• All biopsied embryos remain in culture for up to six days Those that develop into  healthy expanded blastocysts (the most  advanced state of pre-implantation development) are vitrified (i.e ultra-rapid freezing-see below) and then cryobanked.  The remainder are discarded.  
• Fully developed blastocysts derived from embryos that are determined through CGH  to have all twenty three pairs (46) chromosomes intact, are retained in the vitrified state (i.e. cryostored), while those derived from chromosomally abnormal, day 3  embryos, are discarded.
• The patient is subsequently informed of the CGH test results and a date is set for the transfer of up to two euploid embryos.
Second Segment:
• Patients who have at least one “competent” embryo in cryostorage return for clinical assessment, ultrasound and hormonal monitoring   in preparation for the embryo transfer.
• The “competent” vitrified blastocyst(s) selected for transfer is/are  warmed (thawed) and, provided of course, that at least one “competent” blastocyst survives, is/are duly transferred  under ultrasound guidance.  

E. What is embryo vitrification?
Cryopreservation of human embryos has been a routine procedure since the early 1980s. Conventional cryopreservation which is still being used by most IVF centers, involves slowly reducing  the embryo’s temperature until it freezes . This unfortunately results in ice crystal formation inside blastomeres, damaging them and reducing embryo “competency”. The relatively recent introduction of ultra-rapid freezing or vitrification has changed all this.  This innovative technology, whereby the embryo is frozen within the blink of an eyelid, is so rapid that there is no time for intracellular crystallization and its resulting cell damage, to occur. The conventional slow freezing method leads to the death of up to 30% of blastocysts so frozen. In contrast with vitrification, less than 10% are lost. The survival rate is likely even higher for “competent” blastocysts.

F. Does a “competent”, (CGH-“normal”) embryo invariably produce a baby?
Unfortunately this is not the case.  St-IVF with ECT, while potentially representing a major advance in the IVF arena, is not a panacea. 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 and, Third, there is a wide variation in technical expertise when it comes to the performance of embryo transfer, a rate-limiting factor in IVF.
1. Misdiagnosis of  embryo “competence”
What is “Mosaicism”?
Mosaicism is the term used to describe an embryo that contains both euploid and aneuploid blastomeres. With ECT, using CGH, we only examine a single extracted cell (blastomere) removed from a 6-9 cell day 3 embryo. We cannot possibly examine all the blastomeres. Following fertilization, the cells of the early embryo along with their accompanying chromosomes, replicate repeatedly. During this process things can, and indeed do, go wrong about 15% of the time with regard to chromosomal arrangement resulting in one or more of the embryo’s blastomeres becoming aneuploid.
Is embryo mosaicism always lethal?
Mosaicism affecting the early embryo:
Because we only perform CGH on one of 6-9 blastomeres of the day 3 embryo, we will in about 15% of cases erroneously deem an embryo to be “euploid” throughout when in fact it might not be. However, in either the short or the long term, aneuploidy involving even one blastomere of a 3 day embryo (i.e mosaicism)  will inevitably prove to be lethal. Accordingly an embryo that was initially regarded through single blastomere CGH analysis to be “competent” may in fact  be “incompetent”. The explanation for this is as follows: With repeated cell division, one aneuploid blastomere out of 6-9 (in the day 3 embryo) will ultimately translate into such a high percentage of the overall total number of blastomeres in the embryo being aneuploid that the remaining normal euploid cells are incapable of insuring proper function and survival. The lethality associated with  “incompetence” of an early day 3 embryos manifests as :
• Arrested division (cleavage): In such cases, the embryo stops dividing and fails to reach the advanced (blastocyst) stage of development over  the next 2-3 days in culture.
• Unexpanded blastocyst: A fully “competent” blastocyst, given enough time (an extra day) to develop in culture, will always expand due to the formation of a fluid filled center (blastocele). Failure to expand indicates “incompetence”.
• Failure of a blastocyst to survive the freeze/thaw (vitrification/warming): Upon freezing (vitrification) the cellular content of the blastocyst “contracts”. Upon thawing the viable, competent blastocyst will rapidly re-expand to its full pre-frozen state. Failure to do so indicates “incompetence” and such embryos are not eligible for transfer to the uterus. Since failure of a blastocyst to survive the freeze/thaw process is a last minute discovery, it often proves to be highly bewildering and most distressing to the patient, and her partner as well as the treating physician who by this time were all  fully  anticipating that the  embryo  transfer would proceed..
• Preclinical and clinical miscarriage: An “incompetent”, expanded blastocyst that survives vitrification/warming and is transferred in its expanded state to the uterus, will usually fail to gain proper attachment to the uterine lining. In such cases, the pregnancy hormone (hCG) may either be undetectable in urine or blood (i.e. a negative pregnancy test), be temporarily present without there being subsequent ultrasound evidence of a pregnancy (i.e. a chemical pregnancy) or may attach long enough to result in a detectable pregnancy  by ultrasound only to abort spontaneously a few days/ weeks later (i.e. miscarriage).  
Mosaicism in the blastocyst:  
Mosaicism is common in advanced embryos (the 100+ cell morula or blastocyst stage) and when it does occur, unlike the result of mosaicism in a day 3 embryo, it is not necessarily prejudicial to its function and/or its survival. The reason is that in such cases the ratio of aneuploid versus euploid component cells is so small, that the impact on overall functionality is usually negligible. It should be realized that most implantation dysfunction is due to embryo aneuploidy.
2. Uterine Receptivity:
As stated at the beginning of this presentation, when it comes to human reproduction we are dealing with a “seed-soil” relationship. It is important to recognize that given our current state of knowledge, it is not possible to diagnose and treat all causes of decreased uterine receptivity. Often elusive factors, such as: a) dysfunctional immunologic implantation; b) anatomical pathologic endometrial factors c) genetic and poorly understood molecular factors, all too often contribute to failure of a St-IVF procedure.
3. Technical expertise.
Embryo transfer is a critical rate limiting step in IVF. However the level of expertise in performing this important procedure varies widely. It is difficult to standardize a procedure that combines dexterity and skill, with know how. Alarmingly, there are still IVF practitioners who routinely perform ET without using ultrasound guidance to make certain that the embryo(s) is/are deposited in the uterus with precision. (In some cases, where the uterine cavity cannot be adequately visualized, the experienced physician can get by doing  the embryo transfer by feel, but this represents the exception rather than the rule.)

G. Conclusion:
St-IVF represents an important advance in the ART arena. We believe that for the following reasons St-IVF could re-fashion the entire IVF panorama in the years to come.
a. St-IVF produces about a 60%-70% viable pregnancy  rate per embryo transfer. This is approximately double the national average
b. With St-IVF the implantation potential of each embryo is markedly increased to above 50%  per “competent” blastocyst transferred
c. St-IVF markedly reduces the risk of commonly occurring miscarriages and birth defects due to abnormal number of chromosomes (aneuploidy).
d. St-IVF markedly reduces the incidence of IVF high order multiple births by making it possible to transfer up  to two  competent embryos for a remarkably high success rate.