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.  

For many people, the dream of having a family also includes the dream of having children of both sex. Since most families today are much smaller than in generations past, the odds of having two or three or even four children of the same sex is fairly high.

Throughout human history, there always has been interest in methods to sway the chances of conceiving a child of a particular sex. Today, in the 21 st century, it is quite clear that many of these sometimes bizarre and sometimes simple home remedies have no basis in fact.

There are ways to significantly shift the odds of having a child of one sex or another. Sex is conferred on an embryo by whether an X-bearing sperm (for a girl) or a Y-bearing sperm (for a boy) enters the egg. Unfortunately, despite highly publicized claims, there are no proven effective “at home” methods of sperm separation. Nor does timing of intercourse relative to ovulation affect the 50:50 sex ratio. By natural methods, the ratio remains a flip of the coin.

The only commercially available method for sperm separation that appears to be effective is the sperm sorting process available through Microsort.net. This method involves using a fluorescent DNA dye that attaches to either X or Y chromosomes. The sperm then passes through a cell sorter that separates the sperm based on the fluorescence. This method is still under FDA investigation for safety and efficacy but does appear to do a reasonable job in separating sperm, especially if the desired sex is female.

Mirosort reports a 90% success rate with separating X-bearing sperm and a 73% success rate in separating Y-bearing sperm. There have been only a few hundred babies born thus far, but there does not appear to be any increase in birth defects. Because this process is still considered “experimental,” couples wishing to participate, will have to travel to either Fairfax, Virginia (Microsort headquarters) or an affiliated clinic in Southern California for fresh sperm insemination.

Unfortunately, after Microsort processing, the number of sperm available for insemination is severely decreased. Freezing and thawing of sperm, which would allow the sample to be shipped to another location, reduces these numbers even further. Because sperm counts are so low after sorting, it is usually necessary to do in vitro fertilization with sperm injection (IVF-ICSI) to significantly improve the fertilization in the IVF laboratory. PFC is a participating site in the FDA investigation for Microsort. We have used sperm specimens that had been previously Micro-sorted for IVF-ICSI.

Researchers at UC Irvine recently published a study describing the use of lasers to “trap” the heavier and slower moving X-bearing sperm to separate it from the lighter Y-bearing sperm. In the future, this process may provide an alternative to Microsort. However, it is not yet commercially available.

Beyond the Microsort technique, the only way to improve the odds of selecting one sex over another at close to 100% accuracy is to undergo Pre-Implantation Genetic Screening (PGS). PGS uses a DNA-binding technique to determine if there are a correct number of chromosomes in the embryo at the time of IVF. To complete this screening, embryos on Day 3 of culture (5-10 cells) undergo a biopsy to remove a single cell. The rest of the embryo remains in culture in the IVF laboratory. The removed cells are analyzed for the correct number of chromosomes. Currently, PFC with its cytogenetic partner, Genetics and IVF Institue screen embryos for 3-12 chromosomes. This screening is called “aneuploidy screening.” We allow our patients to know and select the sex of their normal embryos for transfer if they so wish.

Although IVF with PGS is the most effective method for sex selection, it is certainly the most expensive and there is no absolute guarantee that the transfer of the screened embryos will result in pregnancy. A PFC physician can best discuss the odds of success, based on the woman’s age and the couple’s history of childbirth.

Many couples undergoing PGS are doing so to screen for specific genetic defects or are specifically undergoing sex selection because of their risks of having a genetic disease that only affects males (X-linked diseases).

On the other hand, PGS for elective sex selection, either for “family balancing” or even for having a first child of a particular sex poses difficult ethical issues. Just because we have the ability to choose the sex of a child, should we? What will the couple do with normal embryos of the undesired sex? At PFC, we do not encourage PGS for elective sex selection. However, if a couple is undergoing IVF and wishes to undergo aneuploidy screening, we do allow them to select to transfer embryos by sex. We encourage all patients to consider donating excess embryos of the undesired sex for adoption by other couples.

Women or couples interested in this procedure should discuss it with their Reproductive Endocrinologist. At PFC, we also refer our PGS patients for a special genetic counseling session at California Pacific Medical Center in preparation for this process.

In vitro fertilization (IVF) is perhaps one of the most effective options available for the treatment of infertility. This procedure has been available in the developed world for approximately 30 years, and has been responsible for 1-4% of all conceptions. While IVF was originally developed for the treatment of female tubal factor, it has evolved to include treatment of male factor infertility via intra-cytoplasmic sperm injection (ICSI), as well as oocyte quality factor (Decreased Ovarian Reserve, or DOR). With the development of embryo biopsy techniques, IVF has also grown to incorporate pre-implantation genetic screening of embryos (PGD) to avoid genetic diseases in embryos and to screen for normal chromosomes. In the history of mankind, IVF will undoubtedly remain the greatest development for the treatment of human infertility for the foreseeable future.

Since the introduction of IVF, there has been a directly proportional increase in multiple gestation births. Traditionally IVF centers have measured success as the number of live births, irrespective of outcomes. This increase in multiple births is driven by the clinical incentives for live births, but some may also be driven by patient request. Two studies have shown that 20% of European and US infertile couples wanted a multiple birth^{(1, 2)}. Even after counseling regarding the risks of a multiple gestation, many patients still wanted to transfer 2 embryos. As IVF success rates have increased, and as the embryo freezing technologies have improved, a shift in the philosophy of IVF providers is occurring. Success rates are more likely to be measured as “live birth of a singleton (single baby) pregnancy”—in other words, “one healthy baby at a time”.

As the number of babies born after IVF has grown, there has been increased interest in looking at the pregnancy and birth outcomes in the successful IVF population. While potential complications for mother and babies are increased with any multiple gestation, there may also be an increased risk for complications even with IVF singleton babies. However, it may not be the IVF treatment itself that results in this increased risk for complications. The questions that reproductive endocrinologists and high-risk pregnancy specialists are trying to answer are primarily: 1) Is there a higher risk for a baby of any adverse birth outcome if that baby is conceived in an IVF laboratory? and 2) Is there something inherent about a past diagnosis of infertility which places even a singleton gestation at greater risk of pregnancy and birth complications?

IVF Singletons:

A number of large studies have addressed the question of increased risk to IVF babies ^(3-7). They echo a similar theme concerning birth outcomes, most importantly preterm birth <37 weeks, and low birth weight. One study compares the differences in degree of risk of poor outcomes with IVF babies vs. naturally-conceived babies. There appears to be a 93% increased risk for IVF singletons as compared to naturally conceived singletons, and a 57% increased risk for IVF twins versus naturally conceived twins⁽⁶⁾. Certainly the overall chance of a preterm delivery is much smaller for singletons than twins, and a twin pregnancy carries much greater risks overall.

A review of the US birth registry indicates that the proportion of IVF singleton babies born at full term with low-birth-weight is decreasing, but the proportion of IVF singleton babies born prior to full term with low-birth-weight is stable. In either case, the incidence of low-birth-weight is higher in babies born after IVF when compared with the general population. While outcomes of low-birth-weight babies may be getting better, there are still elevated risks for singleton low-birth-weight babies conceived via IVF.

For most of these studies looking at risks for IVF babies, factors known to influence pregnancy and birth outcomes are taken into consideration in the analysis. These important factors include maternal age and prior birth history. However, other factors may also be important but are not as well accounted for: factors such as previous poor obstetrical outcome, smoking status, socio-economic status, performance of fetal reduction procedure (especially for the analysis of the singleton data), types of ovarian stimulation protocols, media used in the IVF laboratory, and/or use of laboratory techniques (ICSI, etc.).

Infertility per se may itself be a risk factor for poorer pregnancy and birth outcomes. In an attempt to answer this important question, IVF outcomes have been compared with either non-IVF fertility treatments such as ovulation induction (OI) or to spontaneous conception outcomes. Numerous studies ^(8-15) have evaluated this question, and shown a higher risk of preterm birth for both IVF and OI babies as compared to spontaneously conceived singleton pregnancies. When evaluating outcomes for sub-fertile women (infertility for greater than 1 year) who spontaneously conceive, again we see a greater risk of preterm deliveries, obstetrical complications and adverse birth outcomes ^(16-18). These studies strongly suggest that there is an inherent characteristic of infertile patients which place them at greater risk of poorer pregnancy and birth outcomes. Whether this is due to uterine or embryo issues is not yet known.

IVF Twins:

Many studies have compared the outcomes for twins conceived via IVF versus spontaneous conception. These outcomes were summarized and reviewed in a meta-analysis of birth outcomes of IVF twins in studies up to 2003 ⁽¹⁹⁾. The specific findings showed an increase in the chances of a preterm birth (57% increase), admission to the neo-natal intensive care unit (two-fold increase), and Cesarean section delivery (33% increase). No other parameters were significantly different from spontaneously-conceived twins.

These differences between twin gestations conceived via IVF versus spontaneously-conceived twins were similar for cycles of twin gestation conceived via ovulation induction (OI). The rate of prematurity seemed to be higher for the IVF than OI group ⁽²⁰⁾.

In conclusion, when comparing singleton or twin gestations conceived via IVF or spontaneously, the degree of difference in the overall risk is greater for the singleton-baby births than twins. This is especially true with regards to preterm delivery which is increased two-fold in IVF singletons and by 40% (adjusted for age) in twins. While most studies have made adjustment for factors which can affect birth outcomes, such as maternal age, some other potential factors are difficult to measure, such as history of infertility or direct effects of IVF technology itself. It appears as though infertility prior to conception may play a larger role in IVF outcomes, for both singleton and twin gestations.  

1. Thurin A et al. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med 2004; 351:2392-2402.
2. Ryan GL et al. The desire of infertile patients for multiple births. Fertil Steril 2004; 81; 500-504.
3. Jackson RA et al. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004; 103; 551-563.
4. Helmerhorst FM et al. Perinatal outcomes of singletons and twins after assisted conceptions; a systematic review of controlled studies. BMJ 2004; 328; 261.
5. McGovern PG et al. Increased risk of preterm birth in singleton pregnancies resulting from in vitro fertilization-embryo transfer or gamete intrafallopian transfer: a meta-analysis. Fertil Steril 2004; 82; 1514-1520.
6. McDonald SD et al. Perinatal outcomes of singleton pregnancies achieved by in vitro fertilization: a systematic review and meta-analysis. J Ostet Gynaecol Can 2005; 27; 449-459.
7. Bower C et al. Assisted reproductive technologies and birth outcomes: overview of recent systematic reviews. Reprod Fertil Dev 2005; 17; 329-333.
8. French National IVF Registry. Analysis of 1986 to 1990 data. Fertil Steril 1993; 59; 587-95.
9. Frydman R et al. An obstetric assessment of the first 100 births from the in vitro program of Clamart, France. Am J Obstet Gynecol 1986; 154; 550.
10. McFaul P et al. An audit of obstetric outcome of 148 consecutive pregnancies from assisted conception: implication for neonatal services. Br J Obstet Gynecol 1993; 100; 820-5.
11. Tan S et al. Obstetric outcome of In vitro fertilization pregnancies compared with normally conceived pregnancies. Am J Obstet Gynecol 1992; 167; 778-84.
12. Wang JX et al. The obstetric outcome of singleton pregnancies following IVF/GIFT. Hum Reprod 1994; 9; 141-6.
13. Tanbo T et al. Obstetric outcome in singleton pregnancies after assisted reproduction. Obstet Gyncol 1995; 86; 188-92.
14. Rufat P et al. Task force report on the outcome of pregnancies and children conceived by in vitro fertilization (France 1987-1989). Fertil Steril 1994; 154; 550-5.
15. Friedler S et al. Births in Israel resulting from in vitro fertilization/embryo transfer. 1982-1989: National registry of the Israeli association for fertility research. Hum Reprod 1992; 7; 1159-63.
16. Basso O et al. Subfecundity and neonatal mortality: longitudinal study within the Danish national birth cohort. BMJ 2005; 330; 393-394.
17. Basso O et al. Infertility and preterm delivery, birthweight, and Caesarean section: a study within the Danish National Birth Cohort. Hum Reprod 2003; 18; 2478-2484.
18. Pandian Z et al. A review of unexplained infertility and obstetric outcome: a 10 year review. Hum Reprod 2001; 16; 2593-2597.
19. McDonald S et al. Perinatal outcomes of in vitro fertilization twins: a systematic review and meta-analysis. AM J Obstet Gynecol 2005; 193; 141-152.
20. Adler-Levy Y et al. Obstetric outcome of twin pregnancies conceived by in vitro fertilization and ovulation induction compared with those conceived spontaneously. Europ J Obstet Gynecol and Reprod Biol 2007; 133; 173-178.

Question: I am 35 years old and single, but am still hoping to find my life partner. I am getting a little concerned as my gynecologist has asked me about my plans for having children. She mentioned that I might want to consider freezing my eggs for future pregnancies. Is this something I should do?

Answer: Vitrification is a very new process for preserving unfertilized eggs. As noted in this month’s lead article, PFC has successfully been vitrifying oocytes from proven egg donors. Our first birth from this process occurred in October. Three additional pregnancies from this trial are ongoing. PFC undertook this vitrification trial in order to develop expertise with the technology of oocyte vitrification. For this reason, our study population was confined to donor eggs from healthy donors in their mid-twenties who had successfully completed conventional egg donation.

Why do we want to freeze eggs? For the many single young women diagnosed with cancer and facing fertility-threatening chemotherapy, egg vitrification will provide a fertility preservation option. This group of women has a compelling reason to consider undertaking the procedures and costs involved with in vitro fertilization. The potential threat to their ability to have their own biological children in the future may justify the unknowns that are involved with preserving their eggs in this manner. These unknowns include whether their eggs will survive the vitrification process and whether egg vitrification will ultimately prove to be as safe as conventional in vitro fertilization and embryo cryopreservation. The answers to these questions may not be answered until the patient’s eggs are warmed, fertilized and implanted, which may be years later.

We recognize that a much broader spectrum of the population will look upon oocyte vitrification as a way for women to preserve their fertility. Single women, such as you, who have not yet met their life partner, may be particularly interested in this option. In addition, it may also become an option for women in their 30’s who wish or need to delay their childbearing.

Many questions remain unanswered. Will eggs from women in their 30’s do as well as eggs from proven egg donors in their 20’s? Logic suggests older eggs will not do as well, but will the differences be significant? How many eggs would a woman need to preserve in order to have a reasonable chance for one or two children in the future? How many IVF cycles will that take? Is it safe to rely on these preserved eggs? Would having preserved eggs change a woman’s approach to reproductive planning in her life?

These are not trivial issues. They are important, life-changing concerns. For these reasons, we are not encouraging single women to prematurely jump on the egg vitrification bandwagon. Stay tuned. This area is changing rapidly.

Dr. Carolyn Givens

Question: I am 38 years old with age-related infertility (at least that is what my doctor, a Reproductive Endocrinology and Infertility Specialist (REI), thinks). It has been suggested that I undergo super-ovulation with injectable Follicle Stimulating Hormone (FSH) along with intrauterine insemination. I really don’t want to have twins, if possible, and certainly not triplets or more! But ideally, I would like to have more than one child. Even if I am successful in having one baby now, I am worried about trying to have a second child when I am 40 or more. What do you suggest?

Answer: We agree that having one baby at a time is the safest thing for you and your family. However, undergoing FSH super-ovulation is intended to create more eggs in one cycle in order to increase the odds that one or two will fertilize and implant. This helps to overcome the relative inefficiency of conception for women in their late 30’s. The risks are as you stated, twins or more. Luckily, the risks that a woman undergoing this treatment will get triplets or more is really fairly low – on the order of less than 10% of all pregnancies, with careful monitoring. The risk of twins is higher – on the order of 20% of such pregnancies.

If a woman at 38 years old has no identifiable cause for infertility, the goal is usually to get 3-6 follicles. Most of the time, if the treatment is successful, the pregnancy will be a singleton pregnancy (one baby). Your issue of wanting to have a second child and concern for difficulties beyond age 40 is a real one. You may want to discuss with your REI the option of in vitro fertilization. If your doctor thinks you may be a good responder to fertility medications, you could have extra embryos to freeze, which provides some back-up and allows you to preserve some embryos from 38 year old eggs for down the road.

Patients contemplating conception must consider lifespan expectations as part of their decision on whether to conceive. Such considerations are not, however, a reason to withhold treatment, and are ultimately the individual and family should decide.

– Dr. Carolyn Givens

Fresh Embryo at Blastocyst Stage: The cells are elongated and pressed against one another. The inner and outer cells are clearly visible, as is the cavity.

Two Vitrified Embryos at Blastocyst Stage After Warming: Though their appearances differ, both embryos implanted and created viable pregnancies.

This embryo looks perfect, as if it was never frozen. The outer and inner cells are clearly visible, as is the cavity.

This embryo has rounded, more dissociated cells resulting from shrinkage during incubation in cryoprotectant, (as cells shrink they pull away from each other). The cavity is small, but visible.

The first human pregnancy from an embryo that had been frozen and thawed was achieved in Australia in 1984, 6 years after the birth of the first IVF baby in the UK. The method used to preserve that embryo is called “slow freezing” and it is still the preferred method for preserving embryos throughout the world today. Slow freezing is a reliable and established technique that has served the IVF community well for over 20 years. The procedure has been refined throughout those years and it works, with slight modifications, for freezing all embryo stages and for sperm. However, despite many years of trying, slow freezing has never worked very successfully with oocytes. Frustrated by years of failures, scientists turned to an alternative procedure called vitrification in their quest to preserve oocytes. This approach is relatively new, but appears as through it will be preferentially used for oocyte preservation as we go forward. Vitrification kits are just beginning to get FDA clearance following scientific trials, and embryologists are being trained in the use of the new technology.

The main concern during the freezing of any cell is the removal of water without actually killing the cell. Since water expands in volume as it freezes, ice formation inside a cell would cause the cell to rupture and die. Therefore, cell water is traditionally replaced with a cryoprotectant (antifreeze) prior to cooling of the cell. This is achieved by sequentially incubating the cell in increasing concentrations of cryoprotectant. The cryoprotectant draws water out of the cell and itself enters the cell, all by osmosis. Once most of the water has been removed, the cell is cooled at the very slow rate of -0.3° C/minute until it has been cooled to below -30° C and is therefore fully frozen. Thereafter, storage of frozen cells is in liquid nitrogen (-196° C), which is a simple and practical storage medium.

Vitrification still requires the use of cryoprotectants and the cell is also ultimately stored in liquid nitrogen, but the journey from the incubator (at 37° C) to the nitrogen (-196° C) is much faster. The word “vitrum” in Medieval Latin means “glass” and the process turns the cell contents to a glass like substance instead of ice. Since no ice forms, the risk of rupturing the cell is eliminated. For glass to form instead of ice, the rate of cooling must be thousands of degrees per minute instead of the 0.3 degrees/minute that we use in slow freezing. Therefore, the process is sometimes referred to as ultra-rapid freezing, although the word “freezing” is really inappropriate here since the cell is not really frozen (i.e. no ice is created).

One of the big stumbling blocks during oocyte freezing was the sheer size of the cell (the oocyte is the largest human cell by some margin) and therefore its high water content. Just getting the cell to survive, (an oocyte has only one cell), was a huge stumbling block. Studies where 50-60% of the oocytes survived were considered groundbreaking, and still today there are few studies that have done better. Vitrification as a technique had been largely ignored by the IVF community as it was technically more challenging and used much higher concentrations of cryoprotectants. Cryoprotectants were thought to be toxic to cells. Today we know that they are safe and effective and do not contribute to cell death. It is possible that cryoprotectants may have deleterious effects on cells if they are metabolized, but virtually all freezing protocols utilize them at room temperature or below, where cell metabolism is significantly slowed or stopped. So, with success rates using traditional slow freezing failing to improve, vitrification has been given serious consideration as an alternative. In the few years since its introduction, vitrification has shown promising and excellent results in clinical studies (see Oktay et al., Fertility and Sterility, 2006, Vol 86(1), pages 70-80 a comparative review of slow freezing and vitrification results with human oocytes).

Making the transition from slow freezing to vitrification has been a challenge for the IVF community. As already stated, it is a technically challenging procedure, and training of embryologists in the technique has been slow. With slow freezing, embryos are placed in relatively weak solutions of cryoprotectant for as long as 15 minutes at a time. Then, they are usually moved on through slightly stronger solutions before being placed in large straws or vials which are then loaded into a computer controlled freezer for the long journey to -30° C. The embryologist can spend about 30 minutes with a set of embryos from the time that they come out of the incubator until they go into the controlled rate freezer. After 2 or more hours, the embryos can be placed in liquid nitrogen and the process is complete.

During a vitrification procedure, where typically only one oocyte or embryo can be worked on at a time, the transition from incubator to nitrogen takes only a few minutes. The embryo is stepped through solutions containing high and then higher concentrations of cryoprotectants where it shrivels and swirls in the extremely viscous medium. In the final stage, which is measured in seconds, the embryo is placed in an extremely concentrated cryoprotectant solution and then quickly loaded up into a tiny straw that is barely larger than the embryo itself. The straw is then sealed at both ends and plunged immediately into liquid nitrogen. The straw is so fine that it freezes in an instant, an important part of the vitrification process. The loading of the straw occurs at room temperature (25º C in the IVF lab) and it is cooled to -196º C in one or two seconds, giving a cooling rate of 6000-13000º C/min. The faster the straw can be cooled, the more successful the procedure. Performing this final step too slowly or too quickly can be the difference between success and failure and therefore requires extensive training.

At Pacific Fertility Center, we have been working on vitrification for over 2 years. Our initial interest was in oocyte freezing, but we were also interested in extending the technique to be used with embryos, and in particular to blastocyst stage embryos where slow freezing has not always worked well. Slow freezing has served us well over the years for embryos being frozen 1, 2 or 3 days after an oocyte retrieval, but blastocysts (5 or 6 day old embryos) did less well. With an industry wide transition to blastocyst stage embryo transfers, we looked at vitrification as an alternative method of preservation for these precious embryos.

A blastocyst is an embryo that has developed to the stage where it is ready to implant in the uterus. Instead of having a small number of loosely associated cells characteristic of earlier embryonic stages, it has 2 defined cell populations and a fluid filled cavity (or cyst). The cells that surround the cavity will form the placenta, and the cells within the cavity will develop into the embryo proper, or fetus and some of the extraembryonic membranes, such as the yolk sac. It is these interior cells that cause trouble during freezing since they are on the inside and difficult to expose to cryoprotectant. Slow freezing relies on cryoprotectant traveling through the outer placental cells, then the cavity, and finally into the fetal cells while water travels in the opposite direction. Fully dehydrating these fetal cells has always been a challenge and an embryo where these cells do not survive freezing and thawing will not result in a viable pregnancy. And with slow freezing, embryos tend to collapse in on themselves during dehydration, making it difficult to assess survival after thawing.

After investing heavily in vitrification training and implementing a successful oocyte vitrification program, PFC began working on blastocyst vitrification in January of 2007. By March we had a program established and were delighted by how easily blastocysts seemed to tolerate the procedure. Often, blastocysts looked no different after vitrification when compared to how they looked before the procedure. This result was in stark contrast to slow freezing where blastocysts always look shriveled and deflated after coming out of the freezer. By July 2007, we had switched completely to vitrification and currently we are enjoying the successes that it is bringing to our patients and us.

Our vitrification team consists of 3 embryologists: Mariluz Branch, the team leader, with Erin Fischer and Liz Holmes. Because of the technical challenges involved, we have to be cautious with involving other embryologists. So one of the three team members must be on duty every day (our lab is open 7 days a week). I am grateful to this team for their flexibility in accommodating our needs. By the end of the year we expect to have 2 more embryologists on the team, and then the final 3 in 2008.

Vitrification has been an exciting and challenging technique which we have embraced and conquered in 2007. We look forward to the gradual elimination of slow freezing and the successes that vitrification will bring us in the future.

Joe Conaghan, PhD, HCLD

Question: How do I decide which one of your “Shared Risk” plans is best for me?

Answer: It is estimated that there are approximately 2 million infertile patients in the US. Approximately 1.5 million will seek treatment. Of these, about 750 K conceive with standard treatment and 750 K remain infertile. Of the 750 K who remain infertile, 50 K adopt, 50 K undergo IVF, and 650 K drop out of treatment. Only about ¼ to ¹/3 of infertile patients see a Reproductive Endocrinologist; most patients are only treated by gynecologists. The remaining ¹/3 don’t seek treatment because they believe that they can’t afford it, and ¹/3 don’t proceed to adequate treatment secondarily to financial barriers.

Given these daunting statistics, we understand that it is important for IVF clinics to help maximize access to all levels of fertility care, by sharing the financial burden and the risk of an IVF cycle. With these goals in mind, PFC offers two “shared risk” financial plans to our patients. We call our two plans: the Refund Plan and the Option 2 Plan.

When engaged with you in a shared risk plan, we are indicating that if you meet the appropriate medical criteria, we feel confident that we have a reasonable chance to help you achieve your goal of parenthood, and we are therefore willing to share in the financial risk associated with IVF. Sharing in the financial risk of treatment is a statement of confidence in our ability to help you overcome infertility.

As physicians, we are often asked by our patients to recommend a financial plan. Each patient’s case is different, and choosing the best plan for you has to do with your own comfort level in taking financial risk, dovetailed with your emotional comfort and commitment to treatment. These levels of comfort can only be addressed on a personal and individual level.

Some general questions to consider in analyzing the plans are the following:

• • What are your estimated chances of success with your fresh embryo transfer?
• • What are the chances of having any frozen embryos?
• • What are your estimated chances of success with your frozen embryos?
• • If you are not pregnant with this IVF cycle, would you want to proceed to another IVF cycle?
• • If you do not become pregnant after a treatment cycle, would you rather receive a refund and then decide on the next steps for treatment?

Depending on the answers to these questions, you can begin to define which financial plan may make the most sense. For patients who want some financial buffer if their cycle is not successful, the Refund Plan may be the best choice. It allows you to receive a refund and then decide if the next steps will be to do nothing more, to continue treatment, or to use the refund for other options such as adoption. For those who are ready to commit to a second IVF cycle attempt if the first cycle is not successful, the Option 2 Plan may be the preferred choice. This plan provides the option of 2 cycles for only a little more cost than a single cycle. In general, the Option 2 Plan is for those whose chances of having frozen embryos is less—making the probability of needing a second fresh IVF cycle greater. At PFC, we have two financial coordinators dedicated to providing all the information about costs which can then help you to make these important decisions.

As in any financial decision, there is no right or wrong answer, but only an answer which best fits your particular situation. This decision is then matched with your personal comfort level in sharing risk. For some patients, neither of these plans may seem appealing, and the Single Cycle Plan (pay for services as provided) will be most appropriate. For patients with insurance coverage for IVF, the Single Cycle plan is the only option.

While Pacific Fertility Center can not choose a financial plan for you, if you need more information about your specific fertility situation before deciding on a “shared risk” plan, please do not hesitate to discuss this with your physician or your financial coordinator.

– Dr. Isabelle Ryan

The American Society for Reproductive Medicine (ASRM) is the largest organization in the United States for medical professionals in the field of Reproductive Endocrinology and Infertility. In 2002, ASRM published medical and psychological guidelines for ovum donation. The psychological recommendations for donors are general guidelines for addressing moral, ethical and psychosocial issues that may confront ovum donors. Included are standards for what should be included in a psychosocial screening of donors and reasons to exclude donors. A few of the objective reasons for donor exclusion include known substance abuse, positive family history of heritable psychiatric disorders such as schizophrenia or bipolar disorder, or instability in donors’ lives. To determine “inclusion” I take a psychosocial history and administer a psychological test that informs me about the donor’s personality profile, including just how much they are trying to impress me – the “fake good” factor. I am also assessing motivation to donate and the donor’s “need” versus “desire for” the compensation. Stability, stress levels, and reproductive history are also part of an ASRM assessment of donors.

Although I would like to base my entire decision on objective information, much of my decision on donor acceptance must, in fact, be based on intuition. Throughout my 25 years as a therapist, I have found that my intuition is quite accurate, but it is not fool proof.

There is one major controversy in the field that may hinder a psychotherapist’s ability to screen donors. That is the hotly debated topic on compensation of donors. In August 2000, the Ethics Committee of The American Society for Reproductive Medicine concluded that there is no consensus on the precise payment that oocyte donors should receive. It was suggested, “sums of $5,000 or more require justification and sums above $10,000 go beyond what is appropriate.” Due to costs of living and the scarcity of available donors, there are significant regional variations that affect these rates.

The Society for Assisted Reproductive Technology (SART) has attempted to assist clinics and patients by creating a list of Egg Donor Agencies that have signed an agreement stating that they will abide by the Ethics Committee Guidelines governing the compensation of egg donors.

The debate centers on the fact that donors could feel undue inducement and exploitation in the process if the fee is so high as to be coercive. Part of the job of the mental health professional is to provide donors with informed consent. Might donors conceal pertinent medical information that could be important for themselves or offspring if the monetary incentive is so high? Will donors discount risks to themselves? How can the donor actually give informed consent about the medical procedure and pay attention to the risks if $$ signs are floating before their eyes?

Ethicists and some in the field of women’s health advocacy express concern “that lucrative payments are enticing young women with credit-card debt and steep tuition bills to sell eggs without seriously evaluating the risks.” Can the payment cloud someone’s judgment and can we assess that? How much is too much? Where is that line?

When I began working with PFC in 1998, we were paying first time egg donors $2,500. Nine years later, the compensation is $6,500 for a first time donor and $7,000 for any subsequent donations. Even adjusted for inflation, the payment is rising at a phenomenal rate. Competition, supply and demand govern these rises.

As part of my objective/intuitive approach to interviewing donors, I discuss money with them. What would she use the compensation for? How does she support herself? What kind and how much debt does she have? While $25,000 in student loans points to a person’s drive toward positive goals, the same amount in credit card debt speaks to me of impulsive behavior. How much have they educated themselves on ovum donation? Whom have they talked with about their desires? Do they have their own children? Are they science majors who may be more likely to view gametes as DNA and not their own children? Are their answers well thought out?

My goal is to provide the recipients of donated eggs with intelligent, healthy, and thoughtful young women who understand the implications of ovum donation both for themselves and for the recipients. Donors who are motivated by the “need” for money are more likely to provide inaccurate information on their applications, or leave out information that could be pertinent to the recipients or their offspring. It is my job and the job of the Egg Donor Agency to determine motivation.

Although impossible to attain, we would all like donors to be motivated altruistically. We may diminish altruism by making the donation about eggs for money. However, I believe we can increase altruism by helping the donors to have a greater understanding of who the recipients are and what their struggles have been.

Peggy Orlin, MFT

Ms. Orlin served as 2006-7 chair of ASRM’s Executive Committee of the Mental Health Professional Group, is a member of Resolve’s National Mental Health Advisory Board. She co-teaches PFC’s Mind/Body workshops.

To register for the September 8th Mind/Body@PFC Workshop, please phone 415-834-3095.

Question: I am an OB/GYN in the bay area and I have a patient that is interested in having a baby girl. She asked about “sperm spinning” as a method of gender selection and whether it would be useful in her situation.

Answer: Our office receives a lot of questions from patients and members of the public about sex selection. Our location in the very liberal San Francisco may be cause for the increasing demand we see in having a baby of a predetermined gender. People are also well informed about what can be achieved with modern technology, and since sex selection is a reality, there’s definite demand for it.

The procedure that you ask about, “sperm spinning” is better known in the medical and scientific communities as the “Ericsson Method”. The technology was developed by the German scientist Dr. Ronald Ericsson and has been licensed in the US and internationally since the early 1970′s. It takes advantage of the fact that sperm bearing a Y chromosome (that would make a boy) are very slightly lighter than X-chromosome bearing sperm (that would make a girl). The distribution of X and Y bearing sperm in a normal sperm sample is equal, but Ericsson’s method uses gentle centrifugation of sperm through a slightly viscous fluid to segregate the heavier (girl) sperm from the lighter (boy) sperm. Since the difference in the weight of the 2 types is so slight (about a 3% difference in amount of DNA), a perfect separation cannot be achieved. Ericsson’s website (www.childselect.com) claims a 78-85% success rate in couples seeking a boy and a 73-75% success rate for girls. At PFC, we do not endorse or recommend this method of sex selection, nor can we verify the above success rates. As far as we know, couples availing of sperm spinning are not given details of how well purified their samples are prior to using them for insemination.

A more reliable method for separating sperm in our opinion is the “Microsort” technique offered at the Genetics and IVF Institute (www.givf.com) in Fairfax, Virginia. The technique was developed originally by Dr. Lawrence Johnson at the US Department of Agriculture, and was later refined for use in humans in collaboration with GIVF. Microsort also takes advantage of the small difference in DNA content between “boy” and “girl” sperm. The sperm are dyed with a stain that binds to DNA and then an instrument called a flow cytometer can effectively separate populations of sperm based on how much dye they have incorporated. The Microsort scientists test a small aliquot of every separated sample to determine the exact enrichment that they have achieved. According to the latest figures posted on their website (microsort.net) the average enrichment for X-bearing sperm is 88% with 91% (525/574) of babies born being female. The technique is less effective for Y-bearing sperm with an average sample purity of 73% and 76% (127/152) of babies born being male. Bear in mind that the figures for babies born might be distorted since some patients may have terminated pregnancies that were not the gender that they were seeking. You may also have noticed from the GIVF data that there’s more demand for girls than boys. This is likely due at least in part to the fact that X separations work much better and therefore may be used more, but Dr. Ericsson’s website also claims a much stronger female demand even though his technology supposedly works better for boys. We do support the use of Microsort sperm here at PFC but there are limitations on the use of this technology. First, the sperm can only be separated in 2 laboratories in the US, (Fairfax and Huntington Beach in southern California), and the Microsort researchers prefer that you attend in person to give a fresh sperm sample. Second, the technology is currently only offered under an FDA approved clinical trial, and you have to be doing family balancing or trying to avoid a sex-linked disease in your family to be enrolled. For most people, unless you already have a child of a different gender from the one you are seeking, you won’t be able to participate in this FDA study.

Last, but not least is preimplantation genetic screening (PGS) that can be used to tell the sex of embryos created during in vitro fertilization (IVF). We feel that this technology is the most accurate of the sex determining strategies since there’s less than a 3% chance of a misdiagnosis. Embryos generated in an IVF cycle are subject to a biopsy procedure on the third day of growth that allows a single cell from the embryo to be analyzed to see if it has 2 X chromosomes (female) or X and a Y chromosome (male). IVF with PGS is the most accurate method for sex selection, but also the most involved and the most expensive. The Ericsson method is the easiest and the cheapest, but carries a greater risk of being inaccurate.

Joe Conaghan, PhD