ASSESSMENT OF EMBRYO QUALITY: SELECTING THE BEST EMBRYOS TO TRANSFER:

by

Geoffrey Sher MDLevent Keskintepe PhD

“The numerical chromosomal configuration of a cell is referred to as its karyotype or ploidy. A cell with an irregular chromosome number is referred to as aneuploid while one with a normal karyotype, as euploid. It is predominantly the chromosomal configuration of the embryo that determines its subsequent ability, upon reaching a receptive uterine environment, to propagate a normal pregnancy, also referred to as its “competence.” A “euploid (“competent”) embryo transferred to a receptive uterine environment (free of anatomical, molecular or immunologic impediments to implantation” is “highly likely to propagate a “viable pregnancy “

GS

Introduction:

Embryo transfer (ET) is undoubtedlyone of the most important variables that determine IVF outcome. The procedure itself requires gentle placement of one or more embryo(s) near the roof of the uterine cavity under direct ultrasound guidance.Central to successful IVF outcome is the selection of high quality embryosthat upon being transferred to a receptive uterine environmentare capable of propagating a normal pregnancy (i.e. “competent embryos”). The following methods currently in use for differentiating between “competent” and “incompetent” embryos all to a lesser or greater degree, lack both sensitivity and specificity in evaluating “embryo quality/”competence”: (1) Microscopic Embryo Gradingwhich evaluates and grades embryos based upon their structural appearance (morphology). TheGraduated Embryo Scoring (GES),was developed by SIRM lacks both sensitivity and specificity in evaluating “embryo competence”(2) Prolonged embryo culture to the Blastocyst stage, an approach aimed at culling the poorer quality embryos. Thus embryos that survive to the blastocysts stage are more likely than there GES high-scoring day-3 embryos, to be “competent”. (3) The Embryo Marker Expression Test (EMET), introduced by Geoffrey Sher MD and Levent Keskintepe PhD in 2003 was the first prospective test to measure an embryo generated genetic “marker” as an improved method to evaluate “embryo competence”. 4)Preimplantation Genetic Diagnosis/Screening (PGD/S) with Fluorescence in-situ hybridization (FISH) of certain of the embryo’s chromosomes. However (FISH) only assesses 8-12 of 23 chromosome pairs and thus is not very helpful in measuring embryo “competence”. 5) Egg/embryo Competency Testing (ECT) promises to for the first time provide a highly discriminatory method for differentiating between “competent” and “incompetent” embryos. ECT could finally permit identification and the transfer of a single “competent” embryo .ECT with a great likelihood of a viable pregnancy resulting. ECT involveseither removing the First Polar Body (PB-1) of the mature egg (MII) soon after egg retrieval (Polar Body Biopsy) or a blastomere from the day-3 cleaved (divided) embryo and then testing the DNA through a genetic test known as comparative genomic hybridization (CGH) which identifies ALL the chromosomes. Chromosomally normal embryos are about 80-90% likely to be “competent” (see below).

1. Graduated Embryo Scoring (GES):Graduated Embryo Scoring (GES) is a unique microscopic method for assessing embryo quality. With GES, each embryo is separately examined through a series of microscopic assessments throughout a period of 72 hours following egg insemination. The maximum allotted GES score is 100. A four-year evaluation of embryos derived from the eggs of thousands of women under 40 has revealed that embryos with a GES score of 70-100 each have better than a 35% likelihood of implanting successfully as compared to less than 20% when the GES score is below 70. Embryo implantation potential decreases rapidly, progressively, and proportionately to well below 10% per embryo by the time the egg provider reaches 43 years of age.

It was largely the need to grow embryos separately in order to implement GES throughout all SIRM centers that led to our discovering that the presence of sHLA-G in the media surrounding each embryo could predict which embryos were most likely to

Produce a pregnancy (see “The Embryo Marker Expression Test (EMET)” later in this section). When this book was written, almost all other IVF programs were growing embryos in batches (more than one embryo in the same Petri dish).

2. Blastocyst Transfer: The presumption has always been that it is better to transfer healthy embryos into the uterus sooner rather than later. Recent research conducted by Drs Sher and Keskintepe has clearly shown this to be an erroneous belief. In fact, with few exceptions, embryos that that fail to progress to the blastocyst stage in culture are chromosomally abnormal and would not have been capable of propagatinga healthy pregnancy anyway, even if they had been transferred earlier on. Since only about 40% of embryos progress to blastocysts in culture, there might be an advantage in allowing embryos to grow to blastocysts in order to cull out the abnormal ones. Simply stated, the major benefit from extending embryo culture to day 5 is a natural selection of the best-quality embryos. This means high pregnancy rates can be achieved from the

Transfer of fewer embryos than usual, thus reducing the risk of high-order multiple pregnancies. It is

4. The Embryo Marker Expression Test (EMET): By measuring the concentration of a genetic marker known as sHLA-G (soluble human leukocyte antigen-G), which is released into the media in which early embryos are growing after fertilization, it is now possible to identify those embryos more likely to produce a pregnancy. This Embryo Marker Expression Test (EMET) is performed 46 hours after the egg retrieval to identify EMET-positive, or “more likely to be competent” embryos. It has been determined, based upon the performance of EMET in more than 1,000 women undergoing IVF at SIRM, that the transfer of even a single EMET-positive embryo in women under 39 (provided that they had normal uterine linings and, when needed, were treated for immunologic implantation problems) results in better than a 50% chance of a viable pregnancy. In women 39-43 years the viable pregnancy rate when HLA-G +ve embryos were transferred was >35%.

This discovery although not representing a “silver bullet” assists in the selection of better quality embryos for transfer.

5. Preimplantation Genetic Diagnosis (PGD) with FISH:Currently, probes used in FISH only examine 8 or 9 of the 23 human chromosome pairs in the embryo. Thus, most chromosome pairs cannot be examined for numerical chromosome abnormalities (aneuploidy). The 8 or 9 commercially available probes were actually developed to test for the most common aneuploidies that cause chromosomal miscarriages and birth defects. Since FISH as performed commercially will not evaluate for many “lethal aneuploidies,” it is of very limited value in selecting chromosomally normal embryos fortransfer. Even when the results of PGD with FISH are reported on as being “normal, there remains about a 45% in young women and 50-60% in older women) chance that aneuploidy involving those chromosomes not assessed, might still be present.As such PGD using FISH is of limited value as a method for identifying and selecting “competent” embryos for transfer.

6. Egg/Embryo Competency testing (ECT) Using Comparative Genomic Hybridization (CGH):

The prior lack of a reliable method by which to accurately assess embryo competence often prompts even the most well intended IVF practitioner to transfer several embryos at a time in an attempt to optimize the likelihood of a pregnancy resulting. The widespread adoption of such practice, has resulted in a virtual explosion in the incidence of high order multiple births (triplets or greater) in many first world countries. The high costs associated with addressing short term and long term, obstetric, neonatal, and social complications resulting from these high order multiple pregnancies, is one of the main reasons why most health insurance providers in the U.S.A. are presently reluctant to voluntarily cover ART services.

Ready clinical access to a method by which to reliably differentiate between “competent” and “incompetent” embryos would immediately remove the incentive to transfer multiple embryos, markedly improve IVF success rates, reduce the incidence of multiple pregnancies, decrease the cost per IVF baby born, lead to a reduction in reproductive health care costs and in the process, transform the entire ART arena.

MULTIPHASE STUDY: INTRODUCTION OF EGG/EMBRYO COMPETENCY TESTING (ECT): A NEW METHOD FOR GREATLY IMPROVING IVF OUTCOME BY IDENTIFYING AND SELECTING “COMPETENT” EMBRYOS FOR TRANSFER:

Our study revealed the following:

  1. It is the karyotype (ploidy) of the egg, rather than the sperm, that is the main determinant of an embryo’s karyotype and ability to propagate a normal pregnancy(“competence”).
  2. Even in women under 35, at least 60% of all mature eggs are aneuploid
  3. The incidence of egg aneuploidy increases progressively with advancing age such that by the mid-forties it is probably greater than 90%.
  4. Aneuploid eggs consistently (100% of the time) propagate aneuploid and “incompetent” embryos that are responsible failure to attach to the uterine lining, early miscarriages, and chromosomal birth defects such as Trisomy 21 (Down’s syndrome).
  5. In approximately 90% of cases euploid eggs propagated euploid, “competent” embryos.
  6. In the absence of male infertility and untreated immunologic implantation dysfunction, each euploid embryo, upon being transferred to a receptive uterine environment (free of immunologic, anatomical and biochemical impediments), has about a 70-80% chance of implanting and propagating a normal pregnancy.
  7. With few exceptions, appropriately cultured embryos that fail to progress to the blastocyst stage are in fact aneuploid and could not have produced a normal pregnancy. This dispels the erroneous contention that embryos are better off being transferred to the uterus earlier.
  8. When up to two euploid embryos are transferred, the chance of an ultrasound confirmed, viable pregnancy is at least double the national average. .
  9. Since most miscarriages are attributable to embryo aneuploidy, such euploid pregnancies have a much reduced chance of miscarrying or of resulting in an aneuploidy-related birth defect such as Down’s syndrome.

In summary, aneuploid eggs consistently propagate “incompetent” aneuploid embryos which, accordingly, should be identified in advance and discarded. Euploid eggs almost always propagate “competent” euploid embryos that should be selectively transferred to a receptive uterus.

Staggered -IVF(St-IVF) : The preferred application of ECT in IVF:

Step1: harvesting of eggs

Step 2:fertilization by ICSI

Step 3: PGD with blastomere biopsy performed on 6-9 cell day 3 embryos

Step 4:CGH performed on blastomere-derived DNA

Step 5:vitrification (i.e an ultra-rapid freezing method that minimizes the hitherto encountered incidence of damaging to embryos during the freeze/thaw process)

Step 6: banking (cryo-storing) all chromosomally normal (“competent”) blastocystsfor subsequent dispensation

Step 7:selection of embryos for transfer ,based uponmorphologic grade + CGH and finally,

Step 8: FET of one or more such embryos in a future menstrual cycle

We believe the results of our findings once further corroborated through independently conducted studies both in the United States and in the United Kingdom), will almost certainly result in ECT and St-IVF rapidly becoming the preferred method for reliably selecting embryos for transfer and will rapidly cause a paradigm shift that will transform the entire practice of IVF.

NOTE: PGD by way of FISH and CGH can be wrong.

  1. With Fluorescence in-situ hybridization: Preimplantation genetic diagnosis (PGD) of numerical chromosome abnormalities significantly reduces spontaneous abortions and may increase pregnancy rates in women of advanced maternal age undergoing in vitro fertilization. However, the technique has an error rate of around 10% and trisomy 21 conceptions have occurred after normal PGD/FISH.
  2. With Comparative genomic Hybridization: While the transfer of blastocysts derived from fertilization of CGH” normal” eggs/embryos minimizes the occurrence of aneuploidy related birth defects such as Down’s syndrome, it does not definitively preclude their occurrence.

We strongly recommended that Prenatal Genetic Diagnosis, including (but not necessarily limed to) 1st trimester chorionic villus sampling (CVS) and/or2nd trimester amniocentesis be carried out in all cases where pregnancy occurs following the transfer of “CGH-normal” embryos.

1