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

Sperm are clearly sensitive to environmental conditions. It is possible, through changes in lifestyle and activity, to improve sperm health. The studies available to evaluate environmental effects are unfortunately limited, but they offer insight into sperm sensitivity and ways to optimize their performance.

Temperature The scrotum where sperm are produced is 2 degrees lower than core body temperature. Raising the temperature by a few degrees results in a decline in sperm count and motility. Men suffering from cryoptorchidism, where the testicles are located above the scrotum, closer to central body temperatures, frequently suffer from low sperm counts. Infertile men tend to have a higher scrotal temperature⁽¹⁾, a characteristic that seems to be genetically determined⁽²⁾.

Common illnesses and every day activities can be sources of an increase in scrotal temperature. Acute fever associated with illness causes a significant decline in sperm quality⁽³⁾. In one study, total sperm count decreased within two weeks after a fever and required 79 days to return to normal. The DNA component of these sperm showed high levels of DNA fragmentation. Researchers in France installed temperature sensors to nine volunteers, and recorded scrotal temperatures while driving⁽⁴⁾. Scrotal temperature increased gradually over several hours, peaking 2.5 degrees higher at three hours. Another study showed that scrotal temperature was lowest while standing naked, and highest while clothed, seated, with legs crossed⁽⁵⁾. Higher scrotal temperatures have been associated with use of a laptop computer⁽⁶⁾. A group in Germany looked at scrotal temperatures with a variety of underwear⁽⁷⁾. As expected, tight underwear increased the temperature more than loose or no underwear. The effect was most pronounced while walking and less noticeable while sitting, since sitting temperature was somewhat elevated regardless of type of underwear worn.

The common sense approach is to avoid activities which can increase scrotal and testicular temperature, use loose-fitting underwear, and provide adequate ventilation to the scrotum. Exposure to hot tubs or saunas should be avoided. Take showers rather than baths, because heat conductance is lower when the testicles are not immersed in hot water. Sitting or driving for extended periods should be minimized.

Stress The effects of stress on sperm are complex. Under conditions of extreme stress, sperm counts decline. Analyses of prisoners awaiting sentencing have shown complete suppression of spermatogenesis on testicular biopsies⁽⁸⁾. A study of semen characteristics after the Slovenian war in 1991 showed a reduction in sperm count and motility, and a reduction in the proportion of male children born⁽⁹⁾. In 1995 a strong earthquake of magnitude 7.2 on the Richter scale occurred in Kobe, Japan killing 5,502 people. Sperm motility declined immediately, with low motility lasting for months⁽¹⁰⁾. The sperm of a man who lost his home and his father had still not recovered 10 months after the earthquake.

Stress associated with fertility therapy affects sperm and sexual function. Sperm parameters may decline in patients undergoing in vitro fertilization⁽¹¹⁾. Male fertility patients have a higher incidence of erectile dysfunction, ejaculatory disorders, loss of libido and a decrease in the frequency of intercourse⁽¹²⁾. One study of infertility patients showed an increase in burnout in male patients⁽¹³⁾.

Unfortunately, studies of the effect of stress reduction on sperm are rare,^(14)(15) so the treatment of stress has not been conclusively shown to improve sperm parameters⁽¹⁶⁾. In spite of the lack of clear data, stress reduction therapy is recommended for fertility patients and may reduce problems with sexual dysfunction.

Exercise The risk of developing male fertility problems appears to increase with the intensity of exercise. Intense exercise, such as endurance running, will lower levels of luteinizing hormone (LH) and testosterone.^(17)(18) Studies of semen characteristics have shown variable results. DeSouza⁽¹⁹⁾ developed the concept of a training volume threshold, in which running more than 100 km or 62.14 miles per week was associated with decreased levels of testosterone and sperm motility.

A detailed prospective study comparing competitive cyclists and triathletes with sedentary controls⁽²⁰⁾ was unable to show any suppressive effect of competitive exercise on FSH, LH, or testosterone levels. Although those with the highest levels of training had higher levels of circulating testosterone at baseline, these levels did not change with training. Competitive cyclists developed lower sperm motility during competition, however, motility values returned to normal following competition.

The best advice regarding exercise and sperm is moderation. While attempting conception, it is not advisable to undergo high intensity sports training. Good nutritional standards should be always be maintained when following an exercise program. An existing maintenance exercise program may be continued without concern for its effects on sperm.

Diet is a difficult topic to study in isolation, so fertility data is limited. A recent study of beef consumption showed that maternal consumption⁽²¹⁾ of beef resulted in lower sperm concentrations in sons. The proportion of men with low sperm counts was three times higher in the sons of women that consumed high levels of beef. Lifestyle, pesticide exposure, and xenobiotics (chemicals found in organisms that are foreign to them) were all considered potential factors. Heterocyclic amines (carcinogenic chemicals formed from the cooking of muscle meats), which are estrogenic, may also play a role⁽²²⁾.

Alcohol has long been associated with male reproductive dysfunction. Impotence, infertility, and male secondary sex characteristics are all affected by chronic alcohol use. Testosterone levels are lower, sperm production is reduced, and FSH and LH levels are affected⁽²³⁾. A study of chronic alcoholics demonstrated low levels of pituitary and testicular hormones, and significantly decreased sperm concentration and morphology⁽²⁴⁾. Sperm chromosomes are altered in men that consume alcohol⁽²⁵⁾.

Little data exists on the moderate consumption of alcohol. Data from the Ontario Farm Family Health Study did not show an adverse effect of alcohol consumption⁽²⁶⁾. In another study, alcohol or cigarette consumption did not alter sperm parameters, but when patients both smoked and drank alcohol a significant reduction in seminal volume, sperm concentration, percentage of motile spermatozoa, and a significant increase of the nonmotile viable gametes were detected⁽²⁷⁾.

Smoking tobacco affects sperm parameters, with reduced sperm counts, motility, and morphology reported in several studies⁽²⁸⁾. Whether these changes affect the male fertility remains uncertain. According to ASRM, “The effect of smoking on male fertility is … difficult to discern. The available data do not conclusively demonstrate that smoking decreases male fertility… Few studies have or can address the question, because of the confounding effects of partner smoking habits and fecundity. Although sperm concentrations, motility, and/or morphology are often reduced compared to results observed in non-smokers, they often remain within the normal range. Nevertheless, to the extent that the zona-free hamster egg penetration test reflects the ability of sperm to successfully fertilize a human oocyte, the available evidence suggests that smoking may have adverse effects on sperm function.”

Caffeine studies have revealed inconsistent effects on sperm, with at least one study showing no effect⁽²⁹⁾. Caffeine has been used as a sperm stimulant, increasing the motility prior to insemination. There does not appear to be any substantial adverse effect of caffeine on sperm.

Common Medications The list of medications with effects on sperm is long, and worthy of review. Noteworthy medications are the SSRI anti-depressants (Cipramil, Lustral, and Effexor were the reported medications), which were associated with near-azospermia in a case report⁽³⁰⁾. Ibuprofen (Advil, Nuprin) does not seem to cause adverse effects on sperm⁽³¹⁾.

Vaginal lubricants can interfere with sperm. FemGlide, Replens, and Astroglide lubricants demonstrated a significant decrease in motility, whereas Pre-Seed did not affect motility or DNA integrity⁽³²⁾.

Treatments for erectile dysfunction may have an effect on sperm motility. A significant increase in sperm progressive motility was observed after sildenafil (Viagra) administration as compared with baseline; in contrast, a significant decreased motility was observed after tadalafil (Cialis).

Antihypertensive drugs have numerous effects on sperm. Beta-blockers and diuretics have been associated with impotence. Calcium channel blockers (nifedipine, Procardia) have been associated with infertility⁽³³⁾. If you are on heart medications, review them with your physician.

Reports on the effects of marijuana use on sperm are conflicting. Early studies had poor controls, later studies showed reductions in testosterone and sperm quality⁽³⁴⁾ while other studies showed no effect on testosterone levels in chronic heavy smokers⁽³⁵⁾. A recent study revealed a direct effect of THC, the active ingredient in marijuana, on sperm motility and fertilization capacity⁽³⁶⁾. The conclusion of the study was that “the use of THC as a recreational drug may impair crucial sperm functions and adversely affect male fertility, especially in those who are already on the borderline of infertility.”

Conclusion Sperm are a biological substance, produced in a complex interplay of genetic predisposition, specific temperature and pH, and in association with specific cells and secretions. If the system is insulted, problems will often arise. The sheer numbers of sperm in an ejaculate provide a wide margin for maintaining fertility even after such insults occur, but repeated attacks on the reproductive system can ultimately result in male fertility problems.

Philip Chenette, MD

References:

1. Zorgniotti, A.W. and Sealfon, A.I. (1988) Measurement of intrascrotal temperature in normal and subfertile men. J. Reprod. Fertil., 82, 563–566.
2. Hjollund, N., Storgaard, L., et al. (2002) Correlation of scrotal temperature in twins: Brief Communication. Human Reproduction, 17(7):1837-1838.
3. Sergerue, D.E.S.S., et al., (2007) High risk of temporary alteration of semen parameters after recent acute febrile illness. Fertil Steril, In press.
4. Bujan L, et al. (2000) Increase in scrotal temperature in car drivers. Human Reprod 15, 1355–1357.
5. Mieusset, R. et al., (2007). Effect of posture and clothing on scrotal temperature in fertile men. J Androl. 28(1):170-175.
6. Sheynkin, Y., et al., (2006) Increase in scrotal temperature in laptop computer users. Human Reproduction. 20(2):452-455.
7. Jung, A., et al. (2005) Influence of the type of undertrousers and physical activity on scrotal temperature. Human Reproduction. 20(4):1022-1027.
8. Steve, H. (1952) Der ein Fluss de nerven System auf ban und Fatigkeit des Geschlechtorgane des Menschen. Theim, Stuttgart.
9. Zorn, B et al., (2002) Decline in sex ratio after 10-day war in Slovenia. Human Reproduction.17(12):3173-3177.
10. Fukuda, M, et al. (1996) Kobe earthquake and reduced sperm motility. Human reproduction. 11(6):1244-1246.
11. Clarke R.N., et al., (1999) Relationship between psychological stress and semen quality among in vitro fertilization patients. Human Reproduction. 14(3):753-758.
12. Lenzi, et al. (2003) Stress, sexual dysfunctions, and male infertility. J Endocrin Invest. 26(3 Suppl):72-6.
13. Sheiner, et al., (2002) Potential association between male infertility and occupational psychological stress. J Occup Environ Med. 44(12):1093-1099.
14. Pook, M, et al. (1999). Coping with infertility: distress and changes in sperm quality. Human Reproduction. 14(6):1487-1492.
15. Tuschen-Caffier B, Florin I, Krause W, Pook M. (1999) Cognitive-behavioural therapy for idiopathic infertile couples. Psychother Psychosom 68:15–21.
16. Campagne, D.M., (2006) Should fertilization treatment start with reducing stress? Human Reproduction. 21(7):1651-1658.
17. Wheeler, G. D., et al. (1991) Endurance training decreases serum testosterone levels in men without change in luteinizing hormone pulsatile release. J. Clin. Endocrinol. Metab. 72: 422–425.
18. Arce, J. C., et al. (1993) Subclinical alterations in hormone and semen profile in athletes. Fertil. Steril. 59: 398–404.
19. De Souza, M. J., et al. (1991) Gonadal hormones and semen quality in male runners. A volume threshold effect of endurance training. Int. J. Sports Med. 15: 383–391.
20. Lucia, A, et al. (1996) Reproductive function in male endurance athletes: sperm analysis and hormonal profile. J Applied Physiology. 81:2627-2636.
21. Swan SH et al (2007) Semen quality of fertile US males in relation to their mothers’ beef consumption during pregnancy. Human Reproduction. 22(6):1497-1502.
22. Cho E, Chen WY, Hunter DJ, et al. (2006) Red meat intake and risk of breast cancer among premenopausal women. Arch Intern Med 166:2253–9.
23. Emanuele, MA et al. (1998) Alcohol’s effects on male reproduction. Alcohol Health and Research World. 22:195-201.
24. Muthusami, KR et a;, (2005) Effect of chronic alcoholism on male fertility hormones and semen quality. Fertility and Sterility. 84(4):919-924.
25. Robbins, WA, et al. (2005) Effect of lifestyle exposures on sperm aneuploidy. Cytogenetic & Genome Research. 111(3-4):371-7.
26. Curtis KM, et al. (1997) Effects of cigarette smoking, caffeine consumption, and alcohol intake on fecundability. Am J Epidemiol. 146(1):32-41.
27. Martini, AC, et al. (2004) Effects of alcohol and cigarette consumption on human seminal quality. Fertility Sterility. 82(2):374-377.
28. Vine MF. (1996) Smoking and male reproduction: a review. Int J Androl.19:323–337.
29. Klonoff-Cohen, H, et al. (2002) A prospective study of the effects of female and male caffeine consumption on the reproductive endpoints of IVF and gamete intra-Fallopian transfer. Human Reproduction. 17(7):1746-1754.
30. Tanrikut C, Schlegel PN (2006) Antidepressant-associated changes in semen parameters. Fertil Steril. 86(3):S14.
31. Robinson, N, et al. (2005). Regular Use of Ibuprofen Does Not Affect Semen Analysis Parameters, Need for ICSI, or ART Clinical Pregnancy Rate. Fertility and Sterility (84): S14.
32. Agarwal A, et al., (2007) Effect of vaginal lubricants on sperm motility and chromatin integrity: a prospective comparative study. Fertil Steril. In press.
33. Hershlag A, et al. (1995) Pregnancy following discontinuation of a calcium channel blocker in the male partner. Human Reproduction. 10(3):599-606.
34. Kolodny RC, et al. (1974) Depression of plasma testosterone with acute administration. In: Braude MC, Szara S editor. The pharmacology of marijuana. New York: Raven Press; p. 217–225.
35. Mendelson JH, et al. (1974). Plasma testosterone levels before, during and after chronic marihuana smoking. N Engl J Med. 291:1051–1055.
36. Whan, LB, et al., (2006) Effects of delta-9-tetrahydrocannabinol, the primary psychoactive cannabinoid in marijuana, on human sperm function in vitro. Fertil. Steril. 85(3):653-660.

Polycystic ovary syndrome (PCOS) is the most common endocrinologic disorder in women of reproductive age. Approximately 5-10% of reproductive age women have PCOS. The various symptoms of PCOS can be irregular or absent menstrual cycles, infrequent or absent ovulation, excess facial and body hair, obesity, and infertility. The key components defining this disorder are chronic anovulation (inability to ovulate an egg), clinical hyperandrogenism (elevated male type hormones) and more recently discovered, insulin resistance.

Insulin resistance, the precursor state to diabetes, is present in 35-40% of women with PCOS, even if they are not overweight. Insulin resistance is diagnosed by blood testing, either as fasting glucose to insulin ratio, or as a complete glucose tolerance test. Long term follow up of women with PCOS reveals that up to 40% develop impaired glucose processing or diabetes by age 40. The prevalence of diabetes in women with PCOS is seven times higher than for the non-PCOS population. Excessive insulin production is thought to promote excess male hormone production, though the actual mechanism explaining this observation is still unclear. Insulin resistance may increase the long-term risks of heart disease and hypertension.

Interventions that reduce circulating insulin levels in women with PCOS may restore normal reproductive endocrine function. Non-pharmacologic methods, such as weight loss and exercise, have clearly led to reduced insulin and male hormone levels, resulting in resumption of ovulatory function. However, these regimens are at risk for poor compliance and, over time, the benefit of weight loss is rarely maintained.

Insulin-sensitizing (anti-diabetic) medications can be used to decrease insulin levels, which may help restore the normal ovarian hormone profile (i.e. reduce male hormone), thus allowing for spontaneous ovulation to occur in about 75% of patients. The most commonly used medication is metformin (Glucophage®). Side effects of metformin include gastrointestinal symptoms, which are dose-related and tend to resolve after several weeks. While there are no well-controlled studies of safety during pregnancy, metformin has been administered to a small number of women with diabetes throughout their pregnancies, and no fetal abnormalities have been described⁽¹⁾.

Clinical studies have shown that metformin (500 mg three times per day or 850 mg twice daily with meals) administration to women with PCOS increased the frequency of spontaneous ovulation, menstrual cyclicity, and ovulatory response to clomiphene citrate (CC) (Clomid^®). Benefit has been demonstrated with metformin treatment in PCOS patients both with and without insulin resistance⁽²⁾. Metformin alone may be less effective in obese PCOS women.

Women with PCOS are considered to be at increased risk of miscarriage, as high as 30 – 50 %. When women were treated with 1000-2000 mg daily of metformin throughout pregnancy, rates of early pregnancy loss were 11.6% in the metformin group compared with 36.3% in the control group (p < 0.0001). Administration of metformin throughout pregnancy to women with PCOS may decrease miscarriage rates⁽³⁾.

Controversy exists when comparing metformin to clomiphene citrate (CC) for treating infertility. A well-designed study showed metformin is better for ovulation induction than CC alone and equivalent for pregnancy achievement. The authors suggest that metformin can be used first for ovulation induction in patients with PCOS regardless of their weight and insulin levels because of its efficacy and known safety profile⁽⁴⁾. Alternatively, another study found benefit with metformin if obese (BMI >30 kg/m⁽²⁾ subjects and women older than 34 years were excluded⁽⁵⁾. Another paper pooled the results of 6 studies to examine whether metformin is efficacious when given to patients resistant to CC. They found the addition of metformin in the CC-resistant patient is highly effective in achieving ovulation induction⁽⁶⁾. Most studies showing benefit were small with fewer than 100 patients.

Conversely, two large multicenter trials, one conducted in the US (PPCOS)⁽⁷⁾ and one in the Netherlands⁽⁸⁾, have shown no benefit from metformin either as a single agent or as adjuvant therapy in combination with clomiphene for the treatment of infertility in women with PCOS. They found metformin increased the occurrence of ovulation but did not increase the chance of becoming pregnant. The PPCOS study is large and well designed, with 626 participants. It differs from other studies by using the extended release form of metformin. One very notable result was the absence of any statistically significant effect of this extended release form of metformin on insulin levels or insulin resistance. There were none of the expected metabolic effects of metformin. Extended-release metformin has not previously been studied in women with PCOS. Thus, it has not been ascertained that its efficacy is comparable to regular metformin in PCOS⁽⁹⁾.

Additionally, metformin and clomiphene citrate (CC) differ in their therapeutic time frames (the period of time from initiating therapy to achieving maximum effectiveness). CC produces higher rates of ovulation and pregnancy in the early months of treatment than that of metformin and might be preferable to women who wish to become pregnant quickly ⁽⁵⁾. However, a patient with more time to become pregnant may benefit from metformin’s metabolic effects. During the 3 to 6 months that it takes for metformin to become maximally effective, the patient can prepare for pregnancy by losing weight through diet and exercise. Reducing a patient’s weight might considerably optimize her pregnancy⁽⁹⁾.

Metformin induces normal ovulation, and the risk of multiple gestation is no more than that in the general population. Conversely, CC can precipitate the release of multiple eggs in a given menstrual cycle and carries a risk of multiple gestation: in the PPCOS study, multiple gestation was 6% in the clomiphene group and 0% with metformin.

Metformin may significantly increase the incidence of multiple pregnancy when used in combination with gonadotropins⁽¹⁰⁾.

Short-term co-treatment with metformin for patients with PCOS undergoing IVF/ICSI cycles does not improve the response to stimulation but significantly improves the pregnancy outcome and reduces the risk of ovarian hyperstimulation⁽¹¹⁾.

Conclusions:

• PCOS patients should be screened for diabetes before becoming pregnant. Hemoglobin A1c levels should be normal.
• Metformin alone can induce ovulation and may improve the effectiveness of CC. Extended release metformin may not be as effective.
• Metformin may decease miscarriage rates.
• Weight loss may improve the effectiveness of metformin.
• Time to achieve pregnancy may be longer with metformin than CC.
• Metformin may be less effective in older women.
• Metformin does not increase multiple pregnancy rates when used alone.
• Metformin may increase multiple pregnancy rates and decrease ovarian hyperstimulation when used with gonadotropins.
• Long-term benefits of metformin in preventing hypertension and heart disease need further study.

Eldon Schriock, MD

References:

1. The Practice Committee of the American Society for Reproductive Medicine Committee Opinion. Use of insulin sensitizing agents in the treatment of polycystic ovary syndrome. Fertility and Sterility
2. Nawrocka J, Starczewski A. Effects of metformin treatment in women with polycystic ovary syndrome depends on insulin resistance. Gynecol Endocrinol. 2007 Apr;23(4):231-7.
3. Khattab S, Mohsen IA, Foutouh IA, Ramadan A, Moaz M, Al-Inany H. Metformin reduces abortion in pregnant women with polycystic ovary syndrome. Gynecol Endocrinol. 2006 Dec;22(12):680-4.
4. Neveu N, Granger L, St-Michel P, Lavoie HB. Comparison of clomiphene citrate, metformin, or the combination of both for first-line ovulation induction and achievement of pregnancy in 154 women with polycystic ovary syndrome. Fertil Steril. 2007 Jan;87(1):113-20.
5. Palomba S, Orio F Jr, Falbo A, et al. Prospective parallel randomized, double-blind, double-dummy controlled clinical trial comparing clomiphene citrate and metformin as the first-line treatment for ovulation induction in nonobese anovulatory women with polycystic ovary syndrome. J Clin Endocrinol Metab. 2005;90:4068-4074.
6. Siebert TI, Kruger TF, Steyn DW, Nosarka S. Is the addition of metformin efficacious in the treatment of clomiphene citrate-resistant patients with polycystic ovary syndrome? A structured literature review. Fertil Steril. 2006 Nov;86(5):1432-7.
7. Legro RS, Barnhart HX, Schlaff WD, et al. Clomiphene, metformin, or both for infertility in the polycystic ovary syndrome. N Engl J Med. 2007;356:551-566.
8. Moll E BP, Korevaar JC, Lambalk CB, van der Veen F. Ovulation induction in women with polycystic ovary syndrome: A randomized double blind clinical trial comparing clomiphene citrate plus metformin with clomiphene citrate plus placebo. BMJ. 2006;332:1485.
9. Baillargeon JP, Legro RS. Should metformin be used as front-line therapy for fertility in women with PCOS. Sexuality, Reproduction, and Menopause 2007; 5(2):17-19.
10. Shibahara H, Kikuchi K, Hirano Y, Suzuki T, Takamizawa S, Suzuki M. Increase of multiple pregnancies caused by ovulation induction with gonadotropin in combination with metformin in infertile women with polycystic ovary syndrome. Fertil Steril. 2007 Jun;87(6):1487-90.
11. Tang T, Glanville J, Orsi N, Barth JH, Balen AH. The use of metformin for women with PCOS undergoing IVF treatment. Hum Reprod. 2006 Jun; 21(6): 1416-25.

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

Progesterone is the hormone that prepares the uterus and endometrial lining to support an early pregnancy (Progesterone = “Pro-gestation hormone”). Produced in the ovary between ovulation and the following menstrual period, and by the placenta in the early embryo, progesterone stimulates cells in the endometrial lining to become receptive to the early embryo and, after implantation, to support growth of the embryo. Without progesterone, implantation could not occur; if progesterone were to be removed in early pregnancy, miscarriage would be certain to follow.

Hormones are produced in the ovary by the developing follicle, or egg sac. In the first two weeks of the menstrual cycle, as the egg sac matures, stimulated by Follicle Simulating Hormone (FSH) and Luteinizing Hormone (LH) from the pituitary, the follicle increases its production of estrogen to a peak just before ovulation. At the mid-cycle surge of LH, the follicle abruptly shuts down its estrogen production pathway, converting over to producing large amounts of progesterone. The follicle becomes the corpus luteum, a richly vascularized progesterone production factory.

As the pregnancy is established, the placenta produces chorionic gonadotropin, hCG, a hormone that stimulates the corpus luteum to produce additional progesterone. hCG is very similar to LH, binds to the same receptors, and stimulates the ovary much like LH. Rising hCG stimulates rising progesterone, which strengthens the pregnancy and allows it to produce more hCG, again increasing progesterone; this feedback loop is essential to enabling a strong pregnancy.

Progesterone is essential to the development of the early embryo. Progesterone from the corpus luteum circulates through the bloodstream to the uterus, where the endometrium that has been prepared by estrogen starts to change to support the early pregnancy. This change in the endometrial lining, luteinization, is essential for the embryo. The role of the corpus luteum was demonstrated years ago in experiments where the ovary containing the corpus luteum was removed; miscarriage immediately followed. More recently, progesterone antagonists, such as RU-486, which block the progesterone receptor, have been used in animal studies to induce miscarriage when given in early pregnancy.

Progesterone also has effects on the immune system, stimulating protective proteins, such as HLA-G, in the early pregnancy (Yie, Xiao et al. 2006). Without HLA-G the maternal immune system would reject the embryo, therefore, production of HLA-G antigens are critical to protecting the early pregnancy. Progesterone plays an important role in stimulating HLA-G and preventing rejection of the embryo.

Progesterone also acts as a chemoattractant for sperm (Albano, Smitz et al. 1999; Teves, Barbano et al. 2006). Progesterone in tiny amounts will draw sperm, and may attract sperm to the egg after ovulation. Uterine contractions, which play a role in sperm movement, are also controlled by progesterone.

Because it aids in creating a receptive environment for the embryo, insufficient progesterone can be a source of infertility and miscarriage. Low progesterone levels will result in luteal phase defect, a condition in which there is insufficient hormonal support for the early pregnancy. Failure of implantation of an otherwise healthy embryo, or loss of an early pregnancy, may occur with luteal phase defect. Some women do not produce any progesterone at all, for example, after menopause, or when a menopausal state is temporarily induced using medications to prevent ovulation. Without progesterone, pregnancy cannot occur.

The progesterone receptor mediates the action of the hormone and is critically important to pregnancy; some cases of infertility may be related to abnormalities in the progesterone receptor (Spandorfer, Normand et al. 2006). A simple alteration in the genetic code for the progesterone receptor is common in patients with infertility, and appears to be associated with poorer pregnancy outcomes.

The method of In Vitro Fertilization (IVF) is associated with luteal phase defects and low progesterone levels (Albano, Smitz et al. 1999). With IVF treatment, many of the cells that produce progesterone are removed from the ovary in the course of oocyte retrieval. In addition, the use of GnRH agonists and antagonists (leuprolide, ganirelix, cetrorelix) prevent the release of LH and FSH from the pituitary, removing the primary stimulus for progesterone production from the ovary. Progesterone levels may not be adequate to support the pregnancy, resulting in a luteal phase defect, implantation failure, and early miscarriage.

For treatment, progesterone usage falls into two broad groups, progesterone supplementation, where progesterone is produced in the ovary and supplemented with medication, and progesterone replacement, where there is no natural progesterone production. Progesterone replacement would be used in an oocyte donation recipient. Since ovulation occurs in the donor, and there is no natural progesterone in the recipient, all progesterone must be administered. Progesterone replacement is also common for cryopreserved embryo transfers, though natural cycles can also be used in many women with regular menstrual cycles. Medical supplementation might be used in a variety of conditions associated with luteal phase defect or to reduce the risk of early miscarriage associated with low progesterone levels.

Progesterone is supplemented medically to reduce the risk of pregnancy problems arising from low progesterone levels. Progesterone may be given orally, by vaginal supplement, by injection, or its production enhanced by injection of hCG, which stimulates the corpus luteum to produce additional progesterone(Pouly, Bassil et al. 1996).

Oral progesterone is relatively weak in its effect. Absorbed through the upper intestine, progesterone taken orally is metabolized in the liver. This is known as “first pass effect”, because the hormone passes through the liver first before traveling to its site of action. These metabolites are not effective in inducing luteinization and can induce effects on the central nervous system such as sedation. Very little active progesterone is available after oral use (Friedler, Raziel et al. 1999).

Vaginal progesterone, in the form of creams, gels, and suppositories, is highly effective in supplementing or replacing natural progesterone, and has been the most popular form of progesterone supplementation. Progesterone is absorbed through the vaginal wall and moves through local circulation directly to the endometrium. Levels are sufficient to induce the normal changes in endometrial lining to support the early pregnancy (Pritts and Atwood 2002). The primary clinical concern with vaginal progesterone is the variability in absorption. While most women absorb progesterone vaginally without difficulty, some may not; as an indirect mode of administration, one cannot be certain of the amount that is absorbed.

Progesterone by intramuscular injection is well absorbed, and in some ways closest to natural ovarian secretion (Lightman, Kol et al. 1999). High serum levels of progesterone are achieved with effective preparation of the endometrium. Traditional intramuscular injections, in an oil base, require a relatively large needle; local reactions to the oil base at the site of injection are common. Newer preparations of intramuscular progesterone, such as progesterone ethyl oleate, are considerably easier to inject, but still require daily administration. Injectable progesterone remains the primary progesterone for those patients that produce no natural progesterone, such as for a donated oocyte recipient, or for a frozen embryo transfer in a medicated cycle.

hCG, by acting directly on the ovary, is a good stimulant to progesterone production (Herman, Raziel et al. 1996). Its use requires that an active corpus luteum be present, so it can only be used in a natural or stimulated ovulation cycle. It produces good progesterone levels and reduces the risk of luteal phase defect (Mochtar, Hogerzeil et al. 1996). hCG requires periodic injections and may increase the risk of ovarian hyperstimulation syndrome in patients that have been on fertility drugs. As the hormone that is measured in a pregnancy test, hCG causes a false positive pregnancy test, potentially confusing the diagnosis of early pregnancy. hCG is used occasionally to supplement progesterone production in women with an active corpus luteum.

Vaginal and injectable progesterone appear to be similar in actions on the endometrial lining (Khan, Richter et al. 2007); while the amount of progesterone absorbed can be dramatically different, the clinical effects are similar. Progesterone receptors appear to be saturated at fairly low levels of progesterone in the blood, and additional progesterone does not seem to increase pregnancy rates or reduce miscarriage rates. The specific route or agent for progesterone supplementation is probably not as important as assuring that at least some progesterone is present. Equivalent pregnancy rates have been shown using vaginal gels, progesterone vaginal capsules, and progesterone in a dissolving effervescent vaginal tablet (Schoolcraft, Miller et al. 2007). Vaginal and injected progesterone, in general, show higher bioavailability than oral progesterone.

In patients with no ovarian function, recipients of egg donors, or those patients utilizing cryopreserved embryos in medicated (estrogen/progesterone replaced) cycles, all progesterone must be supplied medically. These patients require a reliable source of progesterone, and injectable progesterone has been established as the best standard (Prapas, Prapas et al. 1998). Vaginal progesterone has also been used successfully, though less commonly. In these patients, progesterone replacement must be continued for an extended period. Because there is no corpus luteum in the ovary, the rising hCG from the placenta cannot stimulate progesterone production, as it would in a conventional pregnancy.

In those patients with an active corpus luteum, such as after in vitro fertilization, external progesterone is required for only a limited time period. In the first two weeks after ovulation, the pregnancy is critically dependent on ovarian progesterone. After a positive pregnancy test, progesterone administration can be stopped entirely (Proctor, Hurst et al. 2006), relying on the embryo to stimulate the corpus luteum through the placental hCG effect on the ovary.

Leuprolide, a GnRH agonist, seems to supplement progesterone and its actions. A single injection of a GnRH agonist releases LH from the pituitary, stimulating progesterone production in the ovary, and may act directly on the endometrium and the embryo, enhancing implantation (Pirard, Donnez et al. 2006). With more study, this may prove to be a useful adjunct to use of progesterone.

Philip Chenette, MD

References

Albano, C., J. Smitz, et al. (1999). “Luteal phase and clinical outcome after human menopausal gonadotrophin/gonadotrophin releasing hormone antagonist treatment for ovarian stimulation in in-vitro fertilization/intracytoplasmic sperm injection cycles.” Hum. Reprod. 14(6): 1426-1430.

Friedler, S., A. Raziel, et al. (1999). “Luteal support with micronized progesterone following in-vitro fertilization using a down-regulation protocol with gonadotrophin-releasing hormone agonist: a comparative study between vaginal and oral administration.” Hum. Reprod. 14(8): 1944-1948. Herman, A., A. Raziel, et al. (1996). “The benefits of mid-luteal addition of human chorionic gonadotrophin in in-vitro fertilization using a down-regulation protocol and luteal support with progesterone.” Hum. Reprod. 11(7): 1552-1557.

Khan, Richter, et al. (2007). “Case-Matched Comparison of Intramuscular Versus Vaginal Progesterone for Luteal Phase Support After In Vitro Fertilization and Embryo Transfer.” Fertility and Sterility 87(4): S13-S13.

Lightman, A., S. Kol, et al. (1999). “A prospective randomized study comparing intramuscular with intravaginal natural progesterone in programmed thaw cycles.” Hum. Reprod. 14(10): 2596-2599.

Mochtar, M. H., H. V. Hogerzeil, et al. (1996). “Endocrinology: Progesterone alone versus progesterone combined with HCG as luteal support in GnRHa/HMG induced IVF cycles: a randomized clinical trial.” Hum. Reprod. 11(8): 1602-1605.

Pirard, C., J. Donnez, et al. (2006). “GnRH agonist as luteal phase support in assisted reproduction technique cycles: results of a pilot study.” Hum. Reprod. 21(7): 1894-1900.

Pouly, J. L., S. Bassil, et al. (1996). “Endocrinology: Luteal support after in-vitro fertilization: Crinone 8%, a sustained release vaginal progesterone gel, versus Utrogestan, an oral micronized progesterone.” Hum. Reprod. 11(10): 2085-2089.

Prapas, Y., N. Prapas, et al. (1998). “The window for embryo transfer in oocyte donation cycles depends on the duration of progesterone therapy.” Hum. Reprod. 13(3): 720-723.

Pritts, E. A. and A. K. Atwood (2002). “Luteal phase support in infertility treatment: a meta-analysis of the randomized trials.” Hum. Reprod. 17(9): 2287-2299.

Proctor, Hurst, et al. (2006). “Effect of progesterone supplementation in early pregnancy on the pregnancy outcome after in vitro fertilization.” Fertility and Sterility 85(5): 1550-1552.

Schoolcraft, Miller, et al. (2007). “Efficacy of a Novel Form of Vaginal Progesterone on Continuing Pregnancy Rates in Women Undergoing IVF with Elevated BMI and Advanced Age.” Fertility and Sterility 87(4): S24-S24.

Spandorfer, Normand, et al. (2006). “O-7 A G->A POLYMORPHISM AT POSITION +331 IN THE PROGESTERONE RECEPTOR GENE IS STRONGLY ASSOCIATED WITH IVF OUTCOME.” Fertility and Sterility 86(3): S3-S4.

Teves, Barbano, et al. (2006). “Progesterone at the picomolar range is a chemoattractant for mammalian spermatozoa.” Fertility and Sterility 86(3): 745-749.

Yie, S.-m., R. Xiao, et al. (2006). “Progesterone regulates HLA-G gene expression through a novel progesterone response element.” Hum. Reprod. 21(10): 2538-2544.

Will my fibroids prevent me from getting pregnant?
A recent PFC study can help answer that question.

Every complete infertility evaluation includes a thorough evaluation of the uterus, where embryo implantation is expected to occur. At Pacific Fertility Center we typically start with a vaginal ultrasound to evaluate for the presence of fibroids (benign growths of the muscle layer), polyps (benign growths of the lining of the uterus), measure the lining thickness of the uterus and observe the uterine lining pattern. If uterine abnormalities are noted, a saline hysterogram (saline ultrasound) or hysteroscopy (visualizing the uterine cavity with a thin telescope) may be recommended.

Fibroids (uterine leiomyomas) are present in 20-40% of reproductive age women. The location of the fibroid(s), relative to the lining of the uterus, is important in determining if it will impact chances of pregnancy. Fibroids which distort the uterine lining and cavity are known to decrease pregnancy rates for patients undergoing fertility treatment. Only about 5% of fibroids directly distort the uterine cavity. The influence of fibroids which do not distort the uterine cavity has remained controversial.

To best determine if non-distorting fibroids also may have an impact on fertility treatment, requires the analysis of a large number of fertility cycles following patients who have non-distorting fibroids, and patients who have no fibroids. Most studies have small numbers of observed cycles, making statistical analysis difficult. One strategy for circumventing this problem, and gathering enough treatment cycles to draw meaningful statistics, is to have large IVF centers collaborate and “pool” data. This type of study is called a multi-center study.

Pacific Fertility Center (PFC) and the University of California San Francisco (UCSF) IVF centers collaborated in just such a study; gathering data on past treatment cycles of egg donor recipients with non-cavity distorting fibroids and without fibroids. Analysis of pregnancy (PR) and implantation (IR) rates were assessed. A total of 369 cycles were analyzed, of which 94 were for patients with fibroids. All recipients underwent their first oocyte donor cycle, and a fresh embryo transfer. Any uterine abnormalities other than non-distorting fibroids were excluded from the study analysis. The primary outcome measure was a clinical pregnancy. Implantation rate was a secondary outcome of the study. We also analyzed to see if the fibroid location: subserosal (growing towards the outside of the uterus) versus intramural (confined to the muscle layer) or if the fibroid diameter impacts PR and IR, as well as miscarriage and ectopic rates.

The following results were revealed.

The clinical pregnancy rate (PR) was not different between the two groups (no fibroids vs fibroids) (54% vs 47%). The implantation rate (IR) was also similar between the groups (38% vs 36%). Miscarriage rates were similar (9% vs 15%). Ectopic pregnancy (which is typically a rare outcome) showed results of 1% vs 4%, which also was not statistically significant. Location and diameter of fibroids did not show a significant impact on PR.

When screening ultrasounds identify fibroids, “treatment” of these lesions is tempting to both providers and patients, especially in cases of unexplained infertility. Our data suggest that there is inadequate evidence to conclude that fibroids which do not distort the uterine cavity have a significant effect on clinical pregnancy rates (PR) in patients undergoing IVF. Thus, there is inadequate evidence to support myomectomy for patients with non-distorting fibroids. Myomectomy may unnecessarily place the patient at risk of delayed treatment, as well as possible surgical morbidity. It is also unknown whether surgery itself may have a negative impact on pregnancy outcome- though our data did not show a lower PR in patients who had past myomectomies.

Future collaborative studies will investigate whether the distance of the closest fibroid to the uterine lining may impact PR and IR. Fibroid volume will also be investigated. These studies are currently in the design phase.

Isabelle Ryan, MD

“The effects of fibroids without cavity involvement on ART outcomes independent of ovarian age”, PC Klasky, DE Lane, IP Ryan, VY Fujimoto, Hum Reprod Advance Access, published September 22, 2006.

The American Society for Reproductive Medicine’s (ASRM) annual meeting was held in New Orleans. It is the largest meeting for reproductive medicine specialists and scientists in the world. From our practice, Dr.s Givens, Schriock and Conaghan attended, as well as embryologists Jean Popwell, PhD and Jennifer Andres. Also, PFC nurse Allison Chamberlaine and PFC’s Marriage and Family Therapist Peggy Orlin attended. In addition, the genetics counselor with whom we work closely, Lauri Black from California Pacific Medical Center, was an attendee and active participant.

PFC’s embryologists attending ASRM’s research poster session Jean Popwell, PhD (left) and Jennifer Andres (right).

Single-Embryo Transfer: Minimizing Risks & Maximizing Outcomes
Dr. Givens attended a post-graduate course entitled “Moving Toward Single-Embryo Transfer: Minimizing Risks and Maximizing Outcomes.” This two-day course dealt with a pressing issue in assisted reproduction: the high incidence of multiple gestations. With the ever-increasing success of in vitro fertilization and the significant improvement in embryo implantation rates, the incidence of twin and higher-order pregnancies has risen dramatically in this country. Many countries now regulate the maximum number of embryos that can be transferred into the uterus at one time. The course topics included a summary of optimal medication protocols, several lectures on pre-cycle evaluation and testing and embryo transfer techniques.

Oocyte Freezing, PGS & Blastocyst Embryo Transfers
On the laboratory side, there were several talks on evaluation of eggs and embryo selection techniques, embryo freezing technology, including a debate about the usefulness of pre-implantation genetic screening (chromosome analysis of embryos) embryo selection. The combination was a fascinating mixture of new ideas, refinements in current technology, as well as a welcome opportunity to network and discuss with others the latest developments in reproductive science. Topping the list of presentations in New Orleans were those concerning the continuing refinements in oocyte freezing technologies, the more selective use of preimplantation genetic testing and the ongoing scrutiny of blastocyst stage embryo transfers.

Slow-freeze vs. Vitrification
The traditional slow-freeze technology used so successfully with embryos for many years, has essentially stalled with oocyte freezing. It appears the slow-freeze technology has finally met its successor: a process called vitrification. Slow freezing has had very limited success with oocytes due to their large size, high water content and their extreme sensitivity to cryoprotective chemicals and to changes in temperature and pH.

Vitrification, a technology that cools cells so rapidly that ice does not form, has been such a success for oocyte freezing that many labs are now abandoning slow freezing altogether. Here at PFC, we have been developing protocols for oocyte vitrification throughout 2006 and are actively working on blastocyst vitrification. It was reaffirming to see that this technology has gained wide acceptance, and is showing excellent results.

Preimplantation Genetic Screening (PGS)
While vitrification is on the rise, it was interesting to see that another technology, Preimplantation Genetic Screening (PGS), was lacking in new improvements or viable alternatives. Embryos have been screened for extra or missing chromosomes for over 15 years now, but the technology has not advanced significantly over that time. It is still possible to count only 12 chromosomes in an embryo. Although the error rate per chromosome is very low, the accumulated error rate becomes significant as we count more chromosomes. PGS was “under the microscope” in several presentations in New Orleans and it appears PFC’s limited use of genetic screening is well justified. Specifically, PGS does not improve embryo selection and pregnancy rates in younger patients. Its use is limited in older patients because there are often too few embryos available to justify testing. The patients who benefit most from PGS are the younger patients who experience recurrent miscarriages. However, unless there is evidence that previous pregnancies were genetically abnormal, PGS may provide limited benefit to this group.

Blastocyst stage embryo transfers
While younger patients (those under 35) don’t benefit from PGS, they are the patient population most likely to benefit from blastocyst transfers. Culturing embryos for 5 days to the blastocyst stage, instead of the more traditional day 3 embryo transfer, is one of the main ways in which the laboratory staff can help in selecting the “best” embryo for single embryo transfer (SET) patients. Blastocyst culture techniques are well refined now and support the commitment within the community to transfer fewer embryos at one time. Furthermore, the promise of vitrification can reassure patients that their remaining embryos can be stored indefinitely when preserved at the blastocyst stage. Several presentations showed that blastocysts which were vitrified early, before their cavity (or cyst) had expanded too much, benefited most from the technology. In more advanced blastocysts, artificial reduction of the cavity gave superior results. It may not be long before vitrification is the procedure of choice for preserving all blastocysts.

2006 ASRM guidelines for numbers of embryos to transfer
The new 2006 ASRM guidelines for numbers of embryos to transfer were presented. See Tables 1 and 2 below.

The topic of whether or not federal or state legislation should regulate the maximum number of embryos to transfer was also discussed. Many people in the general public support such legislation but those of us in the field (and most patients) are opposed to the government regulating medical practice and arbitrarily setting limits on embryo transfer. In order to forestall such legislation, it is obvious that we must decrease the number of twin gestations (the number of triplet and higher-order gestations has already dramatically decreased in the last 5-7 years). At Pacific Fertility Center we have instituted a new emphasis on single embryo transfers and expect to significantly reduce the risk of multiples and achieve our goal of “optimal” pregnancy outcomes. (See From Us to You in this issue for a discussion of our 2006 statistics and please see Conception Health in this issue for a discussion of why it is important to try to achieve single baby conceptions.

– Carolyn Givens, MD and Joe Conaghan, PhD

Male factor infertility is quite common, contributing to 40% of infertility diagnoses. Treatment is designed around the particular type of problem and can be remarkably effective. For those with male factor infertility, the initial course of action is to review personal health habits. Stress, poor diet, and alcohol use have all been correlated with male factor infertility. Alcohol use, in particular, has been shown to have a dose-related effect on sperm; the more one drinks, the poorer the reproductive outcome. High temperature exposure from hot tubs or hot baths (immersion in hot water), or heavy exercise, particularly bicycle riding, have been correlated with male factor infertility as well. Resting a laptop computer on one’s lap has also been implicated in raising testicular temperature.

Diagnosis of male factor infertility starts with a semen analysis. The semen analysis should be performed on an ejaculated sample collected on at least two occasions 2-7 days following abstention from sexual activity. Measurement of the sperm count, motility, and volume can reveal production problems as insufficient or poor quality sperm are released from the testes. Table 1 lists the standards for assessing a semen analysis (Source: The World Health Organization, 1992).

Additional tests to evaluate sperm quality include the detailed or Krueger morphology. This entails viewing individual sperm cells under a high-powered microscope. This is a strict test that reveals abnormalities in the shape and size of the sperm heads, mid-pieces, and tails. A normal morphology is present when over 14% of sperm are normal.

Survival of the sperm on extended testing is also a useful diagnostic test. The sperm survival test, or SST, is a method for testing the lifespan of the sperm. At 24 hours, sperm survival should be over 40% (i.e. 40% of the sperm sample should survive); conversely, lower survival rates correspond to lower pregnancy rates.

Additional testing for male factor infertility includes a physical exam, blood tests for FSH, prolactin, and testosterone, and an ultrasonography of the collecting tubes of the male reproductive system. In some cases, an assessment of DNA fragmentation can give an index of sperm quality as well.

One condition we encounter at our clinic is azoospermia, which is the absence of sperm in the ejaculate. This can occur from birth defects, injury or infection, or rare endocrine abnormalities. In azoospermia, a high FSH level indicates testicular failure. Insufficient levels of testicular hormones lead to an increase in the release of pituitary gland FSH to compensate. High levels of testicular hormones are often accompanied by testicular atrophy (small testicles). Testicular biopsy may confirm the clinical findings.

Men with testicular failure (and very low sperm counts) should be tested for Y-chromosome microdeletions and abnormal karyotypes, or chromosomal count. Microdeletions may be transmitted to offspring, resulting in fertility problems for boys born after treatment.

The most common abnormal karyotype is Klinefelter Syndrome, where the male has three or more sex chromosomes, instead of the normal two. Such chromosomal defects can have effects on children born after treatment, and men should receive genetic counseling and risk assessment prior to treatment. Men with testicular failure may still have partial sperm production. Detailed assessment with microscopic surgery may detect a sufficient amount of sperm to use with in vitro fertilization (IVF).

Obstruction is another type of male factor infertility, as potentially normal sperm cannot move from the testes to the ejaculate. Men with a normal FSH may have an obstruction in the vas deferens or any of the other collecting tubes that gather sperm from the testes. Men with congenital absence of the vas deferens (CBAVD) may be carriers of cystic fibrosis, and should be tested. Surgical obstruction, or vasectomy, is readily repaired. Microsurgical techniques, and an experienced surgeon, will increase success rates. The procedure may be attempted for many years after an initial vasectomy. More unusual obstructions can result after infection of the epididymis. Ejaculatory duct obstruction can be treated with a cystoscopic procedure. Obstructions can sometimes be repaired, but often a simple needle aspiration procedure (percutaneous epididymal sperm aspiration, PESA) will yield enough sperm to achieve fertilization with IVF.

The key treatment when working with low sperm numbers, whether in the ejaculate or obtained by needle aspiration or biopsy, is to perform in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). ICSI is when a highly trained embryologist uses micromanipulators to inject an individual sperm into an egg, optimizing for fertilization.

ICSI has become a common procedure, resulting in many pregnancies worldwide for men that otherwise could not have children. Sperm with a variety of abnormalities, ranging from low counts, to extremely low motilities, can be suitable for use. The DNA of the sperm is tightly compacted in ways that protect it from injury, even when the other components of the sperm do not function well. Injecting the sperm into the egg can bypass the barriers separating sperm and egg.

Another condition we encounter which can lead to abnormal sperm parameters is the presence of a varicocele. A varicocele is an enlarged vein along the upper part of the scrotum. The blood carried in these veins may elevate the scrotal temperature, and possibly carry toxic materials into the testicle, affecting sperm production. Only varicoceles that are palpable are thought to contribute to infertility. Ultrasound is sometimes used to confirm an uncertain diagnosis, but there is doubt whether subclinical varicoceles are associated with infertility. Varicoceles can be repaired, or various fertility treatments attempted, including sperm wash and insemination, and in vitro fertilization. The decision of treatment depends on both male and female factors, such as age, tubal disease, and ovulation disorders.

In closing, it is important to remember that infertility is not just a “female” issue and that men should engage in lifestyle habits that will not compromise their fertility. Furthermore, advancements in assisted reproductive technology (ART) have given men with infertility diagnoses newfound hope in their quest to build a healthy family.

– Philip Chenette, MD

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

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

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

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

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

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

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

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

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

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

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

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

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

– Joe Conaghan, PhD

Bibliography:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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