These tests can be done by your primary care physician or gynecologist prior to consulting your Reproductive Endocrinologist:

• Day 3 FSH (follicle stimulating hormone) and Estradiol (Day 2-3 is acceptable)
• TSH (thyroid stimulating hormone)
• Prolactin
• Progesterone: 7 days prior to menses, this test is occasionally helpful
• Semen analysis

These tests may be useful based on each patient’s particular needs:

• Hysterosalpingogram (HSG) or documentation of tubal status
• Hysteroscopy
• Laparoscopy: The surgeon should be able to treat during this procedure, not just diagnosis.

The following treatments may be done, if indicated, for a limited number of cycles:

• IUI (intrauterine insemination)
• Clomiphene citrate (Clomid, Serophene)

At Pacific Fertility Center, we bring a complete team of specialists together to focus on your fertility situation. With extensive backgrounds as REI specialists, embryologists, nurses, marriage and family therapists and financial counselors, we develop a single, integrated solution to your medical, psychological and financial needs.

Please use our Ask the Experts resource if you have further questions.

– Philip Chenette, MD

These tests are best done through your Reproductive Endocrinologist (fertility specialist):

• Strict sperm morphology
  Strict morphology is a very specific method of evaluating the shape of sperm. Most laboratories do not use strict criteria thus potentially missing a sperm problem. Our laboratory is staffed with embryologists trained to analyze sperm with these strict criteria.
• Evaluation of ovarian reserve
  Evaluation of ovarian reserve includes family history, ultrasound to detect the antral follicle count (AFC), a cycle day 2-3 FSH and estradiol level (both must be done at the same time), Anti-mullerian Hormone AMH, and clinical and family history.  An REI can bring all of these assessments together into one consistent picture of a woman’s ovarian reserve.
• Ultrasound
  A pelvic ultrasound is a very useful test when it is done at the appropriate time in the menstrual cycle. A few days prior to ovulation an ultrasound can evaluate ovulation, follicle growth, endometrial thickness and pattern, polyps, and fibroids. During menses is the best time to evaluate the ovary for cysts and endometriosis.
• Genetic testing
  Genetic testing is important in women with premature menopause and multiple miscarriages and men with very low sperm counts.  Patients with a family history of a genetic disease can use genetic testing to determine if they are carriers of the disease.  Universal genetic testing (Counsyl, www.counsyl.com) can be used to assess risk for certain genetic illnesses that run in families. If detected, Preimplantation Genetic Diagnosis (PGD) can help prevent genetic illness in your child.
• Insulin
  Women who have irregular periods and have been told they have Polycystic Ovary Syndrome (PCOS) should be evaluated by an REI.  Testing can lead to more effective treatment.

Treatments by a fertility specialist

The advanced training of an REI is helpful to provide the most successful treatments for infertility.

Some of these treatments include:

• In vitro fertilization with embryo transfer (IVF, or IVF/ET),
• Fertility preservation
• Egg freezing
• Intracytoplasmic sperm injection (ICSI)
• Preimplantation genetic diagnosis (PGD)
• Ovulation induction
• Intrauterine insemination

A specialist is able to evaluate simpler treatments and finely tune them to make them more effective. For example, a specialist can monitor ovulation induction with clomiphene (Clomid) with ultrasound and blood tests. The vaginal ultrasound can be used to assess follicle development and endometrial pattern and thickness. Intrauterine inseminations can be done to bypass hostile mucus caused by clomiphene. The specialist can also help decide when to stop a particular treatment and/or proceed with more.

Alternative medications like letrozole (Femara) are just as effective as clomiphene but have fewer side effects.  Since letrozole is not approved by the FDA for marketing for fertility use, its use is generally restricted to specialty clinics, that is, REIs.

Gonadotropins, the injectable drugs, for example Follistim, Gonal-F, Bravelle, and Menopur, are potent stimulants to the ovary.  They are designed to produce multiple follicles, in order to improve pregnancy rates.  Due to the risk of multiple pregnancy and overstimulation of the ovaries, the medications should be used only by experts in the field.  Most of these treatments are performed by REIs in the United States.

At Pacific Fertility Center, we bring a complete team of specialists together to focus on your fertility situation. With extensive backgrounds as REI specialists, embryologists, nurses, marriage and family therapists and financial counselors, we develop a single, integrated solution to your medical, psychological and financial needs.

Please use our Ask the Experts resource if you have further questions.

– Philip Chenette, MD

One of the biggest challenges we face as fertility medicine specialists is how to do more to help our least-likely-to-succeed patients. What I mean here is the 42-and-over age group, patients with high FSH levels (decreased ovarian reserve), patients with very low responses to fertility medications, or those with very poor quality eggs. Some patients have a combination of the above which leads to a really dim prospect of having a baby with their own eggs.

Some people get the impression that fertility clinics avoid these patients like they have a communicable disease. They get the impression that we try to cherry pick patients to keep success rates high and make the CDC stats look good.  My impression from talking to my colleagues across the country and certainly from our own practice is that we do not try to discourage patients with poor possibilities from making a consult appointment and discussing treatment options. We all have such patients. In fact, we have so many of them at PFC, I don’t think we would have many patients at all if we tried to pre-select our best prognosis patients for IVF. When it comes to treatment, although there are challenges and sometimes the rewards are few, we don’t just throw up our hands and give up. We try to come up with a strategy to achieve the goal, looking at the emotional reserves and financial resources we have to work with, and start by making a plan.

Sometimes that plan will be to try a couple of cycles of low-tech approach, like just intrauterine insemination or Clomid + insemination, or a mid-level approach, like injections of FSH along with  insemination. We would see how things go and play it by ear from there. Sometimes, the plan will be to blast ahead to the big guns, full steam ahead to IVF. Sometimes, it’s counseling with our marriage and family therapist to begin the discussion: are we ready to move on to donor eggs? Sometimes it’s a sequence of all of the above. There really is no one plan for any one person. It’s just too complex to say one size fits all.

A certain percentage, even of the-less-likely-to-succeed patients will get pregnant with their own eggs and go on to deliver a healthy baby. The remainder may be faced with a tough decision. Do we just stop here and live child-free?  There are certain perks to that plan (sleeping in on the weekends, eating in nicer restaurants, adult vacations to name just a couple) but most people want to have a family no matter what or how. So then there is the adoption vs. egg donation question. There is no right or wrong choice here, either: just choices.

At Pacific Fertility Center we aim to help our patients build a healthy family. To build healthy families, maximum pregnancy rates are a goal, but maximum pregnancy rates must be balanced by consideration of risk, the chance of an adverse outcome. High pregnancy rates with minimal risk is PFC’s goal.

The risk of multiple pregnancy has increased as fertility therapy has improved. The wider use of gonadotropins in the 1990s to induce ovulation of multiple follicles, as well as the use of more effective laboratory and clinical IVF methods, resulted in production of more and healthier oocytes and more embryos, and increased the chances of multiple pregnancy. The very dramatic improvement in success rates over this time period resulted in many more children being delivered after fertility therapies, but also more twins, triplets, and higher order multiples.

Over the last twenty years, the incidence of multiple birth has increased nationally. According to the National Vital Statistics Report and the March of Dimes, the incidence of twins has increased by two-thirds, and the number of triplets and quadruplets has increased four-fold since 1980.

It is thought that about one-third of multiple pregnancies arise because women are waiting until later in life to conceive; age is a well-known risk factor for multiples. Another third arise from use of ovulation induction with gonadotropins (Pergonal, Follistim, Gonal-F, Repronex) alone. Less than one fifth of multiples are from assisted reproduction techniques (IVF and related procedures). Assisted reproduction in 2003 accounted for 18% of multiple pregnancies, 16% of twins and 44% of triplets ¹.

The risks to the children of multiple pregnancy are numerous. Low birth weight and very low birth weight are increased in children born as multiples. The chance of low birth weight (<2500g) is increased 8 times in twins. Cerebral palsy is increased 4 times, neonatal death risk by 7 times ^{2, 3}.

The risk to the mother from multiple pregnancy is also increased. Pre-eclampsia, high blood pressure, preterm labor, and premature rupture of membranes are all more common with multiple pregnancy ⁴ .

Multiple pregnancy is also expensive. It is estimated that twins alone cost the healthcare system some $600,000,000. There is clear evidence of an increase in parenting stress and divorce in families of multiples ^{5, 6} .

The need to assure our patients of the highest quality care requires that we bear this in mind – the healthiest pregnancy is a singleton pregnancy.

Pregnancy requires the cooperation of sperm and egg, accurate transcription of the early genetic code in the developing embryo, a fertile spot for attachment to the mother in the uterus, and a route for getting there. All other factors being equal, pregnancy rates almost double when two embryos are transferred instead of one, and increase again when a third and fourth embryo are added. The desire for high pregnancy rates has driven a desire for more embryos to be transferred ⁷ .

Improvements in insemination technique, embryo culture methods, and transfer efficiency have added substantially to pregnancy rates. Each embryo transferred today has a considerably higher chance of producing a pregnancy than an embryo transferred twenty years ago. Such improvements have enabled us to think about ways to reduce the risk of multiple pregnancy by transferring fewer embryos.

The development of blastocyst (day 5 embryo) culture techniques allows the selection of high quality embryos for transfer. The blastocyst stage requires advanced incubation techniques with low oxygen incubators and specialized culture media. A tight quality control system is also required. The blastocyst stage is a more advanced stage in which the genetic code of the embryo is fully activated and working. Only the healthiest of embryos can move to the more advanced stages, allowing selection of the best embryos for transfer.

In 2006 the ASRM published guidelines for number of embryos to transfer:

These guidelines encourage all of us to transfer ‘just enough’ embryos to achieve pregnancy.

Pacific Fertility Center has pioneered techniques of transferring fewer embryos. Last year, in 2007, our program of single embryo transfer in oocyte donation recipients produced a 66% pregnancy rate. The multiple pregnancy rate in this group was minimal. Utilizing a single embryo, two-thirds of patients were able to conceive a singleton pregnancy. This pregnancy rate was very similar to the overall pregnancy rates regardless of the number of embryos transferred.

Today half of our patients using oocyte donation elect to transfer a single embryo. Single embryo transfer is not always possible. Our criteria include age and embryo quality. A young woman (under age 35) with high quality blastocyst stage embryos and a healthy uterus can reliably transfer a single embryo and achieve high pregnancy rates. An older woman (over 40) may need to transfer 3 or more embryos to achieve a good pregnancy rate. Because of the higher number of embryos transferred, the risk of multiple pregnancy remains higher in these older age groups⁹ .

Pacific Fertility Center is very pleased to offer these techniques of single embryo transfer as some of the best and most advanced fertility treatment technology available. We are moving closer to our goal of growing families, one healthy baby at a time.   Philip Chenette, MD

1. Martin, Births: Final Data for 2003. National Vital Statistics Reports, volume 54, number 2, 2005
2. Scher, Ped Res, Vol. 52:671-81, 2002
3. Rutter, J Child Psychol Psych, Vol. 44:326-41, 2003
4. Pinborg, Human Reproduction, Vol. 18:1234-43, 2003
5. Griesinger, Hum Reproduction, Vol. 19:1239-1241, 2004
6. Glazebrook, Fertil Steril, Vol. 81:505-11, 2004
7. Paulson RJ, Fertil Steril., Vol. 53:870-874 , 1990
8. Fertil Steril, Vol. 85, Suppl. 4, 2006
9. Pacific Fertility Center 2007 IVF Statistics

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Every year, several Pacific Fertility Center professionals participate in ASRM’s national meeting. They evaluate the research and share their findings with PFC and Fertility Flash.

Among those attending the conference from PFC were Dr. Philip Chenette and Dr. Isabelle Ryan and Peggy Orlin, MFT. Their reviews cover the following topics: Update #1: Ovarian Stimulation Techniques, Update #2: PGD and Aneuploidy Screening Techniques, Update #3: Egg Freezing, Update #4: Acupuncture, and Update #5: Men and ART.

Update #5 Men and ART

The Mental Health Professional Group (MHPG) course entitled Men and ART: The Missing Voice, blended medical, psychological, ethical and legal information relating to men who participate in Assisted Reproductive Technology (ART).

The legal issues confronting single men and gay men considering the use of egg donors and gestational surrogates continue to be controversial. Adoption legislation in many states prohibits gays and lesbians from adopting. In a study reported in 2005 by Gurmankin, et. al, 44% of ART programs responded that they would not turn away gay couples seeking surrogacy with one partner’s sperm and 48% responded that they would turn them away. This is in contrast to the higher rate of acceptance of lesbian couples. In lesbian couples seeking treatment using donor insemination, 82% of ART programs agreed to treat versus 17% who refused to treat them.

Though often presented exclusively to women, men can also benefit from the use of stress reduction strategies and following a healthy life style which includes regular exercise, normal body weight, no smoking or recreational drug use and avoidance of environmental toxins. In addition, the effects of aging and cancer on sperm quality should not be overlooked when men seek reproduction assistance. (See articles on: Sperm Aging: Fertility Flash Feb. 2004, Sperm Fragmentation: Fertility Flash March 2005, Cancer and Infertility: Fertility Flash Oct. 2004).

The psychological component of this course was compelling. Approximately 50% of cases of infertility involve at least some degree of male infertility. Why is it that most infertility references are traditionally directed at women? By definition, Infertility is “…the inability of a woman to conceive after some months (12-24) without contraception, or the inability to carry a pregnancy to term.” (Institute of Medicine and National Research Council, 1989). Ancient biblical references and popular literature focus on women’s infertility – e.g. Sarah and Hannah in the bible, Sylvia Plath’s Barren Woman, Jane Smiley’s 1000 Acres. The list is long. Google hits by gender for infertility and psychology show 542,000 for men and 700,000 for women.

The cause of this discrepancy is multifaceted. There are fewer psychological studies on men simply because men have a lower study response rate than women. A variety of successful techniques have been developed to overcome male related medical issues. Additionally, most men spend less time in treatment and experience fewer invasive procedures than women. In general, it is more socially acceptable for women to express their feelings regarding infertility. The opposite is true for men whose fertility often is a taboo topic. Furthermore, some cultures protect their men from the unacceptable stigma of infertility and even falsely describe men as having “poor” coping skills.

Despite these discrepancies, men do have feelings about infertility and may need support and assistance to better cope with the diagnosis. A study by Mason MC in 1993 found that men felt guilt, shame, anger, isolation, loss and a personal sense of failure. This is not all that different from what women feel, but each individual’s coping mechanism is unique. We all, however, find ways to protect ourselves from what we perceive as painful information.

These coping skills can be divided along gender lines. There are ways that many, but certainly not all, men commonly protect themselves from the pain related to his or his partner’s infertility diagnosis. Frequently men are able to distance themselves from the feelings. They appear to have the ability to take painful information and put it in a little box that they then file away in the back of their minds. The box stays tightly shut. Other men want to problem-solve for their partner or avoid the topic completely, throwing themselves into work or hobbies. Some men become extremely optimistic to avoid or counter their partner’s pessimism.

These are different styles- not right or wrong. For many of us, particularly women, the closed box technique does not work. The box is opened often, and feelings appear to refuse to stay tucked away. When partners have different coping styles, it’s important to both learn to tolerate and support these differences. Sometimes that is easier said than done…

Peggy Orlin, MFT

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

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

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

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

– Dr. Carolyn Givens

Introduction

Sara (a hypothetical patient) found a breast lump. 36 years of age, she was a single active professional, otherwise healthy, careful about her diet, and carefully evaluating her options after a diagnosis of breast cancer. Along with the discussion on surgery, chemotherapy, and radiation therapy came the question “Were you planning to have children?”

A diagnosis of cancer presents many decisions that must be made quickly. Confirming the diagnosis and planning therapy will be the primary concerns, but the implications of therapy on long-term quality of life must be assessed. One of the primary issues facing women with a diagnosis of cancer is future fertility.

Candidates

Cancer treatment can interfere with future fertility. Toxicity varies by treatment. Cyclophosphamide, an alkylating agent used in many chemotherapy regimens, is highly toxic to sperm and eggs; methotrexate and 5-flouro-uracil (5FU) are not. Medications used for longer time intervals create a higher risk of fertility problems than shorter time intervals; effects on women in older age groups are more severe than younger. Radiation therapy, in high doses, can have effects on eggs and sperm. Surgery and anesthesia are not known to have direct effects.

It is difficult to give specific fertility risks for chemotherapeutic regimens, since studies are not yet definitive. Among the more toxic treatments are stem cell transplantation for leukemia in which total body irradiation and cyclophosphamide are used, beam radiation to a field that includes the ovaries, and extended chemotherapy of up to 6 cycles using cyclophosphamide in combination with other agents. After conventional chemotherapy for breast cancer for women under 40, the chance of infertility is roughly 50%, in older women the risk is over 80%.

Treatment options

What are the options for fertility in patients diagnosed with cancer? The best choices are available to those that have not yet initiated treatment and involve cryopreservation. During treatment, the risk of problems rises, and after treatment, there may not be adequate recovery of fertility to achieve pregnancy.

Cryopreservation allows cells to be stored with great stability for long periods of time. The record time from sperm cryopreservation to pregnancy is 28 years; there probably is no real limit to the time that cells can be stored. To store cells requires technology that reduces the formation of ice crystals, which disrupt cells, and prevents the rapid rise in salt concentration that occurs as water freezes. Cryopreservatives and management of temperature changes (slow freeze or vitrification) are used to reduce the risk of these problems.

Male

The option for fertility preservation in men is straightforward, cryopreservation of sperm. Sperm is obtained by masturbation and frozen in multiple vials in liquid nitrogen. 2-3 sperm samples can be obtained per week, with 2-4 vials stored per ejaculate; two weeks worth of donations could yield 8-24 vials of sperm. Costs vary widely, but would range from $1500-$3000 for processing and 3 years of storage.

Testicular sperm extraction is an option for individuals with azoospermia. Testicular tissue cryopreservation remains a theory that has not yet produced a human pregnancy. It has been proposed as an option for preservation of fertility in children, but has yet to be proven in clinical practice.

Female

Women have the option of cryopreservation of oocytes or embryos. For women without a partner, oocyte cryopreservation holds promise as a means to preserve fertility potential without committing to a specific sperm source or partner. For women with a partner or sperm donor, embryo cryopreservation is a proven technology.

To create cryopreserved oocytes, Follicle Stimulating Hormone (FSH) is administered over a ten day time period to stimulate ovarian follicles. The oocytes are retrieved under sedation with a needle guided by ultrasound and then stored in liquid nitrogen.

Newer techniques of oocyte vitrification secure good pregnancy rates for those with good oocyte quality. Traditional oocyte cryopreservation is performed using a slow freeze technique, but more rapid vitrification procedures optimize results. The trick with cryopreservation is to lower the temperature while avoiding ice crystals that disrupt cell membranes and proteins. Vitrification, an ultrarapid freezing process utilizing a minimal fluid volume, reduces the risk of these problems and optimizes cell quality.

For those women with a partner, or that are willing to commit to a specific sperm donor, embryo cryopreservation is an excellent option. After stimulation and retrieval, oocytes are inseminated and cultured in an incubator for 1-5 days, followed by cryopreservation. The embryos can be thawed and transferred at a later date, after clearance from the oncologist. Embryo cryopreservation is the best established of the fertility preservation techniques, with years of experience in its applications. Good pregnancy rates can be anticipated.

Ovarian tissue cryopreservation, the cryopreservation of whole pieces of the ovary, as opposed to cells, remains experimental. Complex tissues are more difficult to cryopreserve than cells, though rare success has been reported.

Cancer recurrence

Is there risk to the use of fertility drugs in patients with cancer? It does not appear in studies to date that breast or ovarian cancer risk is affected by use of fertility drugs. Studies indicating an increased risk are balanced by other studies indicating a reduction in risk. Studies to date have been limited, and treatment decisions still must be individualized.

Does pregnancy increase the risk of cancer recurrence? In theory, certain types of cancer could be aggravated by the hormones of pregnancy, but studies have not confirmed an overall risk. Certain types of cancer are less common in women that have delivered a pregnancy. Treatment decisions must be individualized, as future studies gather more information.

Pregnancy

Certain cancer treatments create organ toxicity that must be evaluated in considering patients for pregnancy. Heart output is limited in patients that have received doxorubicin. Uterine irradiation is associated with miscarriage and pre-term labor.

Children

Children born after fertility preservation procedures do not carry any increased risk for birth defects. There are hereditary syndromes that can be associated with cancer that could be transmitted to children, but there does not appear to be any other increased risk for cancer or genetic disease in children of cancer survivors.

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

Philip E. Chenette, MD

Resources:

www.fertilehope.org Fertile Hope

www.livestrong.org Lance Armstrong Foundation

www.cryobank.com California Cryobank

www.PacificFertilityCenter.com Pacific Fertility Center

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