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

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

Almost 20 years ago, a paper in a British medical journal Lancet announced the arrival of a new technology: Oocyte Cryopreservation (Chen, C., 1986, Vol 1, Page 884). What was initially thought to be a landmark paper turned out to be the poster child for the procedure, as Chen himself and many others were unable to repeat the process with consistency. Although it is difficult to open any magazine today without reading about this wonderful new technology, less than 1% of eggs that have been frozen and thawed have resulted in live born infants.

We have learned much about the freezing of human oocytes over the years, yet despite a massive and consistent effort by the scientific community, a reliable method to freeze eggs with the same success as embryos and sperm remains elusive.

Our ability to freeze any cell depends on many factors, but most significantly on how much water the cell contains. Because water expands in volume as it turns to ice, cells must be dehydrated prior to freezing to prevent the cell from rupturing. The addition of a cryoprotectant, which does not expand upon freezing, can greatly reduce the risk of cell rupture.

Scientists have been freezing and thawing sperm with good success for over 100 years. In many ways, sperm are ideal for freezing as they exist as individual cells, they are the smallest human cells and they contain very little water. It is thought that sperm can be stored perhaps indefinitely after being added to a solution of cryoprotectant, and then frozen to minus 1960C.

In contrast to the sperm, the oocyte is the largest human cell and it contains much more water. The oocyte is also much more sensitive and is very intolerant of the chemical and physical stresses that are created during freezing and thawing. Further, the availability of oocytes is much more limited. When an oocyte is ovulated, or retrieved from the ovary during an IVF cycle, ideally it is ready to be fertilized by a single sperm. In anticipation of fertilization, the oocyte prepares to discard half of its DNA – a process called meiosis. Any changes in the physical or chemical environment around the oocyte can disrupt meiosis, leading to an oocyte with too much or too little DNA. Even after we overcome the hurdles of sensitivity and cell water content, there are other obstacles to freezing and thawing oocytes successfully.

In scientific literature, most papers that report success with egg freezing involve very few patients and therefore even fewer pregnancies and deliveries. Porcu et al., 1997, Tucker et al., 1998 and Young et al., 1998 are typical examples of papers that report successful deliveries from just one patient’s frozen oocytes. Between them, these authors froze 34 eggs, of which 15 survived thawing. In larger studies, Porcu et al., 2000 and Fabbri et al., 2001 were able to obtain large numbers of oocytes for freezing (1502 and 1769 respectively), resulting in overall survival after freezing at just over 50% for both studies. Just over half of the oocytes that survived freezing fertilized, and about half of these made good quality embryos. Yet the number of babies delivered reported by Porcu was low (9 births plus 7 ongoing pregnancies). Fabbri reported only fertilization and embryo development rates as a measure of success in his study and has not yet reported on pregnancies and births.

Wider application and success with oocyte freezing depends on continued improvements with the technology and on careful selection of oocytes to freeze. While many researchers are continuing to improve the freezing process, much of the success so far has been with the use of good quality or young oocytes. In the Porcu study, most of the oocytes were collected from young women who would presumably have good quality oocytes. We would expect results to be worse if the eggs were from older women, although no such studies have been undertaken. • Despite all the hype, oocyte freezing will fall short of mainstream therapy in the near future until new technologies improve the process. Oocyte cryopreservation may be an especially disappointing prospect for older women. With this in mind, this year PFC will take part in a large scale study involving Japanese IVF centers and other US centers on an alternative technology called vitrification. This involves an ultra-rapid freezing process that we hope will allow more oocytes to be frozen before they are compromised by the effects of the physical and chemical stresses indicative of typical slow freezing methods. Vitrification has shown good success with human oocytes and embryos in recent Japanese studies.

Pacific Fertility Center is now participating in an important program that helps protect the fertility of cancer patients undergoing chemotherapy and radiation. Fertile Hope, an advocacy organization that raises awareness about fertility issues for cancer patients, is partnering with carefully selected clinics throughout the US in a program called Sharing Hope. The program will be open to those who have been diagnosed with cancer, want to preserve their fertility and have limited financial means. Sharing Hope offers qualifying cancer patients significant discounts for fertility-preservation treatments, such as embryo freezing and egg freezing before undergoing chemotherapy, radiation and/or surgery.

Cancer treatments can affect fertility in both men and women. In some cases infertility will be temporary, but in others it will be permanent. Currently, options are limited for cancer patients wishing to preserve their fertility. Men may freeze their sperm prior to cancer treatments to be used for artificial insemination or IVF. This is quite successful and in most cases at least 50% of a man’s sperm will survive freezing and thawing. The best option for women is to freeze embryos (via IVF). Yet this offers a viable solution only to women with partners or those willing to use donor sperm. What is the single woman diagnosed with cancer to do? She has not yet found Mr. Right, or even Mr. Perfect Sperm Donor, but knows she wants to have a child in the future. The bright spot may be egg freezing. Still considered experimental, egg freezing is a relatively new procedure and has much lower success rates than embryo freezing. Some say the numbers for egg freezing are around 1 live birth for every 100 eggs frozen, yet there are clinics around the world claiming to have 1 live birth for every 10 eggs frozen. The success of egg freezing will continue to improve as technology and scientific knowledge develop. PFC will offer egg freezing in the near future.

For some people, the idea of losing their fertility is as devastating as the diagnosis of cancer. Often, cancer patients have little time or opportunity to gather funds for the high cost of cancer treatment, let alone fertility preserving treatments. At PFC we hope to extend a helping hand to cancer patients unable to afford these costly treatments and to provide them with the hope of building a family.

You may find out more about Sharing Hope at Fertile Hope’s website

Human Egg

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

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

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

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

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

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

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

What might a mindful career-oriented 36-year-old woman have in common with a 22-year-old just diagnosed with an unusual cancer and scheduled for radiation or chemotherapy treatment?

• Both may want to carefully chart their course of family planning.
• Both face the loss of their ovarian egg reserves: one from the damaging chemotherapy, the other from age.
• Both may be considering oocyte (egg) freezing.

The idea that a woman can undergo a standard IVF procedure and then freeze individual eggs, instead of having her oocytes inseminated and then frozen as an embryo, is a notion that is capturing the imagination of grandmothers, women and doctors alike. So much so, dozens of infertility clinics are boasting egg cryopreservation as a new service even though most qualify it as “experimental”. Indeed, egg freezing is simply too new, and it has not shown the success rates necessary for widespread marketplace acceptance. This procedure is not a panacea or an insurance certificate for everybody. However, it can be a viable option for women who are aware of its limitations.

What is most important is a patient’s absolute understanding of the challenges of egg cryopreservation. To say women’s oocytes are much more difficult to freeze than male sperm is an understatement. A good quality female egg is essentially a pin head-sized globule of fluid plus the necessary DNA to carry new life into being. It is this sac of liquid that must be carefully drained and then filled with anti-freeze to help the egg freeze and thaw. Accomplishing this without damaging the microcosm of genetic material, as delicate as a spider web, is the main hurdle. When egg quality is compromised, a myriad of problems ensue: failure to fertilize or implant, miscarriage and birth defects.

The race to offer egg cryopreservation was initially fueled by favorable research results from a study that used subjects in their early 20s, and which resulted in >50% chance of a live birth. Yet with only 7 subjects, that study is not statistically significant. In subsequent studies that used women in their early 30s, the success rate dropped below 25%. Currently, most U.S. clinics pioneering this procedure predict only an 8-10% chance of live birth. Also, a side effect of freezing is the hardening of the egg’s outer membrane, known as the zona pellucida, making sperm penetration difficult. However, this is overcome by using ICSI (intracytoplasmic sperm injection).

Those requesting this service need to have all of the facts before making a choice. In particular women in their mid- to late-30s, who tend to be the most enthusiastic candidates, need to weigh other options with higher proven success rates. We at PFC share an understanding with much of the medical community that this procedure may be the right choice for the right person, but only with a full understanding of its limitations. This will be our approach when we start offering egg cryopreservation to our patients later in 2004.