This year Pacific Fertility Center is undergoing a much anticipated structural and design change. The clinic located on the fifth floor at 55 Francisco Street was purchased in 1999 from a previous owner, and after 8 very busy years, we are ready for a facelift. The opportunity to renovate our space came last month when PFC was offered an adjacent suite vacated by another tenant. The suite, adjacent to our laboratory, was perfect for an expansion that we had been considering for some time.

While the additional space is a bonus, the major benefit of the expansion is the ability to install a state-of-the-art air handling and cleaning system for the laboratory. With the addition of special air-lock doors, the laboratory will have improved separation from the rest of the building, ensuring the highest air quality. The air inside the laboratory will have an updated purification system to remove all particles and chemicals. These combined upgrades will further protect the laboratory from outside environmental influences.

The driving force behind the current construction is the air purification system, and isolation of the lab. However with this opportunity we are also expanding our laboratory space, purchasing new equipment, redecorating and painting. In the past, our laboratory was closed for several weeks at the end of each year to allow for major maintenance, equipment servicing, and cleaning related to normal wear and tear. The use of volatile paint and certain cleaning products is prohibited during the year on the entire fifth floor to protect the delicate growing embryos. The cleaning products normally used throughout the year, are ones we know do not affect our embryos.

We do have many other noticeable upgrades planned. We are taking this opportunity to update the rest of our center. The front reception and lobby are undergoing a significant renovation, as are our exam rooms and some of our offices.

All materials being used in the remodel are organic and toxin free. PFC continues to promote a safe and clean environment. Special paints will be used that are plant derived and free of volatile organic compounds (VOC’s). The chairs and furniture we purchase will also be VOC free. New carpets and flooring are already laid out in warehouses so that any chemicals used in their manufacture can dissipate before being installed.

As a further precaution, to ensure that all furniture, equipment, flooring and paint are guaranteed VOC free, we’re going to “cook-off” the lab by raising the temperature to about 120 0F continuously for 4 days prior to reopening. The high temperatures drive out any residual VOC’s and chemicals which will then be removed by the charcoal and potassium permanganate filters in the air handling system.

All of these changes will cause some minor disruption in our office during the next few weeks. To minimize the amount of disturbance we have construction crews working around the clock. In addition, we have dedicated some of our staff to assist patients in navigating the new space and construction areas.

Pacific Fertility Center is making a clean start. We look forward to welcoming you to our newly renovated clinic. We anticipate even greater success in the years to come. Thank you for your understanding. Please let us know if we can assist you in any way during this time of transition.

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.  

Question: I am an educator for a human sexuality class. A student asked me an interesting question that I was unsure how to answer. Given that we know sperm can survive about 72 hours in a woman’s body, how is it possible to keep sperm viable by freezing them?

Answer: Sperm can survive for a long time under the right circumstances. In a woman’s body we think that 72 hours is approximately correct, but the data supporting this estimate is not conclusive. In the lab, sperm can live 5 days or more provided they are removed from the seminal fluid and placed in a more nurturing environment. Seminal fluid contains many enzymes that first clot and then liquefy. This change in the fluid allows the ejaculated sperm to stay in the vagina initially, but then swim out as the seminal fluid becomes more liquid. These enzymes quickly destroy any sperm that can’t swim out of the semen within a few hours.

It takes approximately 72 days for sperm to mature in the body. During the last 14 days of this process, the sperm are very much alive and swimming. They are alive a long time prior to leaving a man’s body.

During freezing, sperm are cooled to a very low sub zero temperature (minus 196 degrees Centigrade). At that temperature, all biological activity is effectively stopped. The sperm cells are not metabolizing or depleting their energy reserves. They are truly in suspended animation. Bacteria or other microbes cannot attack or degrade the sperm in any way because they are also unable to function at such a low temperature. Everything is on hold.

Biologists believe that correctly frozen cells in long term storage can literally last forever, as long as the temperature is properly maintained. It is believed that constant exposure to normal levels of background radiation is the only thing that could cause loss of viability and this effect is difficult to measure. Studies done in the 1970’s, exposing frozen mouse embryos to the equivalent of 2,000 years of background radiation, showed no measurable mutagenic effects in offspring.

Cryobiology is a relatively new science, and human fertility treatments are newer still. Consequently, in humans there are no long term results with frozen sperm or embryos. There are a handful of reports showing babies born from embryos that had been frozen for 12-15 years. A couple in New York had a child in 2005 from sperm that had been stored for 28 years. Sperm frozen for domestic animal species have a longer record because samples frozen in the 1950’s are still viable.

The process used for freezing is very precise and works best when cells exist individually (such as sperm) or in very small groups (such as an embryo). Larger masses of cells, tissues or even whole bodies cannot be frozen and subsequently thawed alive. It is not currently possible to freeze and thaw a whole ovary or kidney.

To successfully freeze cells we must remove cell water (water expansion during freezing would burst the cell) and replace the water inside the cell with antifreeze. This is done by incubating the cells in a solution of antifreeze. The water and antifreeze swap places through the process of simple osmosis. In a complex tissue like an ovary, there is no way to get all the water out of all of the cells so easily, thus a whole ovary cannot be frozen. If the ovary is chopped up into tiny pieces however, more water can be extracted. Some success has been reported with freezing ovarian pieces in this way.

The following student experiment demonstrates the challenges of freezing. Place a whole peach into your freezer for 24 hours and then thaw it out and see what a mess you have. If however you slice the peach up and mix the slices with sugar for 15 minutes (the sugar will draw out water from the cells), you can freeze the peach quite successfully. If the technology is used correctly, you can keep your peach (or your sperm) for leaner times.

Joe Conaghan, PhD, HCLD

While it has been possible to preserve sperm for many years (the famed Dutch microscopist Anton von Leeuwenhoek allegedly cooled and then recovered sperm using snow and ice in the 17th century), reliable methods for oocyte preservation have been elusive.

We previously discussed some of the problems with oocyte freezing (see Fertility Flash, January 2005, Volume 3, Issue 1), and now report our success in overcoming some of the obstacles.

Traditionally, preservation of sperm and embryos has been achieved with the use of a technique called slow freezing. This process incubates the sperm or embryos in low concentrations of cryoprotectants (antifreeze) to draw water out of the cells. After this incubation, they are cooled very slowly to sub zero temperatures. Typically this slow freezing technology just works for cells that exist individually (such as sperm), or together in small numbers (embryos), as the water must be extracted from every cell. Tissues, which are made up of many hundreds of thousands of cells, cannot be dehydrated successfully and therefore cannot be frozen intact. Cells in the tissue can burst when the water remaining in the cells expands as it turns to ice. For example, it is not possible to freeze a whole ovary, but some success has been achieved with ovaries that were cut into tiny pieces.

Frustrated by the lack of progress with slow freezing, scientists have more recently moved towards a technology called vitrification for oocyte preservation. Vitrification, which was described in detail in September’s Fertility Flash (Volume 5, Issue 8 ) works by using higher concentrations of cryoprotectants and much faster cooling rates. Cells are typically cooled in tiny straws (see article heading). This process allows us to achieve cooling rates of several thousand degrees per minute.

When vitrification straws and cryoprotectants were first approved by the FDA for human embryos, PFC began the process of adapting the technology to oocytes. Our embryologists attended training courses and became proficient with the technology by practicing on mouse and hamster oocytes and embryos. Even though we have been handling oocytes and embryos for many years, this technology provided many new challenges, mainly due to the tiny size of the straws and the speed at which the cells had to be cooled. Once we became proficient with the procedure, we began to freeze high quality oocytes from donors who had proven fertility. In this way, we knew that if the procedure did not work, it would be the vitrification technology and not the oocytes that were to blame. In addition, we satisfied ourselves that the technology was safe by looking at the exhaustive work by Dr. Gary Smith at the University of Michigan, which showed that vitrified/warmed oocytes were both physically and genetically normal and that the resulting pregnancies and babies were healthy.

We recruited five oocyte donors and vitrified all of their oocytes immediately after their oocyte retrieval procedures. We then offered the oocytes to individuals who were on our waiting list to accept donated embryos. Typically, these individuals were unable to get pregnant with their own oocytes or financially unable to proceed to an egg donor cycle. The availability of the vitrified oocytes was a great alternative to accepting donated embryos as it allowed couples to choose their own sperm source. Furthermore, the immediate availability of vitrified oocytes was an attractive alternative to what may be a very long wait for donated embryos.

Pacific Fertility Center had immediate success with the first recipient. We had vitrified 16 oocytes from the first donor, and for the first recipient we warmed only 7 of these. Four hours later we injected a single sperm into each of the 6 oocytes that appeared alive and healthy (1 oocyte had not come through the process successfully). The next morning, 3 of the oocytes fertilized normally. After 2 more days, we had 3 nice embryos for transfer. The positive pregnancy test 11 days later, and a singleton pregnancy confirmed by ultrasound at 7 weeks were great rewards for our efforts and thrilling news for the recipient. Our second recipient used a different donor and although her pregnancy started out well, she miscarried in the first trimester. Our disappointment over this loss was compounded when we discovered the oocytes from 2 of the donors did not survive well when warmed. In these particular donors, we recovered high numbers of oocytes (each had close to 40) and for unknown reasons their oocytes were overly sensitive to vitrification. The next three donor cycles proceeded well and resulted in pregnancies. These 3 pregnancies are all ongoing at the time of writing. We will update readers with their outcomes at a future date.

Although we were warming relatively small numbers of oocytes (typically 6 or 7), we began to have more embryos than could be safely transferred to recipients. Our first pregnancy had been achieved after transferring 3 embryos. It is more typical, however, to transfer only 1 or 2 embryos when donor oocytes are used. Even when using only 2 embryos, multiple pregnancy rates were unacceptably high. Understandably, few patients are willing to risk a decreased chance of conceiving by transferring only a single embryo. In order to avoid high multiple pregnancy rates in a typical IVF cycle, embryos are usually cultured for 5 days to determine which embryos in a cohort have the best chance of establishing a pregnancy. However, if a patient has only a few embryos, the benefits of extended culture are less, and the transfer is typically done after only 3 days growth. With our recipients of the vitrified oocytes, we began by doing 3-day transfers. Once high success rates were evident, we elected to implement day-5 transfers, in an effort to decrease high order multiples. The last 2 pregnancies both resulted from day-5 transfers of 2 embryos each, and they are both twin gestations.

In summary, we have had 7 out of 10 embryos implant after transfer (excluding the 2 failed donors with the high oocyte numbers). This implantation rate (70%) is comparable to the implantation rates that our patients have when using fresh embryos from donor oocytes.

We are moving forward cautiously with our oocyte vitrification program and hope to use the remaining oocytes soon. While these results are encouraging and have brought great joy to a small number of our patients, there are more issues to resolve before we declare complete success. The 70% success rate was obtained with the use of the highest quality oocytes from young donors who were known to be fertile and healthy. We have already seen that some oocytes are less tolerant of the procedure, as evidenced by the results from the 2 donors with high oocyte numbers. We also fully anticipate that the results for older women using their own oocytes will be worse, as they are for these same patients using a fresh IVF cycle. In fact, at this time, we do not have any idea if the oocytes from women in their 30’s will be able to tolerate vitrification.

Going forward, we will offer oocyte vitrification unconditionally to women with cancer who are likely to be left sterile by their treatment. For these women, and for others who elect to vitrify oocytes for social reasons, we will exercise great caution in our estimates of future pregnancy potential with the warmed oocytes. Until we have more data with oocytes from a variety of women, we will have no way of telling if there is any hope from anything other than donor oocytes. That data will accumulate more slowly because women who elect to preserve oocytes are not likely to be using them for some time. For now, until there is more data, we continue to believe that embryo freezing has the greatest potential for those wishing to preserve future fertility. However, for those who are single and in their late 30’s, we will be reluctant to recommend oocyte vitrification as a reliable fertility preservation method. Hopefully, they will find Mr. Right before we have objective data.

Joe Conaghan, PhD, HCLD

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: 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

Many patients receiving medical care for infertility will use cryopreserved (frozen) sperm, oocytes and/or embryos at some time during their treatment. Here in the PFC laboratory, we routinely cryopreserve sperm and embryos. We also receive specimens from sperm banks nearly every day. All of these specimens are stored on-site in our secure tanks with continuous monitoring. All specimens are stored in liquid nitrogen at -196ºC. Movement in or out of the tanks only happens when specimens are transferred post freezing or retrieved for thawing or shipping. We store sperm and embryos for our patients for an annual fee as long as we are able to maintain yearly contact with them and the annual storage agreement is renewed.

The shipping of tissues that are frozen and stored at such a low temperature is not easily accomplished. The liquid nitrogen in which they are stored is not toxic in any way, but it is extremely dangerous and can cause serious injury and even death if not handled properly.

In attempting to transport tissues that are normally stored in liquid nitrogen, we have to use a device that will keep the tissues in their same deep frozen state. This is accomplished using a “Dewar” which resembles a large thermos. A Dewar is a vacuum insulated container, mostly filled with an absorbent lining that soaks up liquid nitrogen. The Dewar is “charged” prior to use by filling it with liquid nitrogen over successive days until it will absorb no more. Once saturated, the excess liquid is poured off and the Dewar is then ready for use. Specimens are loaded into the hollow core and they are maintained in their frozen state by the cold nitrogen vapor evaporating from the surrounding absorbent layer. The Dewar holds an appropriate temperature for as long as nitrogen remains inside. Loss of nitrogen by evaporation happens continuously. Typically a fully charged Dewar maintains temperature for between 7 and 30 days depending on its size, how often it is opened and how well it was charged before use. With any Dewar however, loss of refrigeration occurs after a certain period of time, unless more nitrogen is added. In addition, dropping the Dewar or otherwise damaging it in any way can crack the container and this will result in instant failure of the vacuum seal with subsequent loss of nitrogen and thawing of the contents.

When we receive a shipment of sperm from a bank, there is always a risk that the Dewar was damaged or that there was a shipping delay that was longer than the life of the liquid nitrogen in the tank. If the specimens have thawed, typically the sperm bank will replace them at no cost. However, their liability is limited to replacing the sperm, and if you just lost the last 3 vials of your favorite donor, you’ll have to choose a new donor.

Shipping of embryos is a much more risky proposition. Embryos can’t be replaced in the same way that a sperm sample can be replaced, if they can be replaced at all. The major shipping companies such as FEDEX, UPS and DHL will not knowingly accept embryos for transport and therefore would not have any liability for loss. At PFC we discourage shipment of embryos due to the risks involved. We will not ship embryos from our laboratory on your behalf, however you can come and collect your embryos in person and ship them yourself. We will ask you to sign papers releasing us of any liability once the embryos leave our office. We cannot accept any liability for embryos that are being shipped in from elsewhere; it is a practice that we discourage.

If you absolutely must ship embryos, we suggest that you contact a company that has the expertise and the experience to make this type of shipment as safe as possible. Locally, we recommend “Swift Stork Courier” (www.swiftstork.com) who will arrange collection and delivery of the embryos and ensure appropriate and safe handling during transport. For long distance shipments, we put patients in contact with “Kynisi Courier Systems” (email: kosta@kynisi.com), a company based in the UK that specializes in shipping embryos. If you want to send your embryos from

San Francisco to Detroit, or Dublin or Dubai, Kynisi is the only company we know that can get embryos on airplanes without being x-rayed in security. They also get advance clearance to make sure that embryos don’t get delayed in customs as they cross international borders. Kynisi can also arrange for an embryologist to travel with your embryos, and they can organize for the embryos to travel in the passenger cabin of the aircraft, as opposed to being thrown in the luggage compartment with the other cargo. This is important, as a Dewar left lying on its side will lose nitrogen more rapidly than when upright. Kynisi’s services aren’t inexpensive, but considering that the embryos are priceless, there really isn’t a good alternative.

For those patients considering moving their frozen tissues to a facility that offers long-term storage at reasonable costs, we recommend “ReproTech” (www.reprot.com) in Reno, NV. ReproTech is experienced and knowledgeable, and gives great customer service. They too can arrange safe movement of your tissue from us to them, and back again with minimal inconvenience. They often take the extra precaution with embryos by splitting them into 2 groups that are then shipped separately. ReproTech shares the PFC philosophy of thinking of embryos as irreplaceable, and they take every known precaution to ensure a safe and efficient shipment. However, despite the good work of ReproTech, Kynisis and others, I recommend that you do not ship your embryos. The risks are too great.

Joe Conaghan, PhD

ICSI Overview: Intracytoplasmic sperm injection (ICSI) is a technique used in the IVF laboratory to inject individual sperm into eggs. The procedure was developed in Belgium in the early 1990′s (Palermo et al., 1992) and it revolutionized the treatment of male factor infertility. Prior to ICSI, men with moderate and severe fertility issues had little or no chance of having their own genetic children. ICSI has so revolutionized the treatment of infertility that it is used in the majority (55.6%) of assisted reproductive technology cycles in the United States (CDC National Summary and Fertility Clinic Reports, 2003).

When IVF is performed without ICSI, it is common to incubate individual oocytes in a petri dish with about 100,000 sperm. Usually these sperm have been obtained by processing the semen in such a way as to be able to isolate sperm that look normal and swim energetically. Only the best 10% of the sperm in a normal semen sample are used, and in the petri dish, these compete for the honor of fertilizing the oocyte.

When ICSI is employed, individual sperm are isolated and forcibly injected into the oocyte by an embryologist. The oocytes have to be incubated in the enzyme hyaluronidase to remove the cumulus cells that surround them (naturally, these cells would be dislodged by the many sperm that try to penetrate the oocyte). Prior to injection, the sperm may be processed (as above) but often there are so few sperm available that processing is minimal. Once selected, the sperm is immobilized by breaking its tail. This is accomplished by dragging the injection needle across the tail until a visible kink or break can be seen. The immobilized sperm is then aspirated into the needle, which is pushed through the shell surrounding the oocyte and then through the cell membrane. The elasticity of the oocyte membrane is such that the embryologist must be rough with it to get through. Piercing the membrane is usually achieved either by poking it several times or by aspirating the membrane into the needle until it breaks. Once the membrane breaks, the sperm can be dropped inside the oocyte.

Technically, ICSI is one of the most difficult procedures to perform in the IVF laboratory and it requires a talented embryologist to do it well. As well as being responsible for choosing “the sperm”, the embryologist must work quickly and be firm enough to break the sperm tail and oocyte membrane while not being so aggressive as to kill the oocyte. ICSI has been so successful as a technique that it is now widely used in cases where there is no male factor infertility. In fact, of all the ICSI cases performed nationally in 2003, only 53% had a male issue (CDC, 2003). While ICSI is absolutely indicated for low sperm counts, decreased sperm motility, abnormal sperm morphology (size and shape) and surgically retrieved sperm, its use has expanded to include cases with anti-sperm antibodies, previous low fertilization with IVF, low oocyte numbers, frozen-thawed sperm and ejaculatory dysfunction such as retrograde ejaculation. In addition, ICSI is being widely used for patients having preimplantation genetic testing because it avoids DNA contamination during embryo biopsy by the many sperm that are usually attached to the shell of the embryo.

ICSI Risks: In assessing the risks of ICSI, we must first look at the procedure itself. In piercing the cell membrane, our greatest concern is in avoiding the area within the oocyte where the DNA is located. This is done by orientating the oocyte such that the polar body (a small packet of discarded DNA) is placed at the 12 or 6 o’clock position and the needle inserted at 3 o’clock. The polar body is the most practical indicator of where the oocyte DNA is located since it is created by the division of the oocyte’s total DNA just prior to ovulation. However, the DNA may not always be in the assumed place so a theoretical risk of damage exists, and chromosome breakage has been observed as being higher in ICSI-derived embryos when compared to conventional IVF embryos (Bergere et al., 1995; Edirisinghe et al., 1997).

In addition to DNA disruption or damage, the physical and biochemical disturbance that occurs could be significant. The injection procedure could introduce foreign material into the oocyte such as culture medium, seminal fluid with or without bacteria (Michelmann et al., 1998), viruses (Brossfield et al., 1999), or in theory, even prions (Lacey & Dealler, 1994) or foreign DNA.

Following the ICSI procedure, the fertilization process is known to be different than with conventional IVF with atypical decondensation of the sperm head resulting in delayed replication of the male genome. This is thought to result from the injection of the intact sperm into the oocyte since such sperm retain their acromosomal cap and perinuclear theca, both of which are normally lost as the sperm penetrates the shell of the oocyte. There is marginal evidence that the sperm sex chromosome is preferentially located in the anterior head and therefore might be impacted by the delayed decondensation caused by retention of the cap (Luetjens et al., 1999).

Currently there is no evidence that the miscarriage rate is different between ICSI and IVF pregnancies, and the incidence of prematurity and low birth weight babies (7.6% and 10.3% respectively for ICSI) is similar to that for IVF in large studies (Wisanto et al., 1995; Aytoz et al., 1998), but slightly higher than rates found in natural pregnancies. These outcomes have been confirmed in a large US-based study (Schieve et al., 2002) showing overall lower birth weight and higher perinatal mortality in children conceived with the help of reproductive technologies, but no significant differences between ICSI and IVF.

In the mid 1990′s ICSI had become a routine procedure in the world of assisted reproductive technology (ART) and was being widely used. However, reports surfaced indicating that the resulting children had a high incidence of chromosomal abnormalities (In ‘t Veld, 1995; Van Opstal et al., 1997). The immediate response from the ART community was a flurry of scientific papers refuting the findings, but ultimately the conclusions of the studies were confirmed by large scale, prospective systematic follow up studies on the ICSI children. Instrumental in these studies was the Brussels University where ICSI was invented. Thorough pre- and postnatal testing showed an abnormal karyotype in 2.6% of the ICSI pregnancies (Bonduelle et al., 1999) and in a subsequent study, 3% showed a chromosomal abnormality (Bonduelle et al., 2002). Novel chromosome abnormalities increased threefold (1.6% in ICSI vs. 0.5% in the general population) and these were mostly comprised of sex chromosome aneuploidies with a smaller number of autosomal structural anomalies. Inherited chromosomal abnormalities increased fourfold in ICSI pregnancies (1.4% compared to 0.3% in the general population) and this was related to the higher rate of existing chromosome abnormalities seen in the parents (mainly the fathers). It is important to point out that the incidence of these sex chromosome aneuploidies and structural abnormalities is inversely related to the number of sperm in the ejaculate and is therefore higher in ICSI fathers (4.8% vs. 0.5% in the general population), and interestingly also higher in ICSI mothers (1.5%: Van Assche et al., 1996). The structural chromosome abnormalities include deletions of sections of the Y chromosome in some men with low sperm counts which will be passed directly to sons created by ICSI.

We are fortunate that the children of ICSI are being widely followed and many solid studies have appeared and continue to appear on the incidence of congenital abnormalities (these are problems that cause impaired function and require medical or surgical intervention). The most common abnormality appears to be hypospadias (a urological condition where the urethra opens under the penis instead of at the tip, and which is correctable with minor surgery) which is increased in ICSI births (Wennerholm et al., 2000). However, when evaluating these cases, the increased risk for congenital abnormalities is often reduced or eliminated when confounding factors (maternal age, infertility, multiple pregnancy, familial and pregnancy history) are factored in (Ericson & Kallen, 2001). Nonetheless, it does appear as though ICSI and IVF children do have an increased odds ration (2.77 and 1.8 respectively) for malformations that need medical or surgical intervention in early life when compared to naturally conceived children (Bonduelle et al., 2005).

Concerns have also arisen about developmental delays in ICSI children as a result of a single paper (Bowen et al., 1998) that had them scoring lower on the Bayley Scales of Infant Development at 1 year of age when compared to IVF and naturally conceived infants. However, a good number of solid papers have since been published indicating that this finding is not holding up and that ICSI children are performing normally in psychological testing as well as in their cognitive and verbal skills using the Bayley and other scales of intelligence (Bonduelle et al., 1998; 2003 Ponjaert-Kristoffersen et al., 2004; 2005).

Finally, it is worth asking if gene expression is normal for ICSI children and are problems likely to arise as the children get older? In looking at gene defects, there is emerging evidence that ART children might be at a higher overall risk for genomic imprinting errors when compared to naturally conceived children. Genomic imprinting is a process that silences one gene from a parent, specifically so that the gene inherited from the other parent can do the work. The classic example is placental growth, which is controlled largely by paternal genes. Maternal genes for placental growth are deliberately inactivated since it is considered a conflict of interest for Mom’s genes to be involved in the regulation of how much of her resources the fetus gets. Problems arise when an imprinted gene is defective, because the perfectly good copy of the gene from the other parent has been switched off and therefore cannot work. Diseases such as Beckwith-Wiedmann and Angleman’s syndromes result from not having a functioning copy of a gene and preliminary evidence suggests that these might be more prevalent in IVF children (Gosden et al., 2003). Abnormal spermatogenesis is associated with an increase in defective genomic imprinting (Marques et al., 2003), but it is probably too early to tell if imprinting errors will occur more frequently in ICSI children. Angleman’s syndrome for example occurs at most at a rate of 1/200,000 IVF births, so the impact of ICSI will be difficult to measure. Similarly, retinoblastoma (a type of cancer of the eye that is caused by a genetic defect similar to what causes imprinted diseases) has been reported as slightly higher in IVF children (Moll et al., 2003) but further studies will be required to substantiate this observation and to ascertain the specific risk of ICSI.

ICSI is an aggressively invasive procedure that deposits a single sperm, usually from an infertile father, into the oocyte of a woman who has undergone fertility treatments. The specific risk of ICSI in offspring is an increased incidence of chromosomal abnormalities which may be caused by the procedure or by the parents, or both. ICSI is a routine and overly used procedure and patients should be educated as to the risks. Of the studies cited here, none of the children examined were older than 5 years. The long term hazards of the procedure, if any, remain to be determined. See below for the complete bibliography.

– Joe Conaghan, PhD

Bibliography:

Aytoz A, Camus M, Tournaye H, Bonduelle M, Van Steirteghem A, Devroey P. Outcome of pregnancies after intracytoplasmic sperm injection and the effect of sperm origin and quality on this outcome. Fertil Steril. 1998 Sep;70(3):500-5.

Bergere M, Selva J, Volante M, Dumont M, Hazout A, Olivennes F, Frydman R. Cytogenetic analysis of uncleaved oocytes after intracytoplasmic sperm injection. J Assist Reprod Genet. 1995 May;12(5):322-5.

Bonduelle M, Wilikens A, Buysse A, Van Assche E, Wisanto A, Devroey P, Van Steirteghem AC, Liebaers I. Prospective follow-up study of 877 children born after intracytoplasmic sperm injection (ICSI), with ejaculated epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod. 1996 Dec;11 Suppl 4:131-55.

Bonduelle M, Aytoz A, Van Assche E, Devroey P, Liebaers I, Van Steirteghem A. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod. 1998 Apr;13(4):781-2.

Bonduelle M, Camus M, De Vos A, Staessen C, Tournaye H, Van Assche E, Verheyen G, Devroey P, Liebaers I, Van Steirteghem A. Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod. 1999 Sep;14 Suppl 1:243-64.

Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirteghem A, Liebaers I. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod. 2002 Oct;17(10):2600-14.

Bonduelle M, Ponjaert I, Steirteghem AV, Derde MP, Devroey P, Liebaers I. Developmental outcome at 2 years of age for children born after ICSI compared with children born after IVF. Hum Reprod. 2003 Feb;18(2):342-50.

Bonduelle M, Wennerholm UB, Loft A, Tarlatzis BC, Peters C, Henriet S, Mau C, Victorin-Cederquist A, Van Steirteghem A, Balaska A, Emberson JR, Sutcliffe AG. A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod. 2005 Feb;20(2):413-9.

Bowen JR, Gibson FL, Leslie GI, Saunders DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet. 1998 May 23;351(9115):1529-34.

Brossfield JE, Chan PJ, Patton WC, King A. Tenacity of exogenous human papillomavirus DNA in sperm washing. J Assist Reprod Genet. 1999 Jul;16(6):325-8.

Centers for disease control and prevention. Assisted reproductive technology success rates. National summary and fertility clinic reports 2003 2005 Dec; United States Department of Health and Human Services.

Edirisinghe WR, Murch A, Junk S, Yovich JL. Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study. Hum Reprod. 1997 Dec;12(12):2784-91.

Ericson A, Kallen B. Congenital malformations in infants born after IVF: a population-based study. Hum Reprod. 2001 Mar;16(3):504-9.

Gosden R, Trasler J, Lucifero D, Faddy M. Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet. 2003 Jun 7;361(9373):1975-7.

In’t Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet. 1995 Sep 16;346(8977):773.

Lacey RW, Dealler SF. Vertical transfer of prion disease. Hum Reprod. 1994 Oct;9(10):1792-6.

Luetjens CM, Payne C, Schatten G. Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection. Lancet. 1999 Apr 10;353(9160):1240.

Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet. 2004 May 22;363(9422):1700-2.

Michelmann HW. Influence of bacteria and leukocytes on the outcome of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Andrologia. 1998;30 Suppl 1:99-101.

Moll AC, Imhof SM, Cruysberg JR, Schouten-van Meeteren AY, Boers M, van Leeuwen FE. Incidence of retinoblastoma in children born after in-vitro fertilization. Lancet. 2003 Jan 25;361(9354):309-10.

Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992 Jul 4;340(8810):17-8.

Ponjaert-Kristoffersen I, Tjus T, Nekkebroeck J, Squires J, Verte D, Heimann M, Bonduelle M, Palermo G, Wennerholm UB. Collaborative study of Brussels, Goteborg and New York. Psychological follow-up study of 5-year-old ICSI children. Hum Reprod. 2004 Dec;19(12):2791-7.

Ponjaert-Kristoffersen I, Bonduelle M, Barnes J, Nekkebroeck J, Loft A, Wennerholm UB, Tarlatzis BC, Peters C, Hagberg BS, Berner A, Sutcliffe AG. International collaborative study of intracytoplasmic sperm injection-conceived, in vitro fertilization-conceived, and naturally conceived 5-year-old child outcomes: cognitive and motor assessments. Pediatrics. 2005 Mar;115(3): 283-9.

Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med. 2002 Mar 7;346(10):731-7.

Van Assche E, Bonduelle M, Tournaye H, Joris H, Verheyen G, Devroey P, Van Steirteghem A, Liebaers I. Cytogenetics of infertile men. Hum Reprod. 1996 Dec;11 Suppl 4:1-24.

Van Opstal D, Los FJ, Ramlakhan S, Van Hemel JO, Van Den Ouweland AM, Brandenburg H, Pieters MH, Verhoeff A, Vermeer MC, Dhont M, In’t Veld PA. Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection. Hum Reprod. 1997 Apr;12(4):682-6.

Wennerholm UB, Bergh C, Hamberger L, Westlander G, Wikland M, Wood M. Obstetric outcome of pregnancies following ICSI, classified according to sperm origin and quality. Hum Reprod. 2000 May;15(5):1189-94.

Wisanto A, Magnus M, Bonduelle M, Liu J, Camus M, Tournaye H, Liebaers I, Van Steirteghem AC, Devroey P. Obstetric outcome of 424 pregnancies after intracytoplasmic sperm injection. Hum Reprod. 1995 Oct;10(10):2713-8.

For those of us with an interest in human reproduction, scarcely a day goes by without us hearing or seeing something related to oocyte freezing. The topic has generated a lot of hype and it is difficult to avoid the frequent magazine and newspaper articles, advertisements and TV features that generate excitement on the subject.

We have already discussed oocyte freezing in a previous newsletter article (Keeping Egg Freezing in Perspective; January 2005) and readers unfamiliar with the technology are encouraged to visit our website where they can read this in the newsletter archive. Having already discussed the methods for freezing, and their merits, we now address the achievements of oocyte cryopreservation on this, the 20-year anniversary of the first success.

There are two technologies used in oocyte freezing, and the primary aim of each is avoiding ice formation within the cell. The first is the slow freeze method (used so successfully with embryos) that dehydrates and cools the cells gradually, over three hours. The second is an ultra-rapid procedure that is performed so quickly that the cell contents turn to a glass-like substance. This latter method is called vitrification and it is gaining in popularity for oocyte and embryo freezing. And since no ice forms, the cells are technically not frozen, but “vitrified.”

In reviewing the scientific literature since the first success in 1986, the importance of oocyte freezing is apparent by the sheer volume of publications on the subject. For the purpose of this article, the many papers that report on the technique only have been excluded, and here we will only report on the pregnancy outcome data. However, even this is difficult since some patients may have become pregnant from the first few thawed oocytes, leaving us with no data on the many oocytes still frozen on their behalf. Also, even though there are reports that detail only one or two pregnancies, there are probably many other isolated successes around the world that have gone unreported in the scientific literature.

Most of the pregnancy outcome data has been pulled together in a single review paper by Dr. K. Oktay and colleagues at Weill Medical College in New York (Fertility & Sterility, 2006, Vol 86 (1), pages 70-80). The 47 papers reporting outcome data for slow freezing were analyzed and from these, only 26 provided sound usable data. The others were excluded either because sub-optimal procedures were used, the pregnancies had not yet delivered or the authors could not be reached to clarify the data. The 26 useful papers collectively documented the freezing of 4,564 oocytes from which 4,000 had been thawed in 397 patient cycles. Out of 95 pregnancies, 76 resulted in live births, and since some of these were multiple pregnancies, the total number of children born was 97. If we add in the excluded data, the number of pregnancies becomes 170, resulting in 106 live births and 11 ongoing pregnancies. Because of ambiguities in the excluded data, a final number of children is not stated. However, the data suggest that the number of children that are alive today as a result of 20 years of slow freezing of oocytes is approximately 200. Taking all the data into account, the clinical pregnancy rate per thawed oocyte was a mere 2.3%. The live birth rate in the 26 usable papers was 1.9% per oocyte thawed.

Unfortunately it is not possible to give rates per oocyte frozen since some papers are not complete, but more importantly because many oocytes are still in the freezer.

Vitrification, which is a technology that came late to oocyte preservation, is quickly gaining ground on the slow freezing method. By June of 2005 there were only 10 reported births following oocyte vitrification, but a year later the numbers reported by Oktay are 61 pregnancies from which 42 have delivered live infants and 7 are ongoing. With limited data, vitrification appears to be a more highly efficient preservation method than slow freezing. The latest numbers, based on admittedly limited data, shows that >90% of oocytes survive and about 90% of these fertilize. Overall, 50% of vitrified oocytes make blastocysts in culture which is as efficient as fresh oocytes. These numbers are reported by Masa Kuwayama at the Kato Ladies Clinic in Tokyo. Also, from 29 embryo transfers, 12 pregnancies have yielded 7 live infants with 3 not yet delivered at publication time (Kuwayama et al., 2005, Reprod Biomed Online, Vol 11 (3) pages 300-308). We can compare this data to the latest results with slow freezing where the experience of 20 years has been incorporated. Using sodium-depleted medium, in which oocytes are slow cooled and frozen, 59% of oocytes survived and 68% of these fertilized. Nine pregnancies were established in 28 thaw cycles from which 5 delivered and 1 was ongoing (Boldt et al., 2006, Reprod Biomed Online, Vol 13 (1) pages 96-100). For those women who want to rely on oocyte cryopreservation to postpone motherhood, these data should be sobering. While we don’t expect the technology to ever be 100% successful, it currently offers no guarantees.

Expecting too much from today’s procedures could leave many women very disappointed. Further, many of the pregnancies reported in these studies were achieved by preserving the oocytes from young women. Since oocyte quality declines as a woman ages, the success rates for older women are likely to be less than reported here. Women considering oocyte preservation will need careful counseling and a good understanding of the success rates before putting their eggs in this basket.

– Joe Conaghan, PhD