In January, Dr. Carolyn Givens and I attended a meeting in Hawaii organized by the American Board of Bioanalysts (ABB). This organization board certifies and licenses embryologists, andrologists, and a number of other laboratory specialists in the United States. Our meeting was under the direction of the College of Reproductive Biology, a special interest group within the ABB and for which I am the immediate past Chair.

The meeting was small and intimate, a situation always welcomed among reproductive biology professionals. The location allowed for good interaction with embryologists from Japan who have always been a great source of ideas and innovation within our specialty.

In fact, the highlight of the meeting was a series of videos shown by Dr. Yasuyuki Mio from the Mio Fertility Clinic in Yonago, Japan. He was able to take time-lapse cinematography of human embryos in culture, and as a result reported some novel observations on how oocytes fertilize and how embryos develop. The actual moment of sperm entry into the oocyte was recorded and it was possible to see that human oocytes form a fertilization cone (a membrane that helps bring the sperm into the oocyte), shortly after sperm entry. The events that follow (2nd polar body extrusion, which is the egg extruding a set of chromosomes, and pronuclear formation, alignment of the nuclei from the egg and sperm) occurred as expected, but for the first time the male and the female nuclei could be distinguished from each other.

After fertilization, the embryos were seen to change dramatically as they developed. In particular, they appeared more disorganized and untidy immediately after a cell division event and more symmetrical and organized several hours later. This discovery has implications for those embryos that sometimes may appear poorly. It suggests that they may look better later in the day when they are clear of the cell division process. Another important observation regarding blastocysts, is that those that develop 2 inner cell masses (ICM: the precursor cells of the fetus) do so in a predictable way. At PFC, we avoid using embryos with two ICMs whenever possible, as they are likely to lead to the formation of identical twins. A normal embryo should have only a single ICM. Currently, it is possible that one of the ICMs may be small enough to avoid detection. The observation was made that the fine cellular bridges within the embryo cavity appear to correlate to the presence of an extra ICM.

Another notable presentation was that of Dr. Tetsunori Mukaida, of Hiroshima HART Clinic, on sperm morphology. He demonstrated that observing sperm under ultra-high magnification can show structural defects that are not always visible when using standard microscopes. While magnifying sperm thousands of times has its difficulties, Dr. Mukaida reported that sperm with subtle physical defects have a much lower chance of making an embryo that can become a baby. Sperm that are close to perfect in size, shape and structure are difficult to find in any sperm sample and it can take hours just to find a few ideal sperm. However, the extra effort may be worthwhile, especially in patients that have had a previous IVF cycle where the embryos did not develop well or implant after transfer. PFC is currently looking into this technology and we will report more details in a future issue of Fertility Flash.

Attending meetings like this and keeping up with the latest developments in our field is an important part of the culture at PFC. We share the load of traveling to educational events and are always excited to bring home ideas and thoughts to share with our colleagues. PFC is committed to implementing the latest technology and innovations to maximize pregnancy rates for our patients. We will continue to stay updated with all of the research and development in our specialty.

Both Dr. Givens and Dr. Conaghan contributed to this article.

	Joe Conaghan, Ph.D., HCLD is PFC’s laboratory director. Dr. Conaghan is internationally recognized for his work on improving embryo culture conditions. His interests include developing programs for the treatment of severe male factor infertility; diagnosis of genetic disease in embryos; and improved embryo culture. See more posts
	Carolyn Givens, M.D. was the first in San Francisco to successfully initiate a pregnancy using intracytoplasmic sperm injection (ICSI). She currently co-directs the Bay Area Pre-Implantation Genetic Diagnosis Program (PGD) and is director of PFC’s PGD program. See more posts

For patients having their embryos transferred at the blastocyst stage, the grading procedure used to assess the embryos can seem complicated. However, we simply look to see that the embryos are developing normally, are not slowing down, and are preparing for implantation in the uterus.

In the 2 days following fertilization, embryos go through 3 rounds of cell division. The fertilized oocyte divides in 2, these cells each divide again to give 4, and then these divide to give 8. In the resulting 8-cell embryo, each cell should be 1/8 the size of the original oocyte since there is no growth in size, and each cell should be intact and symmetrical. When we assess embryos at this stage, we first count the number of cells and we then assign a grade based on how good the embryo looks. Embryos that have disintegrating or asymmetrical cells are assigned a lower grade.

At this early stage, the individual cells stay together because they are contained within a shell called the zona pellucida. However, as the embryo progresses past the 8-cell stage, dividing to 16 and then 32, the cells attach to each other and cooperate to form a tight ball called a morula. At the morula stage, the cells are pressed so tightly together that individual cells cannot easily be identified or counted. Once the attachments between cells are formed, the cells begin to pump fluid into the center of the ball, giving rise to a tiny fluid filled cavity or cyst. As long as the junctions between cells hold, no fluid can escape from the cyst, and the cyst grows larger as more fluid is pumped in.

These are critical days for the embryo. In addition to forming the central cyst, the embryo is also busy organizing its cells into two distinct populations. As the embryo moves beyond the 8-cell stage, some cells stay on the outside of the ball and some are pushed to the inside. In the typical 16-cell embryo, there are 12 outer and 4 inner cells. At the 32-cell stage, 22 of the cells are outer cells and 10 are inner cells. Creating more outer cells is deliberate, because these cells are needed to maintain the integrity of the cavity as it becomes larger. More importantly however, these cells will become the placenta, and having enough cells to establish the placenta is critical to successful implantation in the uterus. Once the placenta is established, it can feed the inner cells which become the developing fetus.

The appearance of the cyst at the center of the morula marks the next embryo stage, the blastocyst. In assessing the blastocyst, we look at the size of the cyst and the integrity of the outer and inner cells. Depending on the size of the cyst, the blastocyst is referred to as early, expanding or fully expanded. If the cyst has become large enough to cause the embryo to burst through its shell, we call it a hatching blastocyst. Occasionally, we even see fully hatched blastocysts. Hatching is a natural process that frees the embryo from its shell to allow implantation to occur. The more expanded the cyst has become, the more we favor the embryo for transfer.

In addition to looking at cyst expansion, the grade of the blastocyst is further determined by the integrity of the inner and outer cells. Embryos with more cells are better, and the best blastocysts are well expanded with distinct inner and outer cell populations. In poor quality blastocysts, there can be few cells in one or both populations, and/or the cavity can be small. And sometimes, even in embryos with beautiful outer cells, we cannot see any inner cells at all. These embryos are destined to fail since a full blastocyst with 32 cells is incapable of making inner cells if they do not already exist.

The embryos that are most difficult to assess are those where the cavity has just begun to open up, but has not expanded sufficiently to allow us to see inside. These early blastocysts are usually assigned lesser grades as we are unable to determine whether any inner cells are present. We often look at these embryos again several hours later to see if further expansion has revealed the presence of those critical inner cells. We would then re-grade the embryo, if appropriate.

All of this development, from fertilization to blastocyst expansion and hatching, normally follows a tight timeline that is independent of cell number. The embryo attempts to hatch from its shell approximately 5 or 6 days post fertilization, regardless of the number of cells it contains. If development is slow, and cell number is consequently low, the outer cells stretch to enclose the cyst and expansion continues. This is important, as the uterus waits only a few days for the embryo to implant. If the embryo takes too long to make the “right” number of cells for expansion and hatching, it may miss the implantation window. The practical result of this is that we still get high implantation rates even if only early blastocysts are available for transfer.

The above phenomenon is relevant to frozen embryo transfer cycles too, because many embryos lose one or more cells as a result of freezing and thawing. Such embryos still try to form blastocysts according to their original timeline, even though they may have less than the ideal number of cells. The consequences of arriving with plenty of cells but too late for the uterus are worse than having a chance to implant even with fewer cells. As a result, frozen-thawed embryos that have lost a cell or two are not assigned a lower grade since we still consider them to have high implantation potential.  

Human Egg

In the fifth week of pregnancy, a female fetus will develop a small structure called the genital ridge (which will evolve into the ovaries), colonized by special cells called primordial germ cells. Multiplying rapidly, these early cells eventually become eggs. About half way through the pregnancy there are about 7 million of these so-called primitive eggs. Their frenetic multiplication tapers off and actually declines down to 2-3 million eggs by the time of birth, the point at which scientists have assumed that a female has developed all the eggs that she will ever have.

Strangely, in competition with this process of germ cell multiplication, there is a remarkable course of cell death, which begins at about 16 weeks into gestation and continues unrelentingly until all the eggs are gone (typically when a woman is in her early 50′s). From the many millions of eggs at the time of birth, the reserve reduces to about 400,000 by puberty. During her lifetime, a woman will ovulate between three and four hundred eggs total. The rest die and are reabsorbed by the ovaries.

The reasons behind this early cell death have remained a mystery. But the facts are staggering. Before a girl even ovulates her first egg (at puberty), she has lost an average of 340 eggs per day since birth. After puberty, the rate averages out at about 25 per day. The rate of depletion then doubles at about age 37, ensuring that the egg supply is exhausted by the early 50’s. Evolutionary biologists have assumed that this is nature’s way of stopping a woman from having more babies than she can raise.

Now this entire foundation of knowledge regarding female egg production and depletion is in question. A recent paper by Johnson et al., (Nature, March11th 2004, Vol. 428, pp. 145-150) has opened up new doors for a drastic revision of the biological theory behind female egg production, and thus her fertility.

The research comes from the laboratory of Jonathan Tilly, an established and highly respected developmental biologist at Harvard Medical School. By studying mice, the researchers determined that germ cells persist after birth; and such stem cells give rise to new eggs throughout a mouse’s life. In a remarkably simple experiment, researchers quantified the rate of egg depletion in the mouse ovaries, and determined that the rate should have exhausted the egg supply much earlier. Yet an unidentified replenishment was taking place. Mice are normally fertile for about a year, but Tilly found that their egg supply would be exhausted in just 2 weeks if it were not somehow being replenished.

Tilly is confident that these same results will be found with human ovaries, despite significant differences in egg biology between the two species. If he is correct, many new therapies could evolve to preserve fertility and stave off menopause in omen. The germ cells for example, might be easier to preserve (freeze) than eggs, and women under-going ovarian irradiation as part of cancer treatment, which destroys eggs, could have their ovaries repopulated. Also, since the ovary creates estrogen-producing granulosa cells to surround each new egg, germ cell transplants could reverse the hormone withdrawal effects of menopause.

A tremendous amount of research will have to take place over many years before any of this conjecture starts to take shape. Meanwhile, one has to be cautious in assuming that human eggs behave like those of mice, because significant differences exist. Mouse eggs, for example, do not display the same age-related problems, such as increased rate of genetic abnormalities and decreased embryo implantation, which are evident in the eggs of older women. But the prospect of a replenishing egg supply is very exciting and provides hope for those of us trying to preserve fertility.