Pollution may play a role in recent population trends of declining semen quality1–3 and in regional differences in time to pregnancy (TTP),4–6 in industrialized societies. One class of environmental compounds suspected of affecting population fertility is endocrine disruptors, which include dioxins.2 Dioxins, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are commonly found in industrialized areas as a waste product of combustion. Dioxins are highly lipophilic and extremely persistent in the human body.7 Few studies have examined the relationship of endocrine disrupting compounds and fertility. Of these, most have suggested an association between higher exposure to polychlorinated biphenyls (PCBs) and longer TTP.8–11 However, most did not specifically examine the dioxin-like compounds or use a direct measure of exposure.
On 10 July 1976, a trichlorophenol-manufacturing plant explosion near Seveso, Italy resulted in the highest TCDD exposure known in human residential populations.12 Twenty years later, the Seveso Women's Health Study followed women living in this area to determine whether TCDD exposure had adverse effects on reproductive health. Individual TCDD exposure was quantified using serum samples collected soon after the explosion. Individual serum TCDD levels were associated with a small increased risk for earlier menarche among women who were younger than 5 years of age at the time of the explosion13,14 and with an increase in menstrual cycle length among women who were premenarcheal at exposure.15 We furthermore observed a nonmonotonic dose-related association of TCDD with earlier onset of natural menopause,16 but we failed to detect associations of TCDD levels with ovarian function17 or spontaneous abortion.18 In the present study, we examine the association of individual serum TCDD with TTP and infertility.
To be eligible for the Seveso Women's Health Study, women had to be newborn to 40 years old at the time of the explosion, living in the most highly contaminated area at the time of the explosion (zones A or B), and have adequate volumes of stored sera collected soon after the explosion (see Eskenazi et al19 for details). Women were interviewed between March 1996 and July 1998. Of the 981 participants, 745 reported having ever been pregnant, and 472 had pregnancies after the explosion. Nine women were excluded because they reported fertility-related problems including history of infertility prior to the explosion, fertility drug use within 12 months of trying, or male fertility problems, leaving 463 eligible women for analysis.
For the main analyses, we included only women who delivered a live birth that was not the result of contraceptive failure (n = 278) (Fig. 1). In other analyses, we included one or more of the following groups: women who delivered a live birth resulting from contraceptive failure (n = 49) or from irregular use of contraception (n = 33); women with a pregnancy not associated with contraception that resulted in a pregnancy outcome other than a live birth (n = 47 [41 spontaneous abortions, 2 voluntary abortions, 4 ectopic pregnancies]); or women who delivered other than a live birth resulting from contraceptive failure (n = 46 [9 spontaneous abortions, 37 voluntary abortions]). In addition, for some analyses, we included 7 women who reported trying for at least 12 months to become pregnant, but who never became pregnant and who did not attribute this difficulty to a male fertility problem.
Study details are presented elsewhere.19 Briefly, participation included signed informed consent, structured personal interview, blood draw, and, for most women, a gynecologic examination and ultrasound. Medical records were requested for all other gynecologic procedures/surgeries. Interviews were conducted in private by trained nurse-interviewers who were unaware of the zone of residence and serum TCDD levels. Detailed information was collected about the first postexplosion pregnancy. TTP was determined from the question, “How many months did it take to become pregnant? In other words, for how many months had you been having sexual intercourse without doing anything to prevent pregnancy?” A calendar and pregnancy wheel were used to assist participants in recollection. If a couple had used contraception or the pregnancy did not result in a live birth, the participant was asked the same questions about the next pregnancy that had ended in a live birth. The Institutional Review Boards of participating institutions approved the study.
TCDD was measured in archived sera by high-resolution gas chromatography/high-resolution mass spectrometry methods.20 Values are reported on a lipid-weight basis in parts per trillion (ppt).21 Details of serum sample selection are presented elsewhere.19 The analytic sample consists of 463 women. We measured TCDD in sera collected in 1976 or 1977 for 431 women; between 1978 and 1981 for 13 women; and in 1996 or 1997 for 19 women with insufficient volume in earlier samples. For women with detectable post-1977 TCDD measurements (n = 27), the TCDD level was back-extrapolated to 1976, using the Filser Model.22 For nondetectable values (n = 58), a serum TCDD level of one-half the limit of detection was assigned.23
Serum TCDD was analyzed both as a continuous variable (log10 TCDD) and as a four-category variable. The cut-point for the lowest group was set at ≤20 ppt (body burden ≈4 ng/kg), because 15–20 ppt was the average TCDD level in serum pools collected from unexposed Italian women in 1976.24 The 3 remaining categories were defined by calculating tertiles of exposure >20 ppt in our main analytic population of 278 women, giving groups: ≤20, 20.1–44.4, 44.5–100 and >100 ppt. For the extrapolated values, 4 quartile groups were created: <6, 6.0–14.2, 14.3–40.7, and >40.7 ppt.
The relevant exposure may be the body burden at the time a woman was attempting to become pregnant rather than the initial dose. We therefore also estimated TCDD levels at the time each woman initiated her attempt to become pregnant by extrapolating from her serum TCDD level measured near the explosion. Specifically, for women who were 16 years old or less at the time of explosion, we used a physiologically based toxicokinetic model,22 and for women who were older than 16 years, we used a first-order kinetic model assuming a 9-year half-life.25 TCDD extrapolated to pregnancy attempt (henceforth referred to as “extrapolated TCDD”) was considered both continuously (log10) and categorically, divided into quartiles based on the main analytic population of 278 women.
“Infertility” was defined as 12 or more months to pregnancy. We employed multiple logistic regression to analyze the association between serum TCDD and infertility. To analyze the association between serum TCDD and TTP, we estimated fecundability odds ratios (fORs) and 95% confidence intervals (CIs) using Cox proportional hazards models adapted for discrete time data. The fOR can be viewed as the odds of conceiving in a given cycle per 10-fold increase in TCDD (log10 TCDD), or for each category of exposure relative to the referent exposure group. A fOR less than 1.0 indicates reduced fecundability or longer TTP. TTP was measured in months with censoring at 13 months, although alternate censoring scenarios were also explored (see below).
To check for possible biases and to investigate the consistency of our findings, we conducted several additional sensitivity analyses, as recommended by Joffe et al.26 Models were repeated: (1) including the 7 women who had not achieved pregnancy by the time of interview, censored at 13 months; (2) excluding women who reported conceiving in the first cycle; (3) expanding the population to include pregnancies resulting from contraceptive failure; (4) expanding the population to include nonlive births; and (5) changing the censoring time to 14, 10, or 7 months. When included in sensitivity analyses, contraceptive failures were coded with a TTP of 0 or 1. For women reporting use of contraception “not quite regularly,” TTP was assigned a value of 0 for some sensitivity analyses and for others was calculated as one-half the reported duration of irregular use.
Covariates were included in Cox models if they changed the coefficient for log10 TCDD by at least 10% or if they were independently associated with TTP (P < 0.10). For simplicity, the same covariates were used in the models for infertility after we checked that no additional covariates were important in these models. Covariates retained include maternal age, maternal smoking in the year before conception, parity, menstrual cycle irregularity, oral contraceptive (OC) use in the year before attempt, paternal age near the time of conception, and history of reproductive and endocrine conditions including pelvic infection, thyroid or urogenital problems. Additional covariates that we did not keep in the final model included marital status, frequency of sexual intercourse, menstrual cycle length, whether actively trying or “not concerned” about becoming pregnant, age at menarche, maternal and paternal education, history of cesarean-section, alcohol and coffee consumption, paternal smoking around the time of conception, and zone of the father at the time of the explosion. For the 7 women who did not become pregnant, we imputed paternal age at the time of the initial attempt to become pregnant, based on maternal age and age married. We evaluated the form of the dose–response curve with fractional polynomials.27
We repeated the main analysis with the following variations: adding frequency of sexual intercourse as a covariate (n = 275); limiting the population to primiparous women (n = 224), to those who were less than 8 years old at explosion (n = 19), to those who were premenarcheal at explosion (n = 59), and to women not using oral contraceptives within a year of trying (n = 201). All statistical analyses were performed using STATA 8.0 (Stata Corp, College Station, TX).
Demographic characteristics of the 278 eligible women who became pregnant after the explosion are presented in Table 1. Women averaged 17 years of age at the time of explosion, 26 years at the time they began trying to become pregnant, and 38 years at the time of interview. At the time of the index pregnancy almost all women (99%) were less than 36 years old and most (81%) were primiparous. About 27% smoked and 28% used OCs in the year before the pregnancy. Fathers of the index pregnancy averaged 30 years of age at the time of trying to conceive, and 23% were reported by their partners to have lived in Zones A or B at the time of the explosion.
For the 278 women, the median TCDD concentration in blood collected around the time of the explosion was 50 ppt (interquartile range [IQR] = 25–117). The median TCDD level extrapolated to the time of conception was 13.4 ppt (IQR = 5.3–38.2). Median serum TCDD levels at explosion were higher in women who were nulliparous and in those who used OCs in the year before the pregnancy (Table 1). Age at explosion for the entire Seveso cohort24 and in this study population is negatively associated with serum TCDD levels, with the youngest women having the highest levels. The associations of TCDD levels with parity and oral contraceptives likely reflect this association of TCDD with age.
The median (IQR) time to the index pregnancy was 2 (1–7) months. Table 1 shows odds ratios for fecundability (monthly probability of conception). Lower fecundability was associated with smoking (fOR = 0.76), having irregular cycles (fOR = 0.63), having a history of reproductive or endocrine conditions (fOR = 0.59), and older paternal age (fOR = 0.94 per year).
Table 2 shows the association of time to pregnancy with initial and extrapolated TCDD levels, with and without adjustment for covariates. In the adjusted analysis with log10 serum TCDD as a continuous variable, a 10-fold increase in TCDD is associated with a 25% decrease in the per cycle probability of conception (adjusted fOR = 0.75 [95% CI = 0.60–0.95]). A fractional polynomial in log10 TCDD indicated that no model (with up to 4 knots) fit better than the linear model (data not shown). In the adjusted categorical model of TCDD, with a reference category of ≥20 ppt, categories of 20.1–44.4, 44.5–100, and >100 ppt were associated with decreases in adjusted fORs (1- adjusted fOR) of 19%, 29%, and 37%, respectively. The results were similar after controlling for frequency of sexual intercourse or eliminating adjustment for parity prior to the explosion, OC use in the year before attempt, or irregular cycles.
The results were similar for analyses with extrapolated TCDD levels. We observed a 27% decrease in fOR for every 10-fold increase in extrapolated TCDD (adjusted fOR = 0.73 [0.58–0.94]). In the categorical analysis, with a reference category of <6 ppt, the categories 6.0–14.2, 14.3–40.7, and >40.7 ppt had adjusted fORs of 1.49, 0.95, and 0.76, respectively. Although there is a suggestion of a rise, then fall, in the dose-response curve, the fractional polynomial model indicated the linear model provided the best fit (data not shown).
Figure 2 shows the fecundability odds ratios from the model with continuous (log10) serum TCDD, adjusted for covariates (also shown in Table 2), for 12 definitions of the population and outcomes. Population 1 is that analyzed in Table 2, referred to as the “main population.” The other scenarios vary in their inclusion or exclusion of women who never conceived; women who conceived in the first month of trying; irregular users of contraception; outcomes other than live births; and month of censorship. Results for all scenarios were similar to those shown in Table 2 for the main population. Ten-fold increases in serum TCDD were associated with decreased adjusted fORs ranging from 0.68 to 0.82. Similar results were observed when extrapolated TCDD was used in place of measured TCDD (data not shown).
We repeated analyses with log10 serum TCDD for other subpopulations. Although numbers were small, the results were generally similar to those above. For example, among women who were premenarcheal at the time of the explosion, the adjusted fOR was 0.78 (0.45–1.35; n = 59); among those who were 8 years old or younger, the unadjusted fOR was 0.70 (0.18–2.67; n = 19). Among primiparous women (n = 224), the adjusted fOR was 0.76 (0.59–0.97); and among women who had not used OCs within the year prior to trying to conceive (n = 201), the results were also similar (adjusted fOR = 0.83 [0.61–1.11]).
Forty-nine of 285 women (17%) reported taking twelve months or longer to conceive their first postexplosion pregnancy. (This numerator excludes women who attributed their delayed conception to reproductive problems in their partners.) Table 3 reports the crude and adjusted odds ratios (OR) for infertility. With a 10-fold increase in serum TCDD level, we found nearly a doubling in the odds of infertility (adjusted OR = 1.9 [1.1–3.2]). When serum TCDD levels were categorized, the trend for increasing odds of infertility had a P value of 0.02. The results were similar for extrapolated TCDD levels.
We found that a 10-fold increase in TCDD was associated with a 25% reduction in the monthly probability of conception and a doubling of odds that the pregnancy took 12 or more months to conceive. We found similar reductions in fertility when we considered dose extrapolated to the time of conception or included different subpopulations in sensitivity analyses. Although the average level of TCDD exposure at the time of the explosion was high, the average levels at the time of conception are at the high end of the range of current background levels reported in Europe.28 Thus, these results suggest that dioxin exposure may play a role in reduced fertility in industrialized areas where dioxins are a widespread contaminant.
Previous studies have examined the relationship of other persistent organochlorines and female fertility, with inconsistent results. Axmon et al8 examined the relationship of serum PCB-153 and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) levels and fecundability in women from Greenland, Warsaw, and Kharkiv, and in a cohort of Swedish fisherman's wives. Neither PCB-153 nor DDE is dioxin-like, but PCB-153 correlates with TCDD in the population. Axmon et al found an association with TTP only in women from Greenland, where both PCB-153 and DDE were strongly correlated; it was not possible to isolate the separate effects of the compounds. In another study, Law et al10 used stored serum specimens from the Collaborative Perinatal Project to examine the association of TTP with serum measures of DDE and 1,1,1-trichloro-2,2′-bis(p-chlorophenyl)ethane (DDT) and 11 PCBs, 2 of which were dioxin-like. They found that TTP increased for women in the highest exposure group for both total PCBs (fOR = 0.65 [95% CI = 0.36–1.18]) and for DDE (0.65 [0.32–1.31]), compared with women in the lowest group, but there were no congener-specific associations or associations with DDT. Other studies have examined women who had high levels of PCBs and other organochlorines because they ate contaminated fish. In the New York Angler Cohort, individual serum levels were not measured, but TTP was associated with frequency and number of years of fish consumption and with PCB intake estimated from species, frequency of consumption, and portion size.9 In contrast to these studies, a study of fisherman wives/sisters in Sweden,29 where PCB-153 serum levels were back-extrapolated to the pregnancy, showed increased fecundability in the highest exposed groups.
We know of no previous study of time to pregnancy and infertility that has studied dioxin. The study most similar to ours examined the women of Yucheng in Taiwan, who were poisoned by PCB-contaminated cooking oil in 1979.11 Serum samples from a subset of the population taken more than a decade later revealed high levels of polychlorinated dibenzofurans and total PCBs. The authors compared 186 Yucheng women with 226 unexposed controls, and found that fecundability decreased by about 10% in the Yucheng women and that the odds of infertility doubled (OR = 2.3). In the small subset of Yucheng women with earlier measured PCB serum levels, there was also a decrease in fecundability in the higher group compared with the lower PCB group.
The results of the present study of women from Seveso are corroborated, in part, by the recent results of semen and hormone analyses of men from Seveso.30 These analyses found decreases in semen quality in men who were less than 10 years old at the time of the explosion. The fact that we do not observe a relationship of fecundability and husbands' residence in Zones A or B (based on the wives' report) may be due to the small sample size. Slama et al31 found that several thousand couples are needed to show moderate declines in sperm concentration sufficient to produce a noticeable change in time to conception.
As was observed for the men in Seveso, women who were younger at the time of TCDD exposure may be among the most susceptible, given that their reproductive systems were not fully mature, and that, in the Seveso cohort, children had the highest serum TCDD levels.24 Nevertheless, we observed little difference in fecundability by age at exposure, perhaps because of the relatively small number of pregnancies in the younger subgroup 20 years after the explosion. (We are currently conducting a follow-up study to add 10 years of reproductive experience for these women.) The population of greatest susceptibility may not be the women but their daughters. Previous studies of other endocrine disruptors reported that exposure during in utero development of the ovary may be the most sensitive stage to affect subsequent fertility.32
An increase in length of time to pregnancy can result from many different mechanisms, including effects on oocyte reserve or on hormones that may influence ovulation or maintenance of the corpus luteum, or an increase in undetected spontaneous abortions resulting from failure in implantation or embryo development. There is evidence that TCDD might affect each of these processes. In animal studies, TCDD has been shown to alter ovarian function, including steroidogenesis and ovulation.33,34 Although we found no relation of TCDD with current ovarian function in the Seveso cohort,17 TCDD exposure in rats has been associated with morphologic changes in the ovary, inhibition of follicular maturation and rupture, and altered cyclicity with disruption of the estrous cycle.33,35–37 Altered hormone levels have also been reported with TCDD exposure in rats and primates.34,35,38 In addition, although we found no relation between TCDD exposure and clinically-recognized spontaneous abortion in Seveso,18 TCDD exposure has been shown to affect early embryo development.39 Thus, the potential for dioxin exposure to influence fertility and fecundability is biologically plausible.
One of the strengths of this study is the robustness of the findings with changes in the inclusion criteria (planning status, contraceptive use, birth outcome, or other criteria). Another strength is the data on individual serum TCDD in samples drawn close to the time of the explosion. Reports of pregnancy outcome were not likely to be biased by knowledge of exposure because participants and interviewers were unaware of TCDD levels, and because all participants lived in exposed zones.
The present study also has a number of limitations. First, although we measured TCDD in blood collected near the time of the explosion, we do not know the exact level at the time the woman attempted conception. We were able, however, to estimate this dose by extrapolation, and the analyses of extrapolated and measured levels yielded similar results. Because we analyzed the first postexplosion pregnancies, the accuracy of the extrapolation could not be affected by lactation or subsequent pregnancies. Extrapolation could, however, be affected by factors we cannot fully consider, such as body weight changes. Second, few women had very low TCDD exposure, because all lived in exposed zones. Moreover, we previously showed that background levels of other dioxin-like compounds were also high.24 Thus, our findings may underestimate the true relationship of TCDD and fecundability and infertility. Third, pregnancy histories were recorded an average of 12 years following the index pregnancy, raising the possibility of inaccurate recall. However, Joffe et al40 found that even more than 14 years after a pregnancy, women can fairly accurately report their time to conception. Inaccuracies in reported times to conception were not likely to be related to exposure because participants and interviewers were unaware of exposure level, as described above.
Retrospective report of time to pregnancy has been used in a number of previous studies examining the potential effects of environmental chemicals.8–11 To address the potential biases and inadequacies of this type of study, Joffe et al41 recommended several steps, including the use of discrete-time survival analysis, sensitivity analyses, a study sample representative of the underlying population, and a well-designed questionnaire. We have attempted to adopt these recommendations.
In conclusion, we found dose-related increases in time to pregnancy and infertility associated with individual serum TCDD levels in women from Seveso, Italy. These findings were robust to inclusion/exclusion criteria. It is unclear by what mechanism TCDD may affect fecundability, because fecundability is the end-product of multiple and potentially unknown factors. Given that the serum levels of TCDD extrapolated to the time of the pregnancy are at the high end of the range observed in some European countries, the results of this study may have implications for fertility in industrialized areas.
We gratefully acknowledge Stefania Casalini for coordinating data collection at Hospital of Desio, and Donald Patterson and Wayman Turner (CDC) for serum TCDD measurements.
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