ASRM 2013 Update: Safety: Effect of the ART lab environment

October 22, 2013Carole No Comments »

If you have been following my series of updates on the 2013 ASRM meeting, you will have read the earlier three posts:

ASRM 2013 Update: Patient communication poster from reader’s feedback.¬† This is all about the poster that you, the readers, helped me create. As a bonus, you get to see a silly picture of me sitting on a sperm shaped bicycle. ūüôā There’s also a picture of the poster along with a link to a pdf of the actual poster. You can zoom in on the pdf to read the poster content if you like.¬† The poster describes what I have learned from my blog usage statistics and directly from my readers over the last 3 years about communication of ART lab topics to patients.

ASRM 2013 Update: ART Safety: the twin effect

ASRM 2013 Update: ART Safety: Singleton pregnancy

So from the last two posts, there seems to be a small but persistent increase in obstetrical complications and birth defects in a small percentage (less than 1%) of ART births. The big question remains as to whether any part of these effects- or perhaps more subtle effects on the genome itself- might be due to differences in the lab environment versus the natural environment (in the maternal body) that an embryo experiences during it’s first 5 days of existence.

I attended a presentation by Dr. Catherine Racowsky. PhD, HCLD on this very topic, namely “ART Lab Techniques and their Impact on Clinical Outcome” last week which tried to answer this big question. I will summarize the highlights of her course in this post.

You might expect that the ART environment could cause problems because we still don’t completely understand the maternal¬† (in vivo ) environment which we are trying to copy in the lab (in vitro) environment.¬† What are some differences between nature and lab nurture?

Superovulation. Use of superovulation hormones (Follistim, Gonal-F, etc.)¬† to drive up estrogen levels and follicle counts is standard to get enough eggs for IVF to work. These elevated hormone levels are not normal(!) –as any patient who has gone through IVF can tell you. Mood swings and generally feeling wretched are one by product of the hormonal hammer that is applied to the ovary to get it going for IVF.

The culture environment. Fertilization and the first five days of pre-implantation development occur inside women under presumably ideal conditions of pH, temperature, nutrients and no light exposure. It may shock you to learn that we don’t exactly know what those ideal conditions are except from what we can extrapolate from animal studies. Our human IVF culture media are based on what worked in animal in vitro fertilization experiments starting with rodents, then moving on to livestock (cattle, pigs and sheep) and maybe some non-human primate work but not much. Human embryos are pretty flexible apparently and once minimum conditions are met, seem to survive and even thrive until they can be transferred to the uterus. But just because human embryos are succeeding, doesn’t mean that the lab conditions are ideal and we are making it easy for them.

Sperm selection.¬† Sperm, when they are ejaculated into the vagina, are subjected to a rigorous natural selection process by virtue of having to pass through the vagina, push through the cervical mucous into the uterus, and finally swim up into the Fallopian tubes in search of an ovulated egg. In the vicinity of the egg, you might end up with only about 200 sperm survivors from an original ejaculate of perhaps 50 million sperm!! Now that’s some selection process! The opposite end of the spectrum is an ICSI case with sperm from a man who has such rare sperm numbers that you are searching through testicular samples for literally hours to find anything that isn’t grossly misshapen, fits into the tiny glass pipette and shows some sign of life- even a twitch will do. The only selection here is on the part of the technician who is just relieved to find anything useable so that she can give the couple some chance at conception.

What kind of issues are we worried about as far as poor outcomes for ART children? Some potential issues that have been raised are:

  • Concerns about premature birth and abnormal birth weight (either too low or too high)
  • Metabolic alterations that may have an effect later in life, causing increased incidence of obesity, type 2 diabetes, and elevated fasting glucose.
  • Increased risk of cardiovascular effects such as elevated high blood pressure and abnormalities in vascular function
  • Increased risk of birth defects.
  • Increased risk of genetic imprinting disorders which means that the wrong genes are told to stay turned on or off . These imprinting concerns are based on animal research data.

Which routine ART protocols could be involved in these effects?

ICSI requires abrupt removal of cell -to- cell communication structures. When we process oocytes for ICSI, we remove the outer layers of specialized cells called cumulus cells¬† which wrap around the egg and come out with the egg when the physician aspirates an egg from the follicle at egg retrieval. These cumulus cells send out little projections toward the egg, that actually penetrate the carbohydrate-protein shell of the egg (called the zona pellucida) and touch the surface of the egg. This forms a communication conduit between the surrounding cumulus cells and the egg nestled within.¬† For ICSI, we want to be able to see inside the egg itself so that we can identify that the egg is indeed mature enough to receive a sperm (it has dumped excess maternal DNA in its polar body) and two, we want to find a relatively empty area of the egg to inject the sperm so we don’t run the needle through any maternal chromosomes that we need later.

To see the egg better and accomplish these two goals, we tear off the cumulus cells using a combination of exposure to enzymes that dissolve the bonds between cells and also by mechanical shearing using a pipette. Ultimately, we disrupt this communication between egg and cumulus prematurely. These cells do eventually fall off¬† even after conventional IVF or in vivo but not until after fertilization is done. Animal studies using mice show that we can change the imprinting of genes by removing the cumulus cells prior to ICSI. The consequence of changes in imprinting programs means that some genes that would have been expressed are turned down or off, others that wouldn’t have been expressed are turned on. What does this mean long term? We don’t know.

The picture below on the top shows an egg still cloaked by a cloud of attached cumulus cells. These cells obscure the view of the polar body which can be seen in the second egg picture on the right. The polar body is the black smaller circle (like a Mickey Mouse ear) sticking out of the cell at about 2 o’clock. If you look at the picture on the bottom, you can almost imagine how long thin tendrils from the surrounding cells could pass through the zona pellucida (the circular shell around the egg visible in the photo on the bottom) and touch the egg.

Image of egg with attached cumulus cells

 

ICSI introduces a miniscule amount of polyvinylpyrrolidone (PVP) into the egg. Sperm is routinely exposed to PVP for ICSI.¬† We need this thick syrupy polymer to slow down the sperm enough to catch them and easily handle them in the pipette. If we use culture medium without PVP , the sperm is impossible to control in the pipette and the technique is impossible to do.¬† PVP Is a large polymer (360kDa) and is used in almost all ICSI cycles. We don’t really know if there is a bad effect of having it on the sperm. Invariably, a small amount of PVP is injected into each oocyte as the sperm is injected. Is this a problem?¬† We don’t know.

Length of time in culture. I have been a supporter of extended culture to blastocyst stage for many years  as a selection method to try to find the embryos that are most likely to implant and make a baby. We were one of the early-adoptors of sequential culture medium for extended cultures and were immediately rewarded with a jump in pregnancy rates. We also loved how nice the blastocysts stage embryos looked, plumped and more differentiated into areas that would become either baby or placenta. It was very satisfying to produce blasts and have a better chance at picking a good one for the transfer. But now, there may be some down sides to extended culture.

Some of the possible down sides to extended culture are:

  • Monozygotic/monochorionic twinning rate is increased when blastocysts are transferred. A monozygotic/monochorionic twin is a twin pregnancy that arises from one fertilized egg (identical twins) but they also share the placental plumbing (chorion) which puts them at far greater risk of obstetrical problems from unequal sharing of resources (blood and nutrients) and cord tangling causing life or limb threatening complications.
  • Increased incidence of male offspring from blastocyst culture. We noted this as well in my Indy lab. This may be due in part to the fact that faster developing embryos are more likely to be male and the embryo scoring systems generally reward the faster embryos with a better score so we may be inadvertently transferring back more male embryos.
  • Practical implications: Some patient’s embryos may not make it to day 5 so their transfers may be cancelled- whether this is really a negative depends on whether you think the same non-progressing embryo would have done better inside the uterus earlier. Likewise, fewer embryos may be frozen overall because non-progressing embryos (presumed dead) are not frozen. Again, not freezing as many embryos might not be a bad thing compared to freezing more embryos sooner, some of which don’t have any potential¬† to implant when given the chance later- because now you have incurred costs for storage and transfer of (ultimately) non-implanting embryos.
  • Obstetrical implications: Some, but not all , studies show an increased risk of preterm births and very low birth weight babies when day 5 blastocysts are transferred compared to day 3 cleavage stage embryos.

As pre-implantation genetic testing of embryos (PGD and PGS) is becoming widespread, there is some debate over the best method to biopsy the embryos to get the best cellular sample for testing with least harm to the embryo. I think the debate is pretty much over and the consensus is that trophectoderm biopsy is clearly better for getting a more robust (more cells)  sample for testing that is less likely to give false genetic test results. In addition, because you are taking a smaller percentage of biomass from the trophectoderm stage embryo (4 cells of 150 cells) compared to cleavage stage (one cell removed of 8 cells),  this should be better for preserving embryo viability and implantation ability. But if we have some concerns about extended culture, are these concerns less than the concerns we have about getting a false test result or damaging the embryo by doing cleavage stage biopsy at the earlier opportunity? This is the kind of complicated risk-benefit analysis that we have to consider as we go forward with ART.

The same sort of risk benefit analysis is happening with vitrification. There is almost universal support for vitrification as the superior freezing method for preserving the viability and quality of an embryo, leading to superior outcomes with respect to survival, fertilization and birth rates. So what are we to make of some animal studies which suggest that vitrification may also alter imprinting in some genes? Few random clinical trials (RCTs)- the gold standard of good interpretable science studies- have been performed and so we don’t really know whether we should be very concerned about this or not. The overall weight of the data still suggests that vitrification is the better clinical tool.

Take home messages:

  • The vast majority of over 5 million ART children born world wide are healthy, based on collected data so far.
  • But there are some increased health risks for ART children, early in life and possibly later.
  • The role that the ART lab environment plays in these increased risks is not understood at this time.
  • We do know that human eggs and embryos are very sensitive to environmental perturbations.
  • We know that current in vitro conditions and manipulations are not identical to nature.
  • Available – though limited-¬† data suggests that some routine aspects of ART (culture systems, blastocyst transfer, vitrification and trophectoderm biopsy) may have some unintended consequences. We need more data and more analysis to see if these risks (if confirmed) exceed the benefits.

 

 

 

 

© 2013, Carole. All rights reserved.

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