Cigarette smoking accelerates the development of diminished ovarian reserve as evidenced by the clomiphene citrate challenge test*

Transcription

1 FERTILITY AND STERILITY 1994 The American Fertility Society Printed on acid-free paper in U. s. A. Cigarette smoking accelerates the development of diminished ovarian reserve as evidenced by the clomiphene citrate challenge test* Fady I. Sharara, M.D.t Scott N. Beatse, M.D.:\: Michael R. Leonardi, M.D.:\: Daniel Navot, M.D. Richard T. Scott, Jr., M.D.II~ National Institute of Child Health and Human Development, National Institutes of Health, and Uniformed Services University of the Health Sciences, Bethesda, Maryland; Wilford Hall Medical Center, Lackland AFB, Texas; New York Medical College, Valhalla, New York Objective: To test whether the reduced fecundity in women who smoke cigarettes may be attributed to the accelerated development of diminished ovarian reserve. Design: Retrospective evaluation of clomiphene citrate (CC) challenge tests in women from a general infertility population who did and did not smoke cigarettes (part 1) and retrospective evaluation of the impact of smoking on pregnancy rates (PRs) in IVF among women with normal ovarian reserve (part 2). Setting: Large military tertiary care center. Patients: Sixty-five women who smoked cigarettes and 145 women who did not smoke cigarettes in the general infertility population (part 1) and women undergoing IVF for strict tubal factor infertility with normal ovarian reserve who did (n = 29) and did not (n = 73) smoke (part 2). Interventions: Clomiphene citrate challenge tests, composed of FSH levels on cycle days 3 and 10 with 100 mg of CC administered on cycle days 5 through 9. Main Outcome Measures: Comparison of the incidence of abnormal CC challenge test results between women who did and did not smoke, and comparison of peak E2 levels, number of mature follicles, number of mature oocytes retrieved, fertilization rates, and total and ongoing PRs. Results: The incidence of diminished ovarian reserve was increased in women who smoked (8 of 65 [12.31%]) when compared with age-matched controls who did not smoke (7 of 145 [4.83%]). Among women with normal CC challenge tests who were undergoing IVF, there were no differences in peak E2 levels, the number of mature follicles, the number of mature oocytes retrieved, fertilization rates, or total and ongoing PRs. Conclusion: Women who smoke have an accelerated development of clinically detectable diminished ovarian reserve. Additionally, the fact that women who smoke cigarettes with normal ovarian reserve have ovarian responses and PRs that are equivalent to age-matched nonsmoking controls suggests that diminished ovarian reserve may be a principal mechanism reducing fecundity among women who smoke cigarettes. Fertil Steril1994;62: Key Words: Ovary, ovarian reserve, smoking, in vitro fertilization, clomiphene citrate, clomiphene citrate challenge test Received December 23, 1993; revised and accepted March 30, * The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other Departments of the United States Government. t Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health. :j: Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Wilford Hall Medical Center. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, New York Medical College. II Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences. 11 Reprint requests: Richard T. Scott Jr., M.D., Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland (FAX: ). Sharara et a1. Smoking and diminished ovarian reserve 257

2 The prevalence of smoking among adults in the United States has decreased from 40% in 1965 to 29% in Although the overall decline in smoking has been more substantial among women than among men, the reduction in the incidence of smoking is less dramatic among reproductive aged women who typically begin smoking at younger ages (1). Although many of the hazards of cigarette smoking such as malignancy, atherosclerotic cardiovascular disease, and shorter life span are common knowledge in the general population, the impact of smoking on reproduction has been significantly less emphasized (2). This is in spite of the fact that epidemiologic studies have revealed a consistent and highly significant incidence of decreased fertility (3). Only limited data are available regarding potential mechanisms by which smoking impacts reproduction. Studies in animals who were exposed to either cigarette smoke or some of its toxic components have shown evidence of increased rates of follicular destruction and an accelerated loss of reproductive function (4-7). In the human, both gametogenesis, as evidenced by decreased fecundity (3-7), and hormonogenesis as evidenced by altered E2 production are impacted by cigarette smoking (8, 9). The specific mechanisms that result in these adverse effects have not been elucidated; however, the fact that both hormonogenesis and gametogenesis are affected suggests that smoking has a significant adverse effect at the level offollicular development and function. Additionally, because women who smoke undergo menopause at an earlier age (10, 11), it is possible that these effects reflect an accelerated rate of follicular depletion with entry into the climacteric at a younger age. If so, then the lower fecundity rates that have been described in these women might be attributable to the development of clinically detectable diminished ovarian reserve at an earlier age. Although this information suggests that diminished ovarian reserve may play a role in the reduced fecundity seen in women who smoke cigarettes, no actual data are available regarding the impact of smoking on the incidence of diminished ovarian reserve. Furthermore, no data are available to assess the impact of smoking on women who still have normal ovarian reserve. If diminished ovarian reserve was the principal mechanism adversely impacting reproduction, then it would be anticipated that women with equivalent ovarian reserve would have similar reproductive outcome independent of their smoking status. The purpose of this study was to address these 258 Sharara et al. Smoking and diminished ovarian reserve two questions. First, by comparing the incidence of diminished ovarian reserve as evidenced by abnormal clomiphene citrate (CC) challenge tests in women in the general infertility population who did and did not smoke cigarettes and second, by comparing the results of IVF cycles in women with strict tubal factor infertility who did and did not smoke cigarettes to determine if a nondiminished ovarian reserve mechanism contributed significantly to the reduced fecundity rates seen in women who smoke. Population MATERIALS AND METHODS The two separate questions being addressed in this study required that two different populations of women be evaluated. To evaluate the impact of smoking on diminished ovarian reserve in the general infertility population, the records of all women between the ages of 35 and 39 who had strict tubal factor infertility and who were participating in a larger clinical trial evaluating the CC challenge test between January 1991 and December 1992 were reviewed. The study population consisted of women in the general infertility population who by definition were presenting for their initial infertility evaluation and who had not been referred by other physicians. Women who had never smoked cigarettes were considered nonsmokers, whereas those currently smoking cigarettes were considered smokers. Patients who had smoked cigarettes in the past but who stopped were not included in the study. Women with any type of prior ovarian surgery were excluded. Only women between the ages of 35 and 39 were considered because this age range represents the threshold where some women will begin to have evidence of diminished ovarian reserve (12). As such, any acceleration in the onset of diminished ovarian reserve would be most likely to manifest itself in this population. The second portion of the study was designed to evaluate the impact of smoking on the reproductive capacity of women with no evidence of diminished ovarian reserve. The population for this portion of the study consisted of women 35 to 39 years of age undergoing IVF for the treatment of strict tubal factor infertility. All of these patients had normal CC challenge tests within 1 year of undergoing their IVF cycle. Fertility and Sterility

3 CC challenge tests within 1 year of undergoing their IVF cycle. Experimental Design Assessment of ovarian reserve was done with the CC challenge test as previously described (13). The patients had a serum sample collected on menstrual day 3. Clomiphene citrate, 100 mg, was taken on cycle days 5 through 9, and serum was collected again on cycle day 10. All samples were allowed to clot, the serum separated, and then stored frozen at -20 C until assayed. Samples were assayed weekly for FSH. Clomiphene citrate challenge test results were considered abnormal if either the day 3 or the day 10 FSH concentration exceeded 10 lull (12). The CC challenge test has been evaluated by several groups and found to have excellent predictive value for subsequent pregnancy rates (PRs) (12-15). These studies emphasize the importance of using a clinically defined end point such as PRs to define a threshold value that will discriminate between normal and abnormal. The value of 10 lull used in this study was determined in a long-term prospective study of over 236 couples from the general infertility population. The same assay in the same laboratory was used to determine the serum concentrations in this study. The concept of applying a critical threshold for defining normal has been described by several authors and validated by that study (12-15). The patients in the second portion of the study who underwent IVF all underwent ovulation induction with exogenous gonadotropins (Metrodin and Pergonal; Serono, Randolph, MA) after down regulation with leuprolide acetate (LA, Lupron; TAP, Deerfield, IL) using established protocols (16, 17). The patients continued their LA until the day of hcg administration. All cycles were monitored with daily serum E2 determinations. Follicular growth was assessed via a baseline ultrasound (US) on the 1st day of stimulation and then daily beginning on the 4th day of stimulation and continuing until the day of oocyte retrieval. All oocyte retrievals were performed under US direction as previously described (18). After retrieval, the oocytes were graded using established criteria (19). Fertilization was assessed the day after insemination, and ET was done approximately 48 hours after retrieval. Laboratory Assays Serum FSH concentrations were determined by a commercially available RIA from Becton-Dickin- son (Orangeburg, NY) on a weekly basis. The interassay and intra-assay coefficients of variation (CVs) were 6.7% and 6.9%, respectively. Estradiol levels were determined using a commercially available RIA (Diagnostic System Laboratories, Webster' TX). The interassay and intra-assay CVs were 7.2% and 6.9%, respectively. Statistical Evaluation In this retrospective analysis, the incidence of diminished ovarian reserve as determined by the CC challenge test was compared among women who did or did not smoke using x2 analysis. Cycle data from the patients undergoing IVF in the second portion of the study were compared using Student's t-tests. Specific data that were compared included peak E2 levels, the number of mature follicles that developed (;:.:15 mm), number of mature (metaphase I or II) oocytes that were recovered, and fertilization rates. Pregnancy rates resulting from these cycles were compared using X2 analysis. a-error of <0.05 was considered significant; and all data are expressed as means with their associated SD. Odds ratios (OR) with their associated confidence intervals (CI) were also calculated using the methods described by Feinstein (20). ~-Error values were calculated where significant differences were not detected. The calculations were done using ExSampIe Statistical Software (Idea Works, Columbia, MO) and were based on the ability to dete~t a 20% difference in outcome between the two groups being compared. RESULTS Incidence of Diminished Ovarian Reserve Two hundred ten women, including 65 women who smoked cigarettes and 145 women who did not smoke cigarettes, completed the CC challenge test with no known adverse reactions or complications. The mean ages of those women who smoked (37 ± 1.1 years) and those who did not smoke (36.9 ± 1.3) were not significantly different. Additionally, there were no differences in the incidence of secondary infertility (42% versus 38%), prior history of pelvic inflammatory disease (11% versus 8%), duration of infertility (1.2 versus 1.4 years), or weight (62.2 versus 59.8 kg) (~-error < 0.20). There was no difference in basal or day 10 E2 concentrations, or basal FSH levels between the women who did and did not smoke cigarettes Sharara et al. Smoking and diminished ovarian reserve 259

4 Table 1 Comparison of the FSH and E2 Levels Obtained During the CC Challenge Test in 210 Infertile Women Table 2 In Vitro Fertilization Cycle Results in Women With Normal Ovarian Reserve Who Did or Did Not Smoke Cigarettes Smokers (n = 65) Nonsmokers (n = 145) Smokers* (n = 29) Nonsmokers (n = 73) Basal FSH (IU/L) Day 10 FSH (IU/L)* Basal E2 (pg/ml)t Day 10 E2 (pg/ml)t 5.3 ± ± ± ± 71 * Smokers> nonsmokers; P < t Conversion to SI unit, ± ± ± ± 114 ({3-error < 0.20) (Table 1). In contrast, the mean FSH concentrations on day 10 were significantly higher in women who smoked cigarettes (7.9 ± 0.4 IU/L) than in women who did not (6.8 ± 0.3 IU/L) (P < 0.05). More importantly, the incidence of diminished ovarian reserve as evidenced by abnormal CC challenge tests was significantly higher in women who smoked cigarettes (8 of 65 [12.31 %]) in comparison with women who did not smoke cigarettes (7 of 145 [4.83%]) (P < 0.05) (Fig. 1). The OR for this increased risk was 2.8 (95% CI, 1.2 to 7.99). Evaluation of the results from just the day 3 sample revealed no difference in the incidence of abnormal results between women who did and women who did not smoke cigarettes (3 of 65 [4.62%] versus 3 of 145 [2.07%], respectively) (OR, 2.29; 95% CI, 0.4 to 11.6). However, it should be noted that assuming a 20% difference in the values, the {3-error for this calculation was high at 0.55, indicating that the sample size may have been too small to detect a difference. The evaluation of the day 10 samples alone did reveal a significant difference with women who smoked cigarettes having a signifi % of 8 Patients 6 Day 3 IOSmokers Day 10 Non-Smokersl * Overall Figure 1 The incidence of diminished ovarian reserve as evidenced by abnormal CC challenge tests is significantly increased in women between the ages of 35 and 39 who smoke cigarettes in comparison with age-matched nonsmoking controls. Age (y) No. of ampules of gonadotropins Peak E2 (pg/ml)t No. of follicles <':15 mm No. of mature oocytes retrieved Fertilization rate (%) No. of pregnancies No. of deliveries 37.8 ± ± 1.1 1,413 ± ± ± ± 11 8 (27.6):j: 6 (20.7) * P = NS for all comparisons. t Conversion to SI unit, :j: Values in parentheses are percents ± ± 0.6 1,375 ± ± ± ± (28.8) 16 (21.9) cantly higher incidence (7 of 65 [10.77%]) of abnormal results when compared with their nonsmoking counterparts (6 of 145 [4.14%]) (OR, 2.79; 95% CI, 1.1 to 8.67) (P < 0.05). Outcome of IVF in Women Witb. Normal Ovarian Reserve Cigarette smoking had no detectable impact on the reproductive performance of women 35 to 39 years of age with normal ovarian reserve. Specifically, there were no differences in peak E2 levels, the number of ampules of gonadotropins used during the stimulation, the number of mature oocytes retrieved, or fertilization rates. The {3"error for these calculations was <0.20 except for the peak E2 comparison where the {3-error was The decreased power for the E2 comparison reflects the large variation in peak E2 levels among the patients, which is reflected in the larger standard deviations. The initial PRs were 27.6% (8/29) and 28.8% (21/73) in women who did and did not smoke, respectively, which were not significantly different (OR 0.96; 95% CI, 0.34 to 2.69). Similarly, there were no differences in the delivery rates in those women who smoked (20.7%; 6/29) and those who did not (21.9%) (16/73) (OR, 0.96; 95% CI, 0.38 to 2.47) (Table 2). Although the PRs appear remarkably similar in these two groups, power analysis reveals a {3-error of 0.38 and 0.42 for the total pregnancy and delivery rate comparisons, respectively. Thus while the ovarian response to gonadotropin stimulation data show that these women stimulate equally well independent of their smoking status, larger numbers will be required to make definitive comparisons of PRs. 260 Sharara et al. Smoking and diminished ovarian reserve Fertility and Sterility

5 DISCUSSION The data from this study demonstrate that infertile women who smoke have a significantly increased incidence of diminished ovarian reserve when compared with age-matched, nonsmoking controls. In the second part of the study, comparison of IVF results revealed that stimulation quality and PRs were not affected by smoking status among infertile women with normal ovarian reserve. Together, these findings support the hypothesis that a reduction in ovarian reserve may be causal in the lower fecundity rates seen among women who smoke cigarettes. Diminished ovarian reserve is a common cause of infertility (12). The term diminished ovarian reserve, coined by Navot et al. (13) in 1987, is manifested by an abnormally elevated FSH level obtained on cycle day 3 or after 5 days of CC therapy on cycle day 10. Women with diminished ovarian reserve have been characterized as having decreased ovarian responsiveness to exogenous gonad- 0tropins and lower PRs in assisted reproduction programs and during spontaneous cycles (12-15, 21). The incidence of diminished ovarian reserve rises among women beginning in their early 30s with steady increases thereafter (12). The factors that control the onset of diminished ovarian reserve are unknown. However, the fact that infertile women between the ages of 35 and 39 who participated in this study and who smoked cigarettes had a 150% increase in the incidence of diminished ovarian reserve strongly suggests that smoking may significantly accelerate this process. Many of the prior studies evaluating the impact of smoking on reproduction have produced findings consistent with the accelerated development of diminished ovarian reserve. These include studies indicating that smoking may have direct oocyte toxicity (5-7). This could contribute to accelerated follicular depletion, a factor that is generally associated with diminished ovarian reserve. An accelerated rate of follicular depletion would also be consistent with the fact that women who smoke cigarettes are more likely to have irregular menstrual cycles, poorer corpus luteum function, and eventually enter menopause up to 4 years earlier than their nonsmoking counterparts (5,8, 11). Although the prior studies evaluating the impact of smoking on fertility rates have produced mixed results, most have showed reduced fecundity rates among women who smoke cigarettes. Specifically, the OR of being infertile varied from 1.3 to 1.6 for the population who smoke cigarettes (4, 5). Another study has shown that women who smoke cigarettes are 3.4 times more likely to be infertile (4). Although these studies found no consistent increase in any given etiology of infertility, they did not screen for diminished ovarian reserve. Prospective studies evaluating the performance of infertile women who smoke cigarettes in assisted reproduction programs have also produced conflicting results. Hughes et al. (22) found no differences in peak E2 production, oocytes retrieved, or clinical PRs. In contrast, Van Voorhis et al. (23) found that women who smoked cigarettes produced lower peak E2 levels, produced fewer follicles, and had fewer oocytes recovered and embryos transferred. These findings were supported by the studies of Harrison et al. (24) and Elenbogen et al. (25) who also found reduced fertilization and PRs among their patients who smoked cigarettes. Although none of these studies screened theirpatient population for diminished ovarian reserve, the fact that three of the four studies mentioned above showed decreased ovarian responsiveness to stimulation with exogenous gonadotropins and subsequently lower fertilization and PRs is consistent with an accelerated onset of diminished ovarian reserve among women who smoke cigarettes. Furthermore, even the study by Hughes et al. (22) is consistent with an accelerated loss of ovarian reserve. The mean age of the patients who smoked cigarettes in that study was only 31.8 years. Given the young age of the infertile women who participated in the study, it is unlikely that a significantly increased proportion would have diminished ovarian reserve even ifthe overall process was proceeding at an accelerated rate. Stated another way, the state of diminished ovarian reserve represents the natural process of follicular depletion and loss of ovarian responsiveness. Even if this overall process is being accelerated, it is likely that until the development of a clinically detectable loss of ovarian reserve, a woman's ovarian responsiveness and reproductive potential would remain normal. Therefore, studies evaluating younger women will likely find no differences in stimulation quality and PRs because these women will not have had time to develop a clinically significant diminution in their ovarian reserve. This concept is supported by the data from the second portion of this study that demonstrated that smoking had no impact on stimulation quality or PRs among infertile women with normal ovarian reserve. It should be noted that an alternate interpretation of these data would be that cigarette smoking Sharara et ai. Smoking and diminished ovarian reserve 261

6 somehow inhibits the FSH response to CC administration. At the current time, the authors are unaware of any data that address this specific question. However, the fact that women who smoke have shown other evidence of diminished reserve such as a reduced response to gonadotropin stimulation makes it unlikely that the findings of the CC challenge test are artifactual. Nevertheless, future studies characterizing the development of diminished ovarian reserve in women who smoke will first need to evaluate this important question. The data from this study indicate that infertile women who smoke cigarettes have a significantly increased risk of having diminished ovarian reserve by the time they reach their late 30s. Additionally, the fact that smoking status had no detectable impact on outcome among infertile women with normal ovarian reserve suggests that this process may be a significant causal factor in the lower fecundity rates described in women who smoke cigarettes. However, additional information is clearly needed to define the role of smoking in the development of diminished ovarian reserve and its ultimate impact on reproduction. Longitudinal studies in which ovarian reserve is assessed repetitively in individuals who do and do not smoke cigarettes are needed. Studies evaluating the dose dependency (i.e., pack years of tobacco abuse and ages of exposure) will also be required. Other studies regarding the impact of smoking cessation may also be useful. In spite of the limitations ofthe current study and the need for further evaluations of these questions, the potential for accelerated loss of ovarian reserve among individuals who smoke cigarettes should be viewed as a significant health risk. As such, diminished ovarian reserve should be added to the already substantial list of adverse effects of which infertile women who smoke cigarettes should be made aware. REFERENCES 1. U.S. Department of Health and Human Services. The health consequences of smoking for women: a report of the surgeon general. Washington, D.C.: U.S. Government Printing Office, Fielding JE. Smoking: health effects and control. N Engl J Med 1985;313: Stillman RJ, Rosenberg MJ, Sachs BP. Smoking and reproduction. Fertil Steril 1986;46: Baird DD, Wilcox AJ. Cigarette smoking associated with delayed conception. JAMA 1985;253: Gindoff PR, Tidey GF. Effects of smoking on female fecundity and early pregnancy outcome. Semin Reprod Endocrinol1989;7: Mattison DR, Plowchalk DR, Meadows MJ, Miller MM, Malek A, London S. The effect of smoking on oogenesis, fertilization, and implantation. Semin Reprod Endocrinol 1989;7: Mattison RD. The effects of smoking on fertility from gametogenesis to implantation. Environ Res 1982;28: Yeh J, Barbieri RL. Effects of smoking on steroid production, metabolism, and estrogen-related disease. Semin Reprod Endocrinol 1989;7: Barbieri RL, McShane PM, Ryan KJ. Constituents of cigarette smoke inhibit human granulosa cell aromatase. Fertil Steril 1986;46: Adeno M, Gallagher H. Cigarette smoking and the age of menopause. Ann Hum Biol1982;9: Jick H, Porter J, Morrison AS. Relationship between smoking and age of natural menopause. Lancet 1977;1: Scott RT, Leonardi MR, Hofmann GE, Illions EH, Neal GS, Navot D. A prospective evaluation of clomiphene citrate challenge test screening of the general infertility papu- 1ation. Obstet Gynecol 1993;82: Navot D, Rosenwaks Z, Marglioth EJ. Prognostic assessment of female fecundity. Lancet 1987;2: Loumaye E, Billion J-M, Mine J-M, Psalti I, Pensis M, Thomas K. Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril 1990;53: Tanbo T, Dale PO, Lunde 0, Norma N, Abyholm T. Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated follicle-stimulating hormone levels. Fertil Steril 1992;57: Neveu S, Hedon B, Bringer J, Chinchole J-M, Arnal F, Humeau C, et al. Ovarian stimulation by a combination of gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil Steril1987;47: Scott RT, Rosenwaks Z. Ovulation induction for assisted reproduction. J Reprod Med 1989;34(1 Suppl): Flood JT, Muasher SJ, Simonetti S, Kreiner D, Acosta AA, Rosenwaks Z. Comparison between laparoscopically and ultrasonographically guided transvaginal follicular aspiration methods in an in vitro fertilization program in the same patients using the same stimulation. J In Vitro Fert Embryo Transf 1989;6: Veeck LL. Oocyte assessment and biological performance. Ann NY Acad Sci 1988;541: Feinstein AR. Clinical epidemiology: the architecture of clinical research. Philadelphia: W.B. Saunders, Scott RT, Toner JP, Muasher SJ, Oehninger SC, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril1989;51: Hughes EG, YoungLai EV, Ward SM. Cigarette smoking and outcomes of in -vitro fertilization and embryo transfer: a prospective cohort study. Hum Reprod 1992;7: Van Voorhis BJ, Syrop CH, Hammitt DG, Dunn MS, Snyder GD. Effects of smoking on ovulation induction for assisted reproductive techniques. Fertil Steril1992;58: Harrison KL, Breen TM, Hennessey JF. The effect of patient smoking habit on the outcome of IVF and GIFT treatment. Austr NZ J Obstet Gynaecol 1990;30: Elenbogen A, Lipitz S, Mashiach S, Dor J, Levran D, Ben Rafael Z. The effect of smoking on the outcome of in-vitro fertilization-embryo transfer. Hum Reprod 1991;6: Sharara et al. Smoking and diminished ovarian reserve Fertility and Sterility