A cohort study of in utero polychlorinated biphenyl (PCB) exposures in relation to secondary sex ratio

Polychlorinated biphenyls (PCBs) are a class of chemically stable compounds that were produced in the United States from the 1920's until 1977, and in Europe beginning in the 1940's or 1950's for several decades, and were widely used in industrial and some consumer products. Each individual PCB congener represents one configuration of 209 that are possible, based on the number and location of attached chlorine atoms. These compounds bioaccumulate up the food chain, such that the greatest human sources are meat, fish, and both animal and human breast milk. Because of their high lipophilicity, PCBs are not readily cleared from the body [1–3].

A number of organochlorines, their metabolites, and their contaminants have been characterized as endocrine-disrupting chemicals. PCBs are structurally similar to thyroxine, which is critical to in utero development [2–4]. Animal studies have demonstrated altered thyroid homeostasis following PCB exposure, although human studies have produced mixed results [5]. Additionally, PCB congeners, their metabolites, or related compounds (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and dichloro-diphenyl-trichloroethane (DDT)) have been shown to have estrogenic, anti-estrogenic or anti-androgenic activity [6–9], and some human studies have found reduced sperm motility among PCB-exposed subjects [10–12].

Following the chemical explosion that resulted in dioxin contamination of residential areas in Seveso, Italy, a deficit in male births of couples with high exposures was observed [13, 14]. A similar finding was reported for the offspring of over 2,000 adults exposed in 1979 to cooking oil in Taiwan that was contaminated with a mix of PCBs, polychlorinated dibenzo furans and polychlorinated dibenzo dioxins [15]. Data from a similar accident in Japan were not consistent with an alteration in sex ratio [16], but this report included a relatively small number of births. Few studies at lower environmental exposure levels have reported on sex ratio, and most have not used direct measurements of individual PCBs.

This paper examines sex ratio in births occurring between 1964 and 1967 in the San Francisco Bay Area in relation to concentrations of serum polychlorinated biphenyls in archived serum specimens that were collected during the pregnancies. The participants were a subset of those enrolled in the Child Health and Development Study (CHDS), a cohort of about 20,000 pregnancies. Because the CHDS was conducted during the 1960's, a time of peak production and use of these compounds, body burdens were higher than those found in recent general population studies in western countries [17, 18], yet lower than some current highly-exposed populations [19, 20].

We observed a reduction in the ratio of male:female births in relation to total maternal PCB concentrations in this population. Inverse associations between all nine PCB congeners and secondary sex ratio were also observed, though not all of these were statistically significant at the 0.05 level. In models adjusted for hormonal medications taken in the periconceptional and early pregnancy period, higher concentrations of total PCBs were significantly associated with male birth ratio (p = 0.02). As PCB blood concentrations have been shown to remain very stable during pregnancy [37], these findings suggest that high maternal PCB concentrations, at least for some congeners, may either favor the fertilization by female sperm, or result in greater male embryonic or fetal losses. Sex ratio alterations or decreased survival of wildlife in association with organochlorine exposures have been well documented [38–41], although not all studies have confirmed such findings [42].

Only a handful of human studies of PCBs with sufficient sample sizes have reported data on sex ratio. At environmental exposures, several provide some suggestion of a reduced proportion of males [43–46]. Weisskopf and colleagues [46] examined consumers of sport-fish caught in the Great Lakes and observed a reduction in male births in association with maternal but not paternal serum PCB concentration. In contrast, another investigation found increased male offspring in relation to paternal PCB exposures, though this analysis may have over-adjusted for multiple exposures correlated with paternal PCBs [47]. Reports from the Yucheng [48] and Yusho [16] accidents showed no alteration in sex ratio, however, serious shortcomings in these studies should be noted. The Yucheng group consisted of a select set of mothers who had registered with the health department as having been affected, and who had at least one live child in 1985. Not all affected subjects were registered, and some of the most severely affected children may not have survived: since either of these factors could have been related to the sex of the child, the sex ratio of the survivors could be highly confounded. Moreover, both the Yucheng and the Yusho studies were small: a total of 137 births in the former and only 67 in the latter. These sample sizes would not be adequate to detect small variations in sex ratio. Analysis of a more complete cohort from Yucheng, numbering 469 births with paternal data and 902 with maternal data revealed reduced male:female sex ratio when fathers were exposed at a young age [15], results similar to those previously reported for dioxin exposures from the Seveso accident. Analyses for maternal exposures also showed a lower than expected proportion of males, but the findings were not statistically significant. The difference in findings using maternal vs. paternal exposure appeared to be due, at least partly, to a higher percentage of male offspring among unexposed fathers than among unexposed mothers, or, than expected in the population. Since every child has both a mother and a father, but fewer fathers were tested, this raises the question of selection bias for children whose fathers participated in the study.

In addition to studies of secondary sex ratio, research on the proportion of Y-bearing spermatozoa in relation to paternal PCBs have produced mixed results, i.e., both positive and negative associations [49, 50]. This would suggest that increased paternal PCB concentrations may also alter secondary sex ratio, but other factors may modify this effect. It is not clear from these studies – due to inadequate sample size – whether changes in Y-bearing spermatozoa resulted in more or fewer male births.

The possibility of bias deserves consideration. Selection factors would have to be associated with sex ratio and PCBs, jointly to have produced artifacts for the relationship of these variables of primary interest. Since demographic variables are not generally associated with sex ratio, they would not bias the results. The slight preponderance of girls followed up at age 5 years (50.5%) as compared to the original cohort (48.8% female), combined with our deliberate oversampling of children with low cognitive scores or hearing difficulties increased the percentage of females to 53.4%, however, our sampling weights accounted for these differences, comparable to their standard use in survey sampling. It is possible that because a small number of deaths occurred between birth and five years of age, part of the shift in sex ratio might be due to far higher deaths among boys with higher PCBs, or among girls with lower PCBs. At these exposure levels, however, which were far lower than occurred in the Yusho or Yucheng incidents, it seems unlikely that PCBs would have had a sufficiently strong association with child mortality to produce the results observed and yet not be related to fetal mortality. If PCBs were strongly associated with unknown or gestational age below 35 weeks, some selection bias could have occurred by our exclusion of these births. However, the literature does not support an association of preterm delivery with PCBs, nor did we find any trend of higher PCBs with earlier gestational age among births at weeks 35–45 [23]. In regard to other variables, the sample was representative of the original cohort. Thus, selection bias seems unlikely to be of sufficient magnitude to explain the findings.

Validity of our data is supported by the confirmation of known risk factors for other outcomes, such as maternal smoking, short stature, and low body mass index in relation to intrauterine growth restriction [23]. High quality PCB determinations were ensured by use of within-batch replicates, standards across batches, and surrogate standards. Thus, in comparison with previous work, our study is unique in that it thoroughly addresses maternal exposures, uses an adequate sample, has high quality direct measurements of PCB exposures close in time to the events of interest, and represents exposures at the upper end of today's environmentally exposed populations.

In this study, we observed a lower secondary male:female ratio in association with increased maternal serum PCB concentrations. If not due to an unidentified bias, these results may indicate that with higher exposures to PCBs comes either greater susceptibility of male embryos or a more favorable environment for X-chromatin-bearing sperm. The association could be due to contaminants, PCB metabolites, or the PCBs themselves.

Although PCB concentrations are slowly declining, studies in historical populations such as the CHDS provide an upper limit on the magnitude of toxic effects expected from today's environmental levels of these same compounds in most parts of the world. Secondly, these investigations are useful for assessing reproductive perturbations in populations currently exposed to higher levels, e.g., those that rely on fish from contaminated lakes and seas or reside near former manufacturing facilities. Thirdly, if the findings are actually due to contaminants associated with PCB mixtures rather than PCBs themselves, then attention should be paid to trends in the contaminants, regardless of their source. Finally, other chemical classes with similar structure, such as PBDEs (polybrominated diphenyl ethers), are widely used in plastic casings and foam products and share many of the biochemical and toxicologic properties of PCBs [51, 52]. As PBDE body burdens are rapidly increasing in wildlife and human populations [53, 54], studies like this one provide an indication of potential effects from these newer compounds.