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

One of the mysteries that confound reproductive biologists is the issue of why human embryos implant into the uterus at relatively low rates as compared to other animal species. This is evident when looking at implantation rates at the time of In Vitro Fertilization treatment. The chance that any one embryo will implant in the uterus varies with female age such that at age 40, only about 5-10% of transferred embryos will successfully take hold and create a viable pregnancy. Even when looking at donor egg-derived embryos from 21-28 year-old donors, the rates of implantation are about 30-45% per embryo. One mechanism to explain the failure of some embryos in implanting – perhaps the primary mechanism – is chromosomal abnormality. If an embryo does not carry a perfect set of 23 pairs of chromosomes, the embryo will likely stop developing, often before implantation can occur.

Implantation of embryos is a complex process. Initially, the embryo has to attach its placental cells to the surface cells of the uterine lining (the endometrium). This is a process that is mediated by a complex of proteins expressed both on the surface of the embryo and on the surface of the endometrium. Expression of the uterine proteins is under the influence of the ovarian hormone progesterone. There are estimated to be over 300 genes that are either turned off or turned on in the endometrium during the “implantation window,” the 3-4 days during which the endometrium is receptive to an embryo attaching. Most of the products of these genes and their role in implantation remain to be identified. In a small percentage of cases, failure to properly secrete one or more of these proteins may be a cause for implantation failure of normal embryos.

One protein produced by the endometrium during the implantation window that has some evidence for a scientific basis for a role in implantation is the cell-to-cell adhesion molecule known as beta-3 integrin. Integrins are a class of cell surface proteins that appear to act in all types of cell-to-cell recognition and adhesion processes. The beta-3 class of these proteins has been shown to be produced in response to progesterone in the endometrium and are purported to be one of the key proteins for adhesion of embryos to the endometrium. Failure to express this protein appropriately has been theorized to be a cause of unexplained implantation failure. Why some women do not produce beta-3 integrins is usually unknown. However, some proposed causes include presence of blocked fallopian tubes filled with inflammatory fluids (hydrosalpinx), endometriosis, and poor progesterone production.

In order to diagnose whether or not a patient is producing beta-3 integrins, an endometrial biopsy must be performed 8-10 days after ovulation, as determined by LH surge testing. The biopsied endometrial sample is then sent to a laboratory that performs immuno-histochemical analysis on the tissue. The tissue is fixed to a slide and treated with antibodies to beta-3 integrins. These antibodies then are further treated with a second color marker antibody, so that endometrium-secreting beta-3 integrins will light up under the microscope. The tissue is scored by manual analysis by a medical technologist specifically trained to analyze beta-3 integrin expression.

In June of this year, I had the opportunity to visit Adeza Biomedical, a Cupertino-based laboratory that offers beta-3 integrin testing. I was impressed with the facility and the scientific integrity of the staff. I was also impressed with the labor-intensiveness of the analysis process. They receive specimens every day from infertility clinics across the country and are usually processing 6-12 specimens daily. They also send a portion of the biopsied tissue to a local pathologist to determine if the configuration (histology) of the endometrial tissue indicates it has been obtained within the implantation window or whether it is “out-of-phase.” As it turns out, a high percentage of tissue samples (40-45%) at Adeza are reported as negative for beta-3 integrins. A smaller percentage of these negative specimens are “out-of-phase”. So most of the specimens failing to show beta-3 integrins production are “in-phase”. It is unclear why this lab finds such a high rate of their test samples showing negative results for beta-3 integrins when the true incidence of lack of beta-3 integrins in most patients should be low. It may be that either the lab is setting the scoring level for a positive result too high or it may be that the patient samples really reflect a selected population of women who truly have low expression of beta-3 integrins. Unfortunately, there is no clear answer to this.

Previously, we had been less inclined to perform endometrial biopsies. Even if we found out there was a lack of beta-3 integrins, we wouldn’t know what to do to induce their expression. However, we are beginning to find that we can often induce the expression by treating beta-3 integrin-negative patients with the aromatase enzyme inhibitor, letrozole (see A Closer Look at Letrozole; May 2006). Many women, especially if the histology on the original biopsy is “in-phase,” will have a positive biopsy result after treatment with letrozole.

Biopsies are typically performed 8-10 days after an LH surge in a natural cycle. Repeat biopsies on letrozole (taken days 3-7 of the cycle) are also performed at this time. We usually will use some local anesthetic in the cervix prior to passing a small plastic tube through the cervix to scrape out some endometrial tissue. Mild cramping may occur. The cost of the biopsy is $125.00 and the cost of the tissue analysis by Adeza is $400.00. It takes about 4-5 working days for the results to be received.

If you would like more information about this test, visit www.adeza.com and select the E-tegrity logo. You can download a patient brochure from this website. If you would like to know if this testing is appropriate for you, please ask your PFC physician.

– Carolyn Givens, M.D.

Pacific Fertility Center is pleased to announce the arrival (or return, if you will) of Mariluz Branch, Laboratory Supervisor to Pacific Fertility Center’s IVF Laboratory. Mariluz brings over a decade of laboratory experience dating back to the early 1990s when she was an embryologist at the San Francisco Center for Reproductive Medicine (SFCRM), now a part of Pacific Fertility Center. She was there during the infant stages of SFCRM, working with Pacific Fertility Center’s Drs. Herbert and Chenette.

At SFCRM, Mariluz played an instrumental role in developing their IVF lab, and, after 11 years, she traveled abroad for several years, moving to London where she held a similar position at an IVF lab. Although there were differences in the processes of working at an IVF laboratory in the UK, she never lost her passion for embryology, and, after moving back to the states earlier this year, she was reunited with many of her former SFCRM colleagues at Pacific Fertility Center. The motivation to work at Pacific Fertility Center was to work in an environment that had good pregnancy rates, trustworthy staff, and high professional standards. Since being at PFC, she has been very impressed with the laboratory’s knowledge, meticulousness, and commitment to do what’s best for the patient.

“Teamwork is one of our team’s biggest strengths,” said Mariluz. “For example, we have a witnessing system during every critical stage in the laboratory. This allows us to run as safe and secure a lab as possible. We also have a large enough staff to prevent us from being overloaded, which helps eliminate any confusion.”

Mariluz has training in preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD) as well as extensive experience with ICSI, cryopreservation and embryo handling. She will bring this and other expertise to her role, which will consist of conducting clinical research studies with the aim of increasing embryo quality and pregnancy rates, and will eventually be overseeing the PGD program. She is also working on developing oocyte (egg) freezing at PFC through a technique called vitrification (versus the more traditional slow freezing method). Mariluz is enthusiastic about the significant and ongoing advancements in assisted reproductive technology (ART) and is pleased to play a role in helping Pacific Fertility Center’s patients realize their dream of building a healthy family.

Question: Can I collect my sperm sample at home?

Answer: Yes, sperm samples can be produced at home and brought into our office provided that you follow some simple guidelines. Most importantly, the instructions for producing a sample must be followed as if you were producing a sample in one of the two dedicated rooms in our office. You should shower in the morning and wash the genital area with soap and then rinse with plenty of water. Most of the samples we receive are produced by masturbation and you should be careful to wash your hands immediately before and after the collection. If you need lubrication and/or a condom to produce the sample, these must be supplied by PFC. Most condoms and commercially available lubricants are toxic to sperm in some way, but we can supply you with materials that we have tested and that we know do not kill sperm. You can take them home if that’s where you’ll produce your sample. Similarly, we must provide the container into which you will collect; again to ensure that it is sperm friendly.

The most important part of producing the sample at home is getting it to our office within 60-90 minutes of collection. Your semen sample contains sperm but also many enzymes that are important in the natural process of reproduction. One part of your reproductive tract, the seminal vesicles, produces enzymes that coagulate the semen immediately upon emission. This allows the viscous sample to remain within the vagina, a process that might be an evolutionary vestige of the copulation plugs that are seen in other mammals and that prevent the female from mating with a second male. Within 5-20 minutes however, other enzymes in the semen (this time from the prostate gland) liquefy the clotted semen, liberating the trapped sperm so that they can enter the cervix. Sperm in the first fraction of the semen are bathed in prostatic secretions and have better motility and survival than sperm in latter fractions which are bathed in vesicular fluid, since the seminal vesicles emissions are last in the ejaculatory sequence. This is why we always ask if any part of the ejaculate was lost during collection. If the first few drops of semen don’t get into the collection cup, we may have lost the best sperm and we may underestimate the quality of your sample.

All of these enzymes in the semen make it a hostile environment. Sperm trapped or left in semen will die relatively quickly, but sperm washed out of this enzyme bath can survive easily for 4 or 5 days in the laboratory. Semen can also cause uterine contractions, which is why we have to process sperm samples and remove it before performing your intra uterine insemination. Getting your semen sample to the laboratory within 60-90 minutes of collection allows us to assess your sperm before the enzymes can do any damage.

It is important that you have an abstinence period of at least 48 hours but not more than 7 days before giving us a sample. Samples produced after 2 days abstinence will usually have the highest numbers of motile sperm with the greatest forward velocity, when compared to samples produced after shorter or longer abstinence. Waiting too long between ejaculates is the biggest mistake we see, possibly because some men think that they can save all their sperm for the day of their big test. However, older sperm begin to die if ejaculations are infrequent and we see the percentage of live sperm decrease with increasing abstinence. Also, please remember that abstinence means no ejaculation, not just no intercourse!

Once your sample has been collected, it is important to avoid exposing it to extremes of heat or cold before bringing it to us in the laboratory. Don’t put it in the refrigerator while you take a shower. Don’t leave it on your dashboard in the sun while you pick up your dry cleaning. And don’t leave it in the glove compartment, forget about it for a week, and then deliver it to the lab. The sample will be fine at room temperature, and you don’t have to break the speed limit in trying to get it to us.

You will need to have made an appointment with us so that we know you will be bringing in a sample, and when you arrive in our office, a member of our staff will check your specimen in. We need to be sure that it is labeled properly and we will get some details from you regarding your abstinence period and how and when you produced the sample. And we will check your identification (usually your driver’s license). This last step is important in establishing the identity of the sample and is part of a “chain of custody” procedure that we use with all samples passing through our facility. We will examine and if appropriate, process the sample within 30 minutes of receiving it, or immediately if the sample is already 1 hour old. Hopefully we won’t be calling you to say that we need to repeat the test!

– Joe Conaghan, PhD, HCLD

Human semen is a complex mixture of cells and fluids produced by the various components of the male reproductive system. The objective of sperm preparation is to remove the vigorously swimming sperm from this mixture, leaving behind the dead, dying or otherwise poorly swimming sperm, additional cells, enzymes and other factors that comprise the seminal fluid. A sperm cell is incapable of fertilizing an oocyte until it has separated from the seminal fluid.

We use a variety of separation techniques in the laboratory that are tailored to the procedure that the sperm will be used for, and modified according to the quality and type of sperm sample we receive. The average man manufactures about 250 million sperm in a 24 hour period. From a single ejaculate, we will only use 100,000 sperm for each oocyte that we have to inseminate in an IVF cycle. But for an intrauterine insemination, we want to get as many motile sperm as possible into the female reproductive tract, so we will therefore be using a much higher overall fraction of the sperm. Alternatively, for men who have no sperm in their ejaculate and for whom we have to retrieve sperm surgically from the testicle, we want to biopsy the minimum amount of tissue that will give us one sperm for every oocyte that has to be inseminated.

There are two general methods that we employ for the vast majority of sperm processing in the laboratory. The first is a density gradient centrifugation procedure in which the sperm sample is gently spun through 1-3 columns of a viscous solution of saline coated colloidal silica particles. The layers of silica are created by delicately layering different silica particle densities on top of each other in a test tube, and then layering neat semen on top. This method takes advantage of the fact that living sperm are dense compact cells that pass easily through the columns, while dead or dying sperm that are less dense due to leaky membranes are trapped with other cells and debris in the interfaces between the layers. The second method for preparing sperm takes advantage of the sperm’s natural swimming abilities by placing neat seminal fluid in proximity to some culture medium and allowing the sperm to swim from one to the other. There are many variations in this technique including the swim-up (semen is layered under the medium), or the converse method called the swim-down, and the actual method used depends mainly on the quality of the sperm sample. The swim-up is primarily used for samples that have good numbers of highly motile sperm from which only a small fraction needs to be recovered. The swim-down technique works better when sperm are swimming weakly and need the help of gravity to separate from the seminal fluid. For an individual with vanishing numbers of sperm (say a few hundred) we may use a swim-out technique. Here, the sperm are placed in the center of a small drop of medium and an embryologist will wait with a needle at the edge of the drop, picking up the first sperm to get there. One of the big criticisms of the ICSI procedure, where individual sperm are injected into oocytes, is that the embryologist chooses the sperm. However, with the use of the swim-out procedure, there is some degree of “natural selection” as we choose the sperm that are quickest in getting to the edge of the drop. We also choose sperm that are the normal size and shape, and that are free from defects (such as a bent neck) if we have the luxury to do so. In rare cases we have to use every sperm we have, so there’s no “selection” whatsoever. In most of the cases where we’re processing samples that have normal numbers of sperm, the sperm isolated by density centrifugation or by swim-up will be “washed” once or twice before being introduced to the oocytes. This involves suspending the sperm in a volume of culture medium and then centrifuging gently so that the sperm can be concentrated and removed from the medium, while leaving behind any trace of the silica particles or seminal fluid that may have carried over from the first processing step. Although sperm can be damaged by centrifugation, these steps are necessary to ensure that the sperm are free of contaminants that could prevent fertilization.

There are many other methods used to process sperm samples but we use them so rarely that they are scarcely worth mentioning. For example, samples with a high amount of debris can be filtered through glass wool or processed by sedimentation to clean them up before they undergo any of the procedures already described. In addition, we can treat a semen sample with chemicals in certain situations, but this again only happens under somewhat desperate circumstances. If a semen sample is extremely viscous or clotted, we can digest it using the enzymes amylase or chymotrypsin. If none of the sperm are moving we can treat them with pentoxifylline or caffeine to try to stimulate movement. When performing ICSI, we need to know that sperm are alive, and movement is our primary indicator. We can try to stimulate movement using drugs, but for the sperm that are to be used to fertilize the oocytes, we prefer to go drug-free. Here, we place the sperm into a hypo-osmotic solution (regular culture medium that has more water than normal) and as water enters living sperm their tails coil. These we can then inject into oocytes.

For patients that purchase frozen sperm from a sperm bank, the bank will usually offer the option of buying the sperm processed or unprocessed†. Processed sperm, usually labeled “IUI sperm”, costs a little more since the sperm bank has already prepared it for use. Unprocessed or “ICI sperm” is essentially neat semen that has been frozen. Women who do their own inseminations at home buy this type of sperm and inject it into their vagina after it is thawed. If you buy ICI sperm with the intention of having an intrauterine insemination, we will process the sperm as above to remove the seminal fluid and dead sperm. ICI sperm cannot be placed into the uterus since semen contains many contaminants such as bacteria, but also because semen can cause painful uterine contractions.

On a given day in our laboratory, one embryologist is primarily responsible for processing sperm samples, and each embryologist is assigned to this task about once a week. Each sample has different characteristics and the individual doing the processing must make informed decisions on the best approach for recovering the sperm that we need. It is an interesting and demanding area of the laboratory, but we enjoy the challenge of maximizing the potential of each sample that we receive.

– Joe Conaghan, PhD, HCLD

† For more information on frozen sperm and the products sold by sperm banks, see the “How do I Buy Sperm?” article in the April 2005 newsletter.

Here are some images from the different techniques that can be used for gender selection

To read more about this process, see our Ask The Experts post

Pacific Fertility Center Team
Left to Right: Front: Philip Chenette, MD, Isabelle Ryan, MD, Carolyn Givens, MD
Back: Joe Conaghan, PhD, Carl Herbert, MD, Eldon Schriock, MD

Question: My husband and I have two boys and want to have a girl.
What are our options?

Answer: Gender selection is a complicated and difficult issue. Ethics aside, there really are only two proven methods. The first is a technique of sorting sperm cells called Microsort. If there is a healthy number of motile sperm present (no significant male infertility), the husband can fly to Southern California to the Microsort lab and have the sperm sorted. That sperm can then be used to inseminate the wife at Huntington Reproductive Center in Laguna Hills or can be frozen and shipped back to PFC for use in IVF. Because the technique results in such poor recovery of sperm, insemination may take several tries. This is why most of our patients will use the sperm in conjunction with IVF, in which case we can inseminate by single sperm injection (ICSI) several of the wife’s eggs, producing and then transferring embryos back to the uterus, and giving the couple a better chance of success. The sperm sorting method is much more efficient if the gender desired is female (see Microsort Facts below). We receive a report from Microsort about the estimated percentage of sperm that are X-bearing (female) vs. Y-bearing (male). Usually, for a female, that is about 85% and most couples interested in a girl are comfortable with those odds. For a male, the odds are lower (about 73%) and therefore, if a boy is really desired, most couples look to PGS (Pre-Implantation Genetic Screening).

With PGS, the patients undergo IVF with ICSI to create the embryos, and when the resulting embryos have 5-8 cells, a single cell is removed and analyzed for a number of chromosomes, including X and Y. If the couple wishes to transfer only the embryos of one gender, they will have to decide what to do with the remaining embryos. The technique is close to 93% accurate, which is a huge advantage over Microsort if male gender is desired. However, our most recent statistics with PGS indicate implantation rates tend to be lower. We do suspect that the procedure of removing a cell from the embryo may be decreasing the chances of successful implantation.

There are many more complex issues involved with PGS so we require our patients considering this process to meet with a genetics counselor (we work with the Perinatal Genetics program at California Pacific Medical Center for this counseling) to discuss the implications of undergoing this process in more depth.

– Carolyn Givens, M.D.

Microsort Facts • Sperm sorting technique

Two locations: Virginia and Southern California

Must be younger than 40 years old (or using egg donor)

Must be for Family Balancing (not first child)

Low % of sperm recovered

Efficiency for a girl is about 85%

Efficiency for a boy is about 73%

For more info, visit the website: www.microsort.net

Many of our patients are undergoing fertility treatment for male factor indications, and undergo insemination therapy. This may be patients who are using donor sperm from a sperm bank, or patients who are using their partner’s sperm, but the sperm has been frozen (partner out of town, or other indications). We are often asked if the success rates will be affected by the use of frozen versus fresh sperm. As well, we are asked if the number of inseminations performed per cycle will affect the success rates. There is a body of studies that have been done to address these specific questions, and our clinic’s interpretation of the literature is the following.

The first consideration addresses which type of insemination provides the best outcome when using frozen sperm. A number of studies have looked at this question, and when all the data from those studies are compiled and analyzed, results indicate that if an intrauterine insemination (IUI) is performed (sperm placed directly in the uterus), the odds are 2.5 times greater that a pregnancy will occur, than if an intracervical insemination (ICI) is performed (sperm placed at the entrance of the cervix) (5% vs. 14% monthly chance of pregnancy) (1, 2). When sperm are placed at the cervix, many of them are “lost” as they travel through the cervix and into the uterus, to then find their way to the fallopian tubes. This dilutes the actual numbers that make it to the egg in the fallopian tube, and therefore decreases chances of success. Performing 2 intracervical inseminations in one cycle (9% chance of pregnancy) did not bring success rates close to what one intrauterine insemination achieved (15% monthly chance of pregnancy) (2).

Next consideration addresses if fresh sperm is better than frozen sperm. Two studies have addressed this best, and indicate that the critical components that will provide comparable pregnancy rates are the performance of an intrauterine insemination (IUI), accurate timing of the insemination (relative to the ovulation event), and adequate concentration of sperm inseminated (called total motile count=TMC) (3, 4). The most accurate way to time the insemination is by using ovulation predictor kits (OPK), or by administration of an HCG injection to trigger the ovulation event. Ovulation predictor kits have been evaluated and the kit we recommend is the Clear Blue Easy ovulation kit. First detection of an LH surge is most likely to occur in the morning, and our recommendation is to do one test/day, in the morning (5). The best timing for an intrauterine insemination using frozen sperm is within 24-48 hours after a positive LH surge as detected by an Ovulation Predictor kit. In a well-designed study, using first positive OPK results to time insemination, 5% of total pregnancies resulted in cycles where the IUI was done within 24 hours of the positive OPK result, 90% of total pregnancies if within 24-48 hours, and 5% of total pregnancies if past 48 hours (5). Quite a few studies have evaluated the minimum number of inseminated sperm required to achieve an adequate pregnancy rate. Most indicate a total motile count between 6-15 million. This means that after thawing the frozen sperm specimen, the lab must recover between 6-10 million moving sperm. Most sperm banks provide a post thaw guarantee of 10-15 million/vial if prepped for an IUI (sperm already washed), or 15-20 million/vial if prepped for an ICI (unwashed sperm).

Next consideration addresses sperm washing techniques. There are a number of different laboratory techniques for washing and preparing sperm for insemination. As it turns out, there is no difference in pregnancy rates based on the sperm preparation technique. This holds for both the freezing technique and the post thaw washing technique (if ICI prepped) (6). This also applies if the sperm is pre-washed by the laboratory prior to freezing (if IUI prepped) (7). As long as an adequate TMC is reached post freeze-thaw, pregnancy rates hold steady.

The last consideration is: would one IUI per cycle reach adequate pregnancy rates, or would 2 IUI’s be better? Many studies have been done evaluating this question, and while individual studies may show different results, the majority of studies indicate that one IUI/cycle is adequate, and 2 IUI’s does not improve pregnancy rates, as long as the IUI is well timed, and the TMC inseminated is adequate (2, 8, 9, 10, 11).

In conclusion: We take guidance from the best published literature, and use the following guidelines for managing frozen sperm intrauterine insemination cycles at Pacific Fertility Center:

• Determine best timing of intrauterine insemination or IUI:
  First positive ovulation predictor kit (OPK) if OPKs are reliable, or HCG injection as administered according to our instructions.
• Do one IUI 24-48 hours after first positive OPK, or 24-48 hours after administration of HCG
• Do intrauterine insemination (not intracervical insemination or ICI)
• Assure insemination with adequate total motile count or TMC
  We will thaw sperm until we have a TMC of 10 million

If attention is paid to these management points during your treatment cycle, you should feel reassured that your chances of achieving a pregnancy is comparable to those if you were using fresh sperm.

– Isabelle Ryan, M.D.

Footnotes

1. Goldberg et al, Fertil Steril. 1999 Nov; 72(5):792-5

2. Carroll et al, Fertil Steril. 2001 Apr:75(4):656-60

3. Subak et al, Am J Obstet Gynecol. 1992 Jun; 166:1597-604

4. Bordson et al, Fertil Steril. 1986 Sept;46(3):466-9

5. Khattab et al, Hum Reprod. 2005 Sep;20(9):2542-5

6. Byrd et al, Fertil Steril. 1994 Oct;62(4):850-6

7. Wolf et al, Fertil Steril. 2001 July;76(1):181-5

8. Centola et al, Fertil Steril. 1990 Dec;54(6):1089-92

9. Lincoln et al, J Assist Reprod Genet. 1995 Feb;12(2):67-9

10. Khalifa et al, Hum Reprod. 1995 Jan;10(1):153-4

11. Matilsky et al, J Androl. 1998 Sept-Oct;19(5):603-7

Once patients have completed infertility treatment, a decision must be made about what to do with their excess embryos. Pacific Fertility Center offers three different forms of embryo disposition, and it is important for patients to know about the different options. Excess embryos can be discarded, donated to our Embryo Donation Program, or donated to research.

Creating embryos is a time-consuming, emotional, and expensive experience; sometimes patients do not want to simply discard their embryos. Instead, they anonymously donate them to our Embryo Donation Program, which provides a registry of embryos available for adoption. Patients are matched based on ethnic preference and length of time they’ve been on the embryo donation waiting list. Many couples on the embryo donation waiting list have been through failed cycles. It is comforting to know that they have other family-building options available. This is made possible through the generosity of fellow PFC patients, who have experienced the emotional roller coaster of infertility, and have donated their excess embryos to our Embryo Donation Program.

Sometimes patients do not feel comfortable donating embryos to the donation program. Another option for them to consider is offering their embryos to research, namely for embryonic stem (ES) cells. ES cells are important for the future of medical research, with many believing that some day ES cells will be able to treat cancer, spinal cord injury, and muscle damage, for example. Currently, the biggest challenge for researchers in this field is the availability of embryos. Should patients decide to donate to research, the embryos are transferred to UC San Francisco, where they are kept in a tissue bank until used.

There are three research options in which the embryos might be used:
Option 1 – allows the researcher to analyze proteins and copy genes from the embryos.
Option 2 – develops human ES cells from the embryos.
Option 3 – permits the investigator to contribute to the creation of immune-compatible human ES cells
using somatic cell nuclear transfer.

Due to the important value of each embryo, there are scientific and ethical boards that review different protocols, ensuring that the embryos are used only in the most important investigations.

About Embryonic Stem (ES) Cells
Embryonic stem (ES) cells are derived from a group of inner cells within the inner cell mass within the 5 or 6 day old embryo. Naturally, ES cells are programmed to continuously divide without developing into specialized tissues, a process called differentiation. These cells are pluripotent – meaning that under the influence of certain physiological signals, they can differentiate to form the three primary germ layers: the ectoderm, mesoderm, and endoderm of the developing fetus. Ultimately, they can form any tissue in the body, excluding the placenta. Because they are so versatile, ES cells are extremely important for research in treating diseases that currently have no solutions.

As more and more embryos become available for research, investigators are able to better explore the current problems with ES cells and are able to develop techniques to maximize successful treatments. Like organ donation, immune rejection poses a threat to potential stem cell therapy patients. The current ES cell lines that are available have a very small window of genetic diversity. This is a problem because it means that the number of patients who will match the cells and be able to use them is also small. By providing a greater number of embryos for research, genetic variety will continue to increase, allowing more patients to avoid immune rejection and benefit from stem cell therapies.

In addition to therapy, ES cells are important in other aspects of research. Because of the complex interactions that occur during human development, different problems can lead to cancers or birth defects. ES cells can be used as a research model to test how the differentiation process occurs. A detailed understanding of how such molecular processes occur would help aid in creating new drugs and therapies for a greater number of diseases. ES cells pose a promising future and it is important to develop as many investigations as possible using these cells.

Whether it be to help a fellow patient conceive or to further medical research, embryo donation provides significant short-term and long-term benefits. We encourage patients to consider the different options for embryo disposition and consult their physician for more information.  Talia Mota

Talia Mota, Laboratory Manager works with intended parents who are participating in Pacific Fertility Center’s Embryo Donation Program by matching them up with embryos and answering their questions. Her strong scientific background includes four years of laboratory research and a B.S. degree in Genetics from UC Davis.

Pacific Fertility Center is pleased to provide its final IVF laboratory statistics for 2005. (This is the most recent data from our clinic and has not yet been reported to CDC/SART. This data includes undelivered pregnancies. The pregnancies counted as positive include all pregnancies with a clear gestational sac on ultrasound examination. We urge caution when comparing these statistics to that of another center. Be advised that a comparison of clinic success rates may not be meaningful because patient medical characteristics and treatment approaches may vary from clinic to clinic.)

Comparing IVF programs by published statistics requires some insight into why programs may or may not be different. We have listed some key points to facilitate your understanding of our statistics.

Some factors associated with Pacific Fertility Center statistics:

1. Pacific Fertility Center does not restrict IVF to only those patients most likely to succeed (a practice which often leads to higher pregnancy rates). Our less restrictive approach is confirmed by our high percentage of DOR patients as described in point #2.
2. Over the years, PFC has treated a substantial number of IVF patients diagnosed with Decreased Ovarian Reserve, DOR (a basal FSH level of 10 mIU/mL or higher). As reported by SART/CDC, in 2003 and 2004, 28% and 22% of PFC patients, respectively, were diagnosed with DOR.
3. PFC performs a substantial volume of IVF and ovum donor cycles. This allows for better statistical accuracy of our data, (the fewer number of patients – the less statistically significant the rates become). We feel it keeps all of us well-attuned to the practice of ART.
4. PFC’s non-donor egg success rates with frozen embryo transfers approaches that of fresh embryo transfers. We have had a very strong embryo freezing program for many years and are proud of this. Our patients can avoid high order multiple pregnancies by transferring fewer fresh embryos and successfully freezing the remaining embryos. They may also increase the odds of having more than one pregnancy from a single IVF cycle.

FRESH EMBRYO TRANSFER CYCLES
Table 1: IVF with Own Eggs

Table 2: IVF with Ovum Donor

FROZEN EMBRYO TRANSFER CYCLES
Table 3: IVF with Own Eggs/Frozen Embryo Transfers

Table 4: IVF with Ovum Donor

An individual’s chances for success are based on a variety of factors including age, diagnosis and choice of treatment. We will be happy to discuss any questions you may have and estimate your individual chances of success.