Influence of smoking on fertility in women attending an in vitro fertilization program

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1 » FERTILITY AND STERILITY Copyright 1996 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Influence of smoking on fertility in women attending an in vitro fertilization program Karl Sterzik, M.D.*t Erwin Strehler, M.D.* Mariella De Santo, M.D.:j: Nicole Trumpp, cando med.* Markus Abt, Ph.D. Bernd Rosenbusch, Ph.D. * Achim Schneider, M.D., M.P.R.II University of Chieti, Chieti, Italy; University of Ulm, Ulm; University of Augsburg, Augsburg; and University of Jena, Jena, Germany Objective: To investigate the influence of cigarette smoking of women on the fertilization and pregnancy rates obtained by IVF treatment. Patients: One hundred ninety-seven infertile, otherwise healthy women who entered an IVF program for the first time. Setting: Fertility unit at the Women's University hospital of the University of Ulm, Ulm, Germany. Interventions: The study population consisted of 197 women (23 to 39 years old) who were divided into the following groups: nonsmokers (n = 68), passive smokers (n = 26), and active smokers (n = 103) according to the cotinine concentration measured in follicular fluid. The reason for infertility was strictly a tubal factor with apparently normal ovulatory cycles. To guarantee an objective recording of tobacco smoke exposure, the smoking habit was not determined by questionnaires, but by cotinine, the principal metabolite of nicotine. Results: There were no significant differences in fertilization and pregnancy rates between the different groups. The E2 serum levels were decreased significantly in women who smoked when compared with the results obtained from nonsmokers and passive smokers. Overall, a strong negative correlation of the cotinine and E2 levels was observed (r = -0.65). Conclusion: The results suggest that there is no clinically detectable impairment offertilization potential due to female smoking and that there is a greater influence on the outcome of IVF by other factors. Fertil Steril 1996;65:810-4 Key Words: Smoking of women, IVF, fertility A negative influence of smoking on female reproduction is postulated in several studies. Because more and more young women are smoking, the number of patients in IVF programs with a history of smoking is increasing. Nicotine and other smokingderived agents may act on the level of hypothalamus Reviewed May 30, 1995; revised and accepted September 21, * Department of Gynecology and Obstetrics, University ofulm. t Reprint requests: Karl Sterzik, M.D., Department of Gynecology and Obstetrics, University of mm, Prittwitzstrasse 43, D mm, Germany (FAX: ). :j: Department of Gynecology and Obstetrics, University of Chieti. Institute for Mathematics, University of Augsburg. II Department of Gynecology, University of Jena. and hypophysis, ovaries, fallopian tubes, oocytes, embryo, and/or the endometrium (1-5). Nicotine and its metabolites possibly may cause lower fertilization and pregnancy rates in IVF. However, studies investigating these problems have been contradictory. In the majority ofthese studies, smoking status is determined subjectively using self-administered questionnaires (2, 4, 6, 7); only a few studies analyze serum concentrations of cotinine, which is considered an objective measurement ofnicotine consumption because of its longer half-life (8,9). It was the purpose of this study to look for possible associations between cotinine concentration in follicular fluid (FF) recovered by follicle aspiration and the fertilization and pregnancy rates in an IVF pro- 810 Sterzik et at. Smoking and fertility

2 gram. To assess a possible influence of passive smoking, patients were stratified as nonsmokers, active smokers, and passive smokers. Patients MATERIALS AND METHODS One hundred ninety-seven patients (age range 23 to 39 years, median age 33 years) who entered an IVF program for the first time were recruited into this study. Entry criteria for this study were a pathologic tubal factor representing the cause of sterility, normal spermiogram in the male partner, duration of sterility> 1 year, and positive follicle aspiration after hormonal stimulation. In Vitro Fertilization For stimulation and induction of ovulation, patients were treated with hmg and hcg. Starting with day 8 of the cycle, follicle diameter and serum concentration of E2 were measured in all patients. Protocols for stimulation, follicle aspiration, insemination, and ET have been described previously (10). Estradiol serum concentration on the day of ovulation induction using hcg was included in statistical analysis. Fertilization was diagnosed 18 to 24 hours after insemination when two pronuclei were visible. Pregnancy was defined by sonographic detection of positive fetal heart movement 20: 28 days after ET. Detection of Cotinine Concentration in FF In all 197 patients, cotinine concentration was measured in the FF. Mter all oocytes had been transferred in the culture medium, the FF of each patient was pooled, a mean cotinine concentration was measured using a competitive cotinine enzyme immunoassay (Biosciences-Corporation, Houston, TX). The interassay coefficient of variation (CV) was < 10%. Based on a previous study of smokers (11), smoking status was classified according to the following cotinine concentrations in the FF: nonsmoker, cotinine concentration :5 20 ng/ml (conversion factor to SI unit, 5.68); passive smoker, cotinine concentration >20 ng/ml and :550 ng/ml; active smoker, cotinine concentration >50 ng/ml. In addition, history of smoking was assessed using interview-based questionnaires. Statistical Analysis Estradiol serum levels were compared with fertilization and pregnancy rates in association with smoking status using X 2 for independence and Wilcoxon test for significance. RESULTS Of 197 patients in an IVF collective, 68 woman (34.5%) were classified as nonsmokers, 26 (13.2%) were classified as passive smokers, and 103 (52.3%) were classified as active smokers. Cotinine values up to 500 ng/ml were measured in FF of active smokers. Age of patients and mean number of recovered oocytes per cycle were similar for all three groups (Table 1). Only one cycle per patient was evaluated. The fertilization rate per cycle was 67.6% for nonsmokers, 57.7% for passive smokers, and 67.9% for active smokers (P = 0.59) (Table 1). The pregnancy rates were 32.6%, 33.3%, and 32.9%, respectively (P = 0.99). A significant difference between active smokers and passive or nonsmokers was found (P < 0.025) only for E2 serum concentration. Between passive and nonsmokers, no significant E2 level differences were assessed. Comparison of fertile (fertilization of at least one oocyte in the IVF attempt) and nonfertile patients (no fertilization occurred) showed no significant differences of cotinine concentration (P = 0.91) (Fig. 1) or E2 serum concentration (P = 0.34). A negative correlation was found between cotinine and E2 values when analyzing all patients (r = -0.65; P < 0.01). DISCUSSION The influence of smoking status (nonsmokers, passive smokers, or active smokers) on fertility in women undergoing IVF was evaluated in this study. We used cotinine concentrations and interviewbased smoking history to classify patients. Assessment of smoking status using self-administered questionnaires is subjective and unreliable. Resorption of nicotine and other smoking-related agents varies according to the different components of tobacco and smoking habits (depth, intervals, and frequency of inhalation per cigarette). In addition, resorption through the mucous membranes and metabolism of nicotine and other smoking-related agents may vary considerably between different individuals. These factors cannot be assessed by using smoking history as an instrument. Thus, objective measurements, using specific parameters such as nicotine or its direct metabolic products, are more specific parameters (9, 12). The half-life of nicotine is short (2 hours) and the measurement of nicotine as an indicator of the ingested amount of smoke is unreliable. In contrast, its main metabolite cotinine has a half-life of 16 to 20 hours and correlates well with the total amount of inhaled cigarette smoke. Sterzik et al. Smoking and fertility 811

3 F Table 1 Treatment Cycle Data for Nonsmoking, Passive Smoking, and Active Smoking Groups* Cotinine (ng/ml)* Nonsmoking 0;;20 Passive smoking 20 < x 0;; 50 Active smoking >50 No. of patients Duration of infertility (y) No. of cycles Age (y) No. of ampules used Peak E2 levels (pg/ml) Day ofhcg No. of follicles aspirated No. of oocytes recovered Fertilization rate (%) No. of embryos transferred Clinical pregnancies (%):j: : : : 3.2 1,658 2: : : : : (32.6) : : : 3.5 1,738 2: : : : : (33.3) : : : : 57.3t 9.6 2: : : : (32.9) * Values are means 2: SD. Conversion factor to SI units are as follows: cotinine, 5.68; E 2, t p < :j: Values in parentheses are percentage of transfers. Therefore, we used cotinine concentration in the FF as our main parameter for definition of smoking status. Results of our study show that fertilization and pregnancy rates in women attending an IVF program are not influenced by smoking. Only a few studies have analyzed this problem and their results are inconclusive. Several authors found a reduction of fertility (2, 9, 13), whereas others could not confirm different fertilization rates between smokers and nonsmokers (4, 6, 8). Data are difficult to com- Cotinine (ng/ml) , * * fertile group non-fertile group Figure 1 Cotinine concentrations of fertile and nonfertile patients (box plots demonstrating median, upper, and lower quartiles and ranges). Conversion factor to SI unit, *Not significant, P = pare because definition of smoking status is heterogeneous in these various studies. No study distinguished between nonsmokers and passive smokers. In the prospective study on 650 women from an IVF-ET or GIFT collective, Harrison and co-workers (6) evaluated smoking status by an interview-based questionnaire. Five hundred forty-two women were nonsmokers and 108 women were smokers. All women were younger than 40 years and comparable with respect to cause of infertility and mode of treatment. Rate of fertilization was higher in smokers compared with nonsmokers, but the difference was not statistically significant. However, clinical pregnancy rates were higher in nonsmokers (22% versus 8%) because of a higher risk of spontaneous abortion in smokers. Fertilization rate was reduced significantly in smoking women in a study reported by Rosevear and co-workers (9). Smoking status was assessed by analyzing cotinine concentration in the FF in 45 women. All patients were younger than 40 years, had normal ovarian cycles, and were classified as smokers when cotinine concentration was >20 nglml (n = 13). Cause of sterility was either tubal or unknown. A normal postcoital test was used to exclude a pathologic-andrological factor. The fertilization rate in smokers was significantly lower than in nonsmokers (44% versus 72%). This was confirmed in a followup study by Rowland and co-workers (13) using an identical design. Smoking habits of the male partners were assessed and a reduced fertilization rate was found when either women or men were smoking. The latest report in this context from Hughes and co-workers (8) found no association between smoking status and rate of fertilization. Serum concentration of cotinine was used to assess smoking status, which correlates well with the cotinine level in FF. Cotinine and E2 serum levels were assessed in two 812 Sterzik et ai. Smoking and fertility

4 subgroups of 52 smokers and nonsmokers. Patients did not differ with respect to mean age. Rates of fertilization and pregnancy did not differ significantly between nonsmokers, exsmokers, and active smokers. When women were stratified according to age, a significantly decreased rate of conception was found in women older than 35 years (P < 0.04). It was concluded that age is a stronger negative prognostic factor for successful IVF treatment than smoking. A reduced ovarian threshold rate is postulated in a study using the clomiphene citrate (CC) challenge test (5). Exclusively, women in the age group between 35 and 39 years were evaluated. One group of women represented normal infertility patients with respect to cause of sterility. A second group of patients was recruited from an IVF program. They were diagnosed with tubal sterility and normal ovarian function verified by a normal CC challenge test. In the group of IVF patients with intact ovarian function, fertilization rate, pregnancies, and births did not differ between smokers and nonsmokers. However, in the first group, ovarian function of smokers was reduced significantly compared with nonsmokers. Clomiphene citrate challenge test was pathologic in 12.3% of smokers compared with 4,8% of nonsmokers. Sharara and co-workers (5) concluded that smoking may reduce the ovarian threshold rate. This has no influence on smoking women who have an unimpaired ovarian threshold rate due to young age. Our results are comparable to the ones reported by Hughes and co-workers (8). Because we selected patients with normal ovarian function but pathologic tubal factor, our data also are identical to those found by Sharara and co-workers (5) in their second group of patients. Discrepancy with the results of Rosevear et al. (9) may be explained by a larger number of patients examined in our study, which to our knowledge is the largest one so far using cotinine measurement to assess smoking status. In addition, inclusion criteria varied between the study of Rosevear et al. (9) and our study: in contrast to a positive postcoital test we used a spermiogram with normozoospermia as our entry criterion. A reduced quality of the oocytes due to smoking may be compensated by a morphologically and functionally intact spermatocyte. Thus, no difference in the fertilization rates between smoking and nonsmoking women may be observed if pathologic-andrological factors are excluded. This putative compensation mechanism, which is not present when the quality of sperm is reduced, could be evaluated in a collective of subfertile men analyz- ing fertilization rates according to the smoking status of women. Several studies confirm the negative influences of smoking on E2 serum concentration (1, 2, 14). We found a significantly reduced E2 concentration in smokers and a significantly negative correlation between cotinine and E2 concentration (r = -0.65). Reduced E2 levels in the first half of the cycle may lead to impairment of proliferation of the endometrium, a decreased preovulatory LH surge, ovarian dysfunction, and irregular cycles. Decreased E2 serum levels probably are due to a nicotine concentration-dependent blockage of aromatase in the granulosa cells (1). Aromatase, which also is present in the placenta, metabolizes T to estrogen. Increased metabolism of estrogen in the liver by enzyme induction may be an alternative mechanism for an E2 decrease. Decrease of E2 concentration was not associated with a negative influence on fertilization and pregnancy rates in smokers in our study. This may be explained by ovarian stimulation in an IVF cycle. Hormonal and morphological changes caused by decreased E2 concentrations have been assessed only in nonstimulated physiological cycles so far. We conclude that no association between active smoking, passive smoking, and nonsmoking and the rates of fertilization and pregnancy was found in women attending an IVF program. This conclusion, however, is valid only for the specific cohort of patients examined, who were of young age, had a normal tubal factor, unimpaired ovarian function, and male partners with a normal spermiogram. Therefore, a general conclusion that smoking has no influence on fertility in women cannot be made. REFERENCES 1. Barbieri RL, McShane PM, Ryan KJ. Constituents of cigarette smoke inhibit human granulosa cell aromatase. Fertil Steril 1986;46: Elenbogen A, Lipitz S, Mashiach S, Dor J, Levran D, Ben Rafael Z. The effect of smoking on the outcome of in vitro fertilisation-embryo transfer Hum Reprod 1991;6: Fredricsson B, Gilljam H. Smoking and reproduction: short and long term effects and benefits of smoking cessation. Acta Obstet Gynecol Scand 1992;71: Pattinson HA, Taylor PJ, Pattinson MH. The effect of cigarette smoking on ovarian function and early pregnancy outcome of in vitro fertilization treatment. Fertil Steril 1991;55: Sharara FI, Beatse SN, Leonardi MR, Navot D, Scott RT Jr. Cigarette smoking accelerates the development of diminished ovarian reserve as evidenced by the clomiphene citrate challenge test. Fertil SterilI994;62: Harrison K, Breen T, Hennessey J. The effect of patient smoking habit on the outcome of IVF and GIFT treatment. Aust NZ J Obstet Gynaecol 1990;30: Sterzik et al. Smoking and fertility 813

5 7. Hughes EG, YoungLai E, Ward SM. Cigarette smoking and outcome of in vitro fertilisation and embryo transfer: a prospective cohort study. Hum Reprod 1992;7:358-6l. 8. Hughes EG, Yeo J, Claman P, Young Lai EV, Sagle MA, Daya S, et a1. Cigarette smoking and the outcomes of in vitro fertilization: measurement of effect size and levels of action. Fertil Steril 1994;62: Rosevear SK, Holt DW, Lee TD, Ford WCL, Wardle PG, Hull MGR. Smoking and decreased fertilization rates in vitro. Lancet 1992;340: Sterzik K, Dallenbach C, Schneider V, Sasse V, Dallenbach Hellweg G. In vitro fertilization: the degree of endometrial insufficiency varies with the type of ovarian stimulation. Fertil Steril 1988;50: Grab D, Lucke R, Benz R, Wenderlein M. Stellenwert des Passivrauchens in der Schwangerschaft. Z Geburtshilfe Perinatol 1988; 192: Benowitz N. Pharmacologic aspects of cigarette smoking and nicotine addiction. N Engl J Med 1988; 17: Rowlands DJ, McDemott A, Hull MG. Smoking and decreased fertilization rates in vitro. Lancet 1992;340: Van Voorhis BJ, Syrop CH, Hammitt DG, Dunn MS, Synder GD. Effects of smoking on ovulation induction for assisted reproductive techniques. Fertil Steril 1992;58: r I, 814 Sterzik et al. Smoking and fertility