Choosing the Optimal Day for Embryo Transfer by Dlugi, MD

Choosing the Optimal Day for Embryo Transfer
By Alexander Dlugi, M.D.

Selecting the correct time for embryo transfer is not always easy.  Historically, embryo transfers were initially performed two days after egg retrieval when the embryo(s) had reached a 2-4 cell stage. While the formative years of IVF held promise, the pregnancy rates were rather disappointing given today's standards.  Indeed in the early 1980s, success rates were no better than 5%-7% despite the transfer of large numbers of embryos (6-8 or higher). Eventually, as techniques and culture media improved, transfers began to be performed 3 days following oocyte retrieval when the embryo(s) ranged from 6-10 cells each. These embryos appeared to have a better chance for success. However, in order to achieve respectable pregnancy rates 3 or more embryos typically need to be transferred. The number to be transferred often depends upon the woman's age with more embryos being transferred with increasing age. The choice of which embryos specifically are to be transferred is based upon the cell number as well as the appearance or morphology of the embryo. Elaborate grading systems have been developed for Day 3 embryos but these fall short of an absolute correlation with a successful conception. For example, three beautiful 8 cell grade 1 embryos have not uncommonly failed to implant while a single 4 or 6 cell grade 3 embryo might result in the desired outcome. Increasingly, IVF programs have resorted to placing more and more embryos into the woman's uterus in order to gain a competitive edge.  This has led to an unacceptable multiple pregnancy rate across the country.

In an effort to reduce the multiple pregnancy rate while maintaining a high overall pregnancy rate, attention was turned to allowing embryos to mature an additional 2 to 3 days in culture achieving a blastocyst stage of development. Blastocyst transfer in human IVF is thought to have several advantages over a Day 3 transfer. First, during natural conception, the early embryo spends several days in the fallopian tube before reaching the uterine environment on the fifth day following conception - the blastocyst stage. Accordingly, it has been proposed that the uterine lining at that point might be more receptive to implantation. Second, premature placement of the embryo into the uterus, such as on Day 3, could possibly result in significant stress and loss viability. The blastocyst is thought to be more tolerant than an earlier stage embryo and better suited to development in the uterine cavity. Third, uterine contractions which have been demonstrated on the day of retrieval may also be present on Day 2 and 3 but are mostly absent by Day 5. Finally, not all day 3 embryos are able to survive to the blastocyst stage. Thus, it has been assumed that those that do achieve this more advanced stage of development are the "hardiest" and represent the survival of the fittest. While this may indeed be true, the issue that arises is that if there are only a small number of 8 cell embryos available on Day 3 in culture, there may be none that survive to Day 5. Indeed, it is not unusual to see the loss of anywhere from 47%-86% of Day 3 embryos by Day 5. Pregnancy outcome with Day 5 transfers appears to be related to the number of embryos available on Day 2 as well as the number of 8 cell embryos on Day 3. Consequently, the criteria used by many IVF programs to determine whether or not to continue to culture to Day 5 or 6 is often based upon the age of the woman (often <35 years old) and having an "adequate" number (usually 6 or more) 8 cell embryos on Day 3. This is despite the fact that numerous studies have demonstrated a higher clinical pregnancy rate with blastocyst transfer.

The expression of the embryonic genome is activated once the embryo passes the 8 cell stage. On Day 3, it has not heretofore been possible to determine which embryos have the highest developmental potential as assessment on Day 3 or earlier appears to reflect oocyte quality.  Recently, however, soluble (s) HLA-G has been identified in the media surrounding the embryo as it grows in culture.  HLA-G is a non-classic type I human leukocyte antigen produced by the embryo. It plays a pivotal role in the development of a pregnancy. Embryos derived from culture media expressing high concentrations of sHLA-G exhibit improved cleavage and implantation rates. In our hands, we test for this embryo marker expression by requiring that each embryo be cultured separately allowing each embryo to be tested independently. This differs from the common practice of culturing embryos in groups or clusters. Furthermore, we move all embryos to blastocyst media, 46 hours post insemination. At this point, we take the original media droplets and test these individually for sHLA-G expression. This allows for selection of fewer embryos for transfer, (only those with the greatest potential for producing a pregnancy), well  in advance of the embryo transfer and in so doing minimizes the risk of high order multiple pregnancies (triplets or greater) while optimizing overall IVF success rate. Indeed, our most recent results indicate that when at least one out of three embryos that are transferred on Day 3 has a positive embryo marker expression test, the pregnancy rate exceeds 70%!

Interestingly, it appears that only about 16% of embryos express sHLA-G. This therefore begs the question of what one should do if all the embryos available for possible transfer are sHLA-G negative.  There is, currently, no answer to this question. There have been numerous pregnancies resulting from the transfer of all sHLA-G negative embryos, though the rates are lower than if there is at least one that is positive. One might argue that in this setting, it might be advisable to continue to culture to the blastocyst stage as these more advanced embryos appear to have enhanced implantation potential as well. Studies are ongoing at SIRM to address this question.

Finally, one needs to consider the high loss of embryos when the culture period is extended for another 2-3 days. The debate rages as to whether embryos would survive better within the uterine environment as opposed to the laboratory Petri dish. The experiment to definitely answer this question cannot be performed as one cannot transfer embryos on Day 3 and then remove them on Day 5 to check for their developmental stage.  However, recent data from SIRM laboratories seems to indicate that embryos that appear healthy and viable on Day 3 in culture but are unable to develop to the blastocyst stage in the laboratory setting are actually aneuploid - that is they have an abnormal number of chromosomes. If this proves to be true, it would imply that these abnormal embryos would either not have survived in the uterus or would have resulted in an early pregnancy loss. Ultimately, a combination of sHLA-G analysis and early embryonic or even oocyte genetic analysis should result in a more accurate determination of which embryo(s) are the proper ones to transfer and on which day.  This capability, currently under active investigation at SIRM, has the potential to revolutionize the way in which IVF is performed.

Alexander Dlugi M.D.
Medical Director
Sher Institutes for Reproductive Medicine - New Jersey

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