Emmet J. Lamb

Cryopreservation of embryos and ova

Development of techniques for cryopreservation of embryos of several species, principally the mouse, laid the foundation for cryopreservation of human embryos. As IVF has become more widely available and the need for the cryopreservation of human embryos has become apparent, pressure for technical development has increased. The ideal method would be simple, inexpensive, and effective. The most effective method for cryopreservation of early human embryos, such as those at the 1-cell pronuclear stage and up to the 4-cell stage, now appears to be stepwise cooling in 1,2-propanediol with sucrose in plastic ministraws. The preferred method for intermediate stage embryos uses DMSO with cooling and thawing at slow rates in a programmed biologic freezer. For the human blastocyst, slow cooling in glycerol and rapid thawing is the only method reported with survival rates comparable to those achieved for intermediate stage embryos using DMSO. The rates of survival from freezing and thawing blastocysts are not sufficiently high, however, to justify the losses associated with prolonged in vitro incubation. Even at the current level of technical achievement, cryopreservation of human embryos provides the clearest opportunity to improve the clinical results obtained with IVF. Research now underway in the modification of methods for vitrification and ultrarapid freezing holds promise for both simplification of technology and improvement of outcome. In view of legal and ethical considerations involved in embryo preservation, the desirability of ova preservation is widely accepted. Although a small number of human unfertilized mature ova have been cryopreserved using various methods, success rates are still low. Methods for the cryopreservation of eggs should be developed, but these methods probably should be proved by animal experiments to be safe, especially with regard to genetic damage, before a policy of transfer of embryos derived from frozen-thawed human ova is applied on a large scale.

OVARIAN CANCER RISK AFTER THE USE OF OVULATION-STIMULATING DRUGS

OBJECTIVE: To assess the long-term effects of ovulation-stimulating drugs on the risk of ovarian cancer. METHODS: A retrospective cohort study of 12,193 eligible study subjects (median age 30 years) who were evaluated for infertility during the period of 1965–1988 at 5 clinical sites identified 45 subsequent ovarian cancers in follow-up through 1999. Standardized incidence ratios compared the risk of cancer among the infertile patients to the general population, whereas analyses within the cohort allowed the derivation of rate ratios for drug usage compared with no usage after adjustment for other ovarian cancer predictors. RESULTS: The infertility patients had a significantly elevated ovarian cancer risk compared with the general population (standardized incidence ratio 1.98, 95% confidence intervals [CI] 1.4, 2.6). When patient characteristics were taken into account and risks assessed within the infertile women, the rate ratios associated with ever usage were 0.82 (95% CI 0.4, 1.5) for clomiphene and 1.09 (95% CI 0.4, 2.8) for gonadotropins. There were higher, albeit nonsignificant, risks with follow-up time, with the rate ratios after 15 or more years being 1.48 (95% CI 0.7, 3.2) for exposure to clomiphene (5 exposed cancer patients) and 2.46 (95% CI 0.7, 8.3) for gonadotropins (3 exposed cancer patients). Although drug effects did not vary by causes of infertility, there was a slightly higher risk associated with clomiphene use among women who remained nulligravid, based on 6 exposed patients (rate ratio 1.75; 95% CI 0.5, 5.7). CONCLUSION: The results of this study generally were reassuring in not confirming a strong link between ovulation-stimulating drugs and ovarian cancer. Slight but nonsignificant elevations in risk associated with drug usage among certain subgroups of users, however, support the need for continued monitoring of long-term risks. LEVEL OF EVIDENCE: II-2

Do the results of semen analysis predict future fertility? a survival analysis study

The authors examined the results of the initial semen analysis from 1089 couples in an infertility clinic. The distributions of the values for various semen variables among couples who remained infertile were very similar to those among couples who later conceived. A cutoff point was selected in each pair of distributions to compare the cumulative probability of conception for couples with higher and lower results. The authors took into account the varying times to conception or loss to follow-up by using life table analysis. No statistically significant differences were found at the P = 0.05 level of significance. Coxs univariate and multiple regression models were then used to investigate the relationship between various semen characteristics and future fertility. Finding no significant influence of any semen characteristic on the cumulative probability of conception, the authors believe that using specific values of the semen analysis to estimate the potential for fertility of infertile couples is not useful.

Causes of Infertility as Predictors of Subsequent Cancer Risk

Background: Although studies have found elevated risks of certain cancers linked to infertility, the underlying reasons remain unclear. Methods: In a retrospective cohort study of 12,193 U.S. women evaluated for infertility between 1965 and 1988, 581 cases of cancer were identified through 1999. We used standardized incidence ratios (SIRs) to compare cancer risk with the general population. Analyses within the cohort estimated rate ratios (RRs) associated with infertility after adjusting for other risk predictors. Results: Infertility patients demonstrated a higher cancer risk than the general population (SIR = 1.23; 95% confidence interval [CI] = 1.1–1.3), with nulligravid (primary infertility) patients at even higher risk (1.43; 1.3–1.6). Particularly elevated risks among primary infertility patients were observed for cancers of the uterus (1.93) and ovaries (2.73). Analyses within the cohort revealed increased RRs of colon, ovarian, and thyroid cancers, and of melanomas associated with endometriosis. Melanomas were linked with anovulatory problems, whereas uterine cancers predominated among patients with tubal disorders. When primary infertility patients with specific causes of infertility were compared with unaffected patients who had secondary infertility, endometriosis was linked with distinctive excesses of cancers of the colon (RR = 2.40; 95% CI = 0.7–8.4), ovaries (2.88; 1.2–7.1), and thyroid (4.65; 0.8–25.6) cancers, as well as melanomas (2.32; 0.8–6.7). Primary infertility due to anovulation particularly predisposed to uterine cancer (2.42; 1.0–5.8), and tubal disorders to ovarian cancer (1.61; 0.7–3.8). Primary infertility associated with male-factor problems was associated with unexpected increases in colon (2.85; 0.9–9.5) and uterine (3.15; 1.0–9.5) cancers. Conclusions: The effects of infertility may extend beyond gynecologic cancers. Thyroid cancers and melanomas deserve specific attention, particularly with respect to endometriosis.

Ovulation-inducing drugs and ovarian cancer risk: results from an extended follow-up of a large United States infertility cohort

OBJECTIVE To examine the relationship of ovulation-inducing drugs and ovarian cancer. DESIGN Retrospective cohort study, with additional follow-up since initial report. SETTING Five large reproductive endocrinology practices. PATIENT(S) In a retrospective cohort of 9,825 women evaluated for infertility at five clinical sites in the United States between 1965 and 1988 with follow-up through 2010, we examined the relationship of ovulation-inducing drugs and ovarian cancer (n = 85). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Hazard rate ratios (RR) and 95% confidence intervals (CI) for ovarian cancer. RESULT(S) Among women evaluated for infertility, there was no association of ovarian cancer risk with ever use of clomiphene citrate (CC) (adjusted RR 1.34, 95% CI 0.86-2.07) or gonadotropins (RR 1.00, 95% CI 0.48-2.08) and no evidence that any of several more detailed subgroups of usage were related to an increased risk with one exception: women who used CC and remained nulligravid did demonstrate much higher risks than those who successfully conceived compared with nonusers (respectively, RR 3.63, 95% CI 1.36-9.72 vs. RR 0.88, 95% CI 0.47-1.63). CONCLUSION(S) Our overall results were reassuring and consistent with other studies. A reason for an association between CC use and ovarian cancer among persistently nulligravid women remains to be determined. Given the large and increasing number of women treated with ovulation-inducing drugs, the increased risk of ovarian cancer among the subset of women who remained nulligravid should be further monitored.

Endometriosis: studies of a method for the design of a surgical staging system.

This study develops a method of identifying factors that predict pregnancy rates in patients with endometriosis. In 123 women, 60% had adhesions, 89% had implants, and 29% had endometriomas. The most common lesions were cul-de-sac implants (60%), ovarian implants (58%), and ovarian adhesions (55%). With no treatment, the life-table-estimated 3-year pregnancy rate was 40%; with oral contraceptives, 33%; and with surgery, 53%. Clustering techniques suggested new combinations of variables to be tested. We identified structures commonly involved simultaneously in a given patient, defined subgroups based on type of lesion, and developed a method to determine which factors were important in predicting outcome. In our patients, neither of two current methods of endometriosis staging predicted outcome. Further testing of the model systems suggested by the data may enable us to develop a staging system more predictive of pregnancy rates in endometriosis patients.

Long-term Relationship of Ovulation-Stimulating Drugs to Breast Cancer Risk

Background: Although fertility drugs stimulate ovulation and raise estradiol levels, their effect on breast cancer risk remains unresolved. Methods: An extended follow-up was conducted among a cohort of 12,193 women evaluated for infertility between 1965 and 1988 at five U.S. sites. Follow-up through 2010 was achieved for 9,892 women (81.1% of the eligible population) via passive as well as active (questionnaires) means. Cox regression determined HRs and 95% confidence intervals (CI) for fertility treatments adjusted for breast cancer risk factors and causes of infertility. Results: During 30.0 median years of follow-up (285,332 person-years), 749 breast cancers were observed. Ever use of clomiphene citrate among 38.1% of patients was not associated with risk (HR = 1.05; 95% CI, 0.90–1.22 vs. never use). However, somewhat higher risks were seen for patients who received multiple cycles, with the risk for invasive cancers confirmed by medical records being significantly elevated (HR = 1.69; 95% CI, 1.17–2.46). This risk remained relatively unchanged after adjustment for causes of infertility and multiple breast cancer predictors. Gonadotropins, used by 9.6% of patients, mainly in conjunction with clomiphene, showed inconsistent associations with risk, although a significant relationship of use with invasive cancers was seen among women who remained nulligravid (HR = 1.98; 95% CI, 1.04–3.60). Conclusions: Although the increased breast cancer risk among nulligravid women associated with gonadotropins most likely reflects an effect of underlying causes of infertility, reasons for the elevated risk associated with multiple clomiphene cycles are less clear. Impact: Given our focus on a relatively young population, additional evaluation of long-term fertility drug effects on breast cancer is warranted. Cancer Epidemiol Biomarkers Prev; 23(4); 584–93. ©2014 AACR.

Fertility drugs and endometrial cancer risk: results from an extended follow-up of a large infertility cohort

STUDY QUESTION Do fertility drugs influence the subsequent risk of endometrial cancer in a manner that is independent of other risk predictors, such as parity? SUMMARY ANSWER In this follow-up of a large cohort of women evaluated for infertility and for whom information was captured on fertility drugs, indications for usage and other risk factors that might influence cancer risk, we found no evidence for a substantial relationship between fertility drug use and endometrial cancer risk. WHAT IS ALREADY KNOWN Although the hormonal etiology of endometrial cancer has been well established, it remains unclear whether the use of fertility drugs has an influence on risk. Results regarding the effects of fertility drugs on endometrial cancer risk have been inconsistent, although several studies have shown some evidence for possible increases in risk. The relationship is of particular interest given that clomiphene, a commonly prescribed drug, is a selective estrogen receptor modulator, with chemical properties similar to tamoxifen, another drug linked to an increase in endometrial cancer risk. STUDY DESIGN, SIZE, DURATION In a retrospective cohort of 12 193 women evaluated for infertility between 1965 and 1988 at five US sites, follow-up was pursued through 2010 via both passive as well as active (questionnaire) means. PARTICIPANTS, SETTING, METHODS Among the 9832 subjects for whom follow-up was allowed and achieved, 259 346 at-risk person-years (i.e. prior to hysterectomy) were accrued, and 118 invasive endometrial cancers identified. Cox regression determined hazard ratios (HRs) and 95% confidence intervals (CIs) for fertility treatments adjusted for endometrial cancer risk factors and causes of infertility. MAIN RESULTS AND THE ROLE OF CHANCE Although we observed slight increases in endometrial cancer risk associated with clomiphene (HR = 1.39, 95% CI: 0.96-2.01) and the less commonly prescribed gonadotrophins (1.34, 0.76-2.37), there were no convincing relationships of risk with either cycles of use or cumulative exposures for either drug. A statistically significant risk associated with the use of clomiphene among women who began use at younger ages (<30) (1.93, 1.24-3.00) may have reflected indications for drug usage rather than the effect of the drug itself. Women who received clomiphene followed by gonadotrophins were at a non-significantly elevated risk (1.77, 0.98-3.19). LIMITATIONS, REASONS FOR CAUTION Like most studies of endometrial cancer, we were limited by sample sizes, particularly for evaluating subgroup associations. We were also unable to follow all women and were not able to obtain complete risk factor information (including hysterectomy status) for the entire cohort. WIDER IMPLICATIONS OF THE FINDINGS Although we found no support for a relationship between fertility drugs and endometrial cancer risk, the association should continue to be monitored given that our study population was still young and had not yet reached the age of peak endometrial cancer incidence. STUDY FUNDING/COMPETING INTEREST(S) This project was supported in part by funds from the intramural research program of the National Cancer Institute, National Institutes of Health. None of the authors has any conflicting interests to declare.

Cancer Risk Among Infertile Women with Androgen Excess or Menstrual Disorders (including Polycystic Ovary Syndrome)

OBJECTIVE To define relationships of androgen excesses to cancer risk. DESIGN Retrospective cohort study. SETTING Five large infertility practices. PATIENT(S) Among 12,193 women evaluated for infertility during 1965-1988 and traced for cancer incidence through 1999, 2,560 had androgen excess or menstrual disorders; among these, 412 met established criteria for polycystic ovary syndrome. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Cancer incidence. Derivation of standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) for cancer risk comparisons with the general population and rate ratios (RRs) for comparisons with other infertility patients. RESULT(S) Androgen excess/menstrual disorder patients showed significant SIRs for breast (1.31; 95% CI, 1.05-1.62) and uterine (2.02; 95% CI, 1.13-3.34) cancers and melanoma (1.96; 95% CI, 1.12-3.18). Significant associations for breast and uterine cancers were restricted to primary infertility patients (respective SIRs of 1.53 and 3.48). After adjustment for other cancer predictors, the only excess risk was for uterine cancer among primary infertility patients. Compared with women with secondary infertility and no androgen excess/menstrual disorder, those with primary infertility and a disorder had an RR of 1.88 (95% CI, 0.82-4.32). Cancer risks among the women with polycystic ovary syndrome or androgen excess disorders appeared to be similar to those in the more comprehensive group. CONCLUSION(S) Previous findings linking androgen excess disorders to elevated uterine cancer risks might largely reflect underlying risk profiles.

Epidemiologic studies of male factors in infertility.

In the US a reduction of mean sperm density and seminal volume has been documented during the past 50 years and an unexplained 3-fold increase in infertility has occurred from 1965 to 1982. The hypothesis that American men have impaired fertility because of exposure to environmental pollution is supported by the facts that: 1) spermatogenesis involves rapid proliferation and may react with particular sensitivity to biological changes 2) that industrialized nations have accepted pollution as a necessity and 3) humans are exposed to an increasing amount of chemicals in the food chain. This paper considers the various methods which epidemiologists can use to study this phenomenon including 1) animal studies to screen for possible toxicity and elucidate mechanisms of actions; 2) semen analysis to screen for exposure to toxicants and to establish regulatory levels for reproductive toxicants; 3) the computer-aided semen analysis (DASA) system to determine characteristics of sperm motion (velocity along a curvilinear path along a straight line and along an average path as well as lateral head displacement); and 4) questionnaires. The complete analysis of fertility data involves 1) organization of follow-up data in a life table 2) analysis of single variable differences (for example exposed and control groups) by log rank tests and 3) analysis of multiple variables by use of the Cox proportional hazards regression model or related models. Cycle fecundability estimates can be obtained by using a single parameter model or a 2-parameter model. 2 survival curves can be compared using the Mantel-Haenszel log-rank analysis (a chi-square technique). Surveillance methods can also be used to replace these analysis techniques. Power curves can determine the sample size required to detect a given exposure-related decrease in fertility. After a general consideration of exposure to toxic substances in the workplace and environment and a specific consideration of exposure to halogen-containing compounds to glycols and glycol ethers to metals to hormones and to heat this paper ends with a discussion of the effects of lifestyle (smoking alcohol consumption and the use of illegal drugs) on fertility.