During recent decades there has been an increase in the incidence of several male reproductive disorders and an increased focus on environmental exposures that may explain some of these trends (1). It has also been suggested that cryptorchidism, hypospadias, poor semen quality and testicular germ cell cancer may be associated through a common etiology involving abnormal fetal development of the male gonads (2–4). In modern societies humans are exposed to numerous industrial chemicals, some of which have documented endocrine disrupting abilities in wildlife and animal studies (5). Examples of such endocrine disrupting pollutants with wide-spread use are phenolic substances as well as benzophenones which are characterized as non-persistent or partly non-persistent because of their short half-lives within the body (6). Fetal life is a critical period for normal development of the male reproductive organs and thus exposure of pregnant women to such chemicals is of concern.

Among the phenols is a group of industrial chemicals which primarily are used as precursors for various types of plastics. This includes bisphenol A (BPA), the most commonly used of the phenols, which is mainly used in polycarbonates and epoxy resins used in some food and drink packaging and also in various electronic equipment, and toys (7). Other phenols, like triclosan (TCS), are known for their antibacterial properties and are commonly used in a range of personal care products (8). Humans are ubiquitously exposed to numerous phenols (9, 10). Besides being detectable in human maternal serum, BPA is also detectable in fetal serum and placenta (11). However, BPA levels in umbilical cord blood are half of those in maternal and placental samples, suggesting some protection by the placenta (12). Studies in rodents have shown that pre- or perinatal exposure to BPA is associated with impaired testis function in offspring, i.e., disrupted spermatogenesis and reduced testosterone level (13–15). However, findings in humans from longitudinal pregnancy cohorts are scarce and the majority is characterized by short follow-up. Some, but not all studies have indicated that prenatal exposures to either BPA or TCS are associated with reduced anogenital distance (AGD), a marker of prenatal androgen action in early life (16–18). To our knowledge, only one study has previously investigated the impact of BPA exposure in relation to markers of reproductive function in adulthood but did not see any clear association (19).

Because of the ultraviolet (UV) absorbing properties, benzophenones are used in a wide range of consumer products, including sunscreen formulations and skin lotions, but also as additives in e.g. plastic food packaging materials, paint, and textiles. In recent years, one of the most used UV absorbers, benzophenone-3 (BP-3) has been detected in most children and adults in US and Danish populations (20–23). Besides being present in pregnant women, several benzophenones have been shown to reach cord blood and fetal blood, although concentrations are lower than in maternal circulation (24). In vitro and in vivo studies on different species have indicated that some UV filters, e.g. BP-3 can interfere with reproductive hormones by exerting estrogenic or antiandrogenic effects (25–27). In humans, studies on benzophenones in relation to adult male reproductive outcomes are scarce and only include cross-sectional studies of young men rather than studies of in utero exposures (28, 29).

In general, human studies on prenatal exposure to environmental chemicals and male reproductive health in adulthood are scarce due to the inherent challenges in performing longitudinal studies with sufficient follow-up time while maintaining an adequate study participation rate. Thus, based on a unique mother-child cohort initiated in 1997 with repetitively, and long-term follow-up of the individuals, the aim in the present study was to investigate the association between maternal serum concentrations of phenols and benzophenones in pregnancy and reproductive function in the adult sons.

Of the included chemicals, only BPA, TCS, 2-phenylphenol, BP-3, 4-hydroxy-benzophenone (4-HBP) and 4-methyl-benzophenone (4-MBP) could be detected in more than 30% of the samples ( Table 1 ). BPA showed a significant weak correlation with 2- phenylphenol (r=0.20, p=0.04) whereas BP-3 was moderately correlated to 2-phenylphenol (r=0.33, p<0.01), 4-HBP (r=0.55, p<0.01) and 4-MBP (r=0.34, p<0.01). Finally, 2-phenylphenol and 4-MBP were moderately correlated (r=0.41, p<0.01) whereas no other significant correlations were observed (data not shown).

No difference in chemical concentrations was observed between mothers of men who did not participate in the follow-up examination and men who did except for TCS ( Figure 2 ). Median TCS levels were significantly higher in mothers of men who participated in the follow-up (5.3 ng/mL vs 3.4 ng/mL, respectively, p=0.015).

The mothers’ median age at delivery was 31 years and their sons were 19 years old at follow-up ( Table 2 ). Their median sperm concentration was 52 million/ml and the median total sperm count was 131 million ( Table 3 ).

In fully adjusted analyses, higher prenatal levels of BPA (evaluated in tertiles, T1-T3) were significantly associated with higher LH levels in sons (T2: 12% (-8%,36%) and T3: 33% (10%,62%) compared to the reference category, T1, respectively) ( Figure 3 ; Supplementary Tables 1– 3 ) but not with total testosterone. Hence, higher BPA levels were associated to lower TT/LH ratio (T2: -20% (-35%,-1%), T3: -23% (-38%,-6%). An inverted U-shaped association was seen for BP-3 exposure and total testosterone level (T2: 16% (3%,31%), T3: -1% (-12%,12%)) and to a lesser degree for free testosterone (T2: 13% (-1%,27%) and T3: 6% (-6%,19%) compared to T1) ( Figure 4 ; Supplementary Tables 4–6 ). Also, a positive association between BP-3 exposure and LH levels was seen (T2: 21% (-2%,49%), T3: 18% (-4%,45%) compared to T1). Finally, a tendency of higher INSL3 levels with higher tertile of BP-3 was seen (p-trend = 0.04). For 4-HBP a tendency was seen of higher exposure levels being associated to higher levels of INSL3 (p-trend = 0.09) as well as lower levels of IGF-1 (p-trend = 0.06). For prenatal TCS exposure, no significant associations were found. However, a tendency of higher levels of FSH with higher TCS exposure was seen. No clear associations were seen for 2-phenylphenol in relation to the observed outcomes.

No clear associations were seen between any of the chemicals and the semen parameters and AGD with exception of 2-phenylphenol that tended to be positively associated to higher sperm concentration and total sperm count (p-trend = 0.17 and p-trend = 0.11, respectively) ( Supplementary Tables 7–12 ).

In subanalyses, further adjustment for maternal age or parity did not alter the observed findings. In general, excluding the one azoospermic man from analyses did not change the results. After adjusting for multiplicity, no associations remained statistically significant (data not shown).

In this long-term prospective study, we observed that young men prenatally exposed to the highest levels of BPA and BP-3 had higher LH levels in adulthood and a lower TT/LH ratio indicating a compensated reduced Leydig cell function. However, no associations were seen to AGD as a marker of prenatal androgen action or to markers of semen quality. Testosterone production in men is characterized by a buffer capacity so that testosterone levels to some degree can be sustained by a compensatory increase in LH stimulation. Although our observational study cannot prove causality, our findings indicate a long-lasting effect of prenatal BPA and BP-3 exposure on Leydig cell function which has been described in experimental studies. A compensated Leydig cell function has been associated with an increased all-cause mortality in a long term follow-up study (40). Thus, we speculate that the observed compensated Leydig cell function in this cohort of young men may become clinically relevant over time and reflect a potential health risk.

Several in vitro studies have shown that BPA has both estrogenic and anti-androgenic properties due to its capability to bind and activate the estrogen receptor although with much lower affinity than estradiol (41, 42) as well as the androgen receptor (43). In addition, BPA can inhibit steroidogenesis via a number of pathways in both human and rodent testis in vitro and thus, exert antiandrogenic effects (44, 45). Rodent studies of postnatal effects of in utero exposure to BPA are conflicting and differences in findings are likely explained by variations in doses and duration of exposure as well as differences between species. However, prenatal and perinatal BPA exposure has in the majority of studies been associated to reduced testis weight and testosterone level as well as a reduction in key enzymes and proteins in steroidogenesis in male offspring, indicating a disruption of Leydig cell function [reviewed in (46)].

Anogenital distance (AGD) is the distance from anus to the genitals and has in rodents been shown to be determined by androgen action in early fetal life (47). Thus, reduced AGD may be indicative of insufficient testosterone during the early stages of reproductive development. Furthermore, in adult men a shorter AGD has been associated with impaired semen quality (48). Few human studies have investigated the association between prenatal BPA exposure and AGD with conflicting results. Several studies found no association between maternal BPA levels and AGD levels in newborns (17, 18, 49) in line with our findings, whereas one study observed that higher maternal BPA levels were associated with shorter AGD among 12-month-old boys (18). Notably, in a real-life scenario, exposure to BPA occurs continuously throughout pregnancy and postnatally, which complicates human observational studies compared to controlled exposure settings in rodents and in vitro experiments.

To our knowledge, only one other longitudinal pregnancy cohort has investigated the association between prenatal BPA levels and reproductive hormones in adulthood. In contrast to our findings, results from that cohort indicated that maternal serum levels of BPA were positively associated with the sons’ sperm concentration and motility in adulthood, whereas no association was seen with levels of reproductive hormones, including LH (19). This difference may be explained by differences in BPA exposure levels which was higher in our cohort compared to the levels observed in the Raine cohort (median (max): 1.7 ng/ml (21 ng/ml) vs. 0.25 ng/ml (10.5 ng/ml), respectively). Cross-sectional studies of young adult men have observed a positive association between urinary BPA levels and LH levels in line with our findings (50, 51). However, one of the studies also observed that higher urinary levels of BPA were associated with higher levels of total testosterone and estradiol suggesting that BPA in adult men could act as a partial antagonist to both the estrogen and androgen receptor (50).

In the present study, higher levels of BP-3 were associated with higher LH levels and consequently a lower TT/LH ratio whereas an inverted U-shaped association was seen in relation to testosterone levels. Maternal levels of BPA and BP-3 were not correlated, indicating different exposure patterns and thus, separate effects may be at play. An in vivo study in rodents showed that male offspring dermally exposed to BP-3 at up to 7 weeks of age and with dermally exposed mothers during pregnancy showed a substantial reduction in plasma testosterone levels (52). This may be explained by a direct adverse effect of BP-3 on the Leydig cells or on steroidogenesis in general. Thus, a decrease in testosterone could subsequently be followed by a compensatory increase in LH in line with our findings. More studies in humans are, however, needed to confirm the associations seen for BP-3 in relation to testosterone and LH, respectively.

To our knowledge, no longitudinal studies in humans have investigated the association between prenatal BP-3 exposure and reproductive hormones in adulthood, however, few cross-sectional studies have been performed with conflicting results. In a cohort of 215 young Spanish men, higher urinary BP-3 concentrations were associated with higher FSH levels, whereas no associations were seen with LH or other reproductive hormones (28). However, in a study of 195 young Danish men, higher urinary BP-3 levels were associated with higher total testosterone and estradiol levels and lower FSH levels but only in men with a loss-of-function mutation in the filaggrin gene, important for normal skin barrier, and thereby probably generally more exposed dermally (29).The levels of BP-3 were in general higher in the Danish men compared to the Spanish men (median levels: 2.8 ng/ml vs. 1.3 ng/ml), and it is possible, that different levels of co-exposures not accounted for in the studies also could contribute to the observed differences in findings.

In contrast to cross-sectional studies, a key advantage of the present study is the use of a prospective birth cohort with information on chemical exposures during a critical period where reproductive development takes place (3) and with detailed follow-up information including a comprehensive andrological examination. Some limitations should, however, be considered when interpreting the results. Multiple factors are known or suspected to affect reproductive outcomes in the young men. This includes adult exposure to phenols and benzophenones as well as exposures from additional EDCs [reviewed in (53)] which was not taken into consideration in this study. We also did not explore simultaneous fetal exposure to other chemicals in this study, although animal studies have shown that mixed exposures to different chemical groups during fetal life may have additive effects on reproductive outcomes (54). Although we adjusted analyses of the association between exposure levels and adult reproductive function for several confounders, we could not control for variations in this over time. Furthermore, we cannot exclude, that our findings are influenced by residual confounding not accounted for in the present study.

Another limitation is the small study size, which limits the statistical power. Furthermore, no associations were significant after adjustment for multiplicity. Thus, we cannot rule out, that our findings are false positive (type I errors) and the data should be replicated in larger studies. This project applied a single prenatal exposure measurement and a single adult reproductive health assessment despite the well-known intra-individual variation in both (55), which increases the risk of exposure and outcome misclassification. However, these would be expected to be random and most likely attenuate any true association. We speculate that the endogenous negative feedback mechanism between LH and testosterone may have been the most sensitive parameter for finding subtle long-term effects of prenatal exposures. In contrast, semen quality shows a high intra-individual variation which could attenuate potential associations (56). Thus, our findings need to be replicated in larger populations. Furthermore, we cannot rule out that genetic factors could play a role for the observed associations which was however, not investigated in the present study. Another limitation is that exposure levels were analyzed in serum samples as maternal urine samples were not available. Urine is considered the preferred matrix to determine exposures of non-persistent compounds due to their short half-lives and subsequent rapid metabolization and excretion into urine (57). Also for bisphenols, urine is the preferred matrix to determine the concentrations (58), although urine alone also has been acknowledged as unsuitable to assess the full toxicokinetics of bisphenols (59). Moderate correlations have furthermore been observed between some benzophenones determined in serum compared to urine (23). Since serum concentrations are lower compared to urine concentrations, a higher proportion of the samples will be below detection level with the risk of underestimating the exposure in the lower range.

In conclusion, our findings indicate that young men prenatally exposed to BP-3 or BPA were characterized by a compensated reduced Leydig cell function in adulthood whereas no association was seen in relation to AGD or semen quality. Larger studies on long-term reproductive consequences of prenatal exposures are warranted to validate our findings.

The datasets presented in this article are not readily available because the data are not publicly available due to ethical restrictions. Requests to access the datasets should be directed to Katharina M. Main, Katharina.Main@regionh.dk.

The studies involving human participants were reviewed and approved by the Regional Committee on Health Research Ethics ((KF) 01-030/97 and H-17011468) and the Danish Data Protection Agency (1997-1200-074 and VD-2018-118/i-Suite 6358). The patients/participants provided their written informed consent to participate in this study.

Conceptualization: SH, A-MA, AJ, JT, NS, KM. Data collection: LS, HF, KM. Data analysis and interpretation: SH, LS, A-MA, LP, NJ, NS, KM. All authors had full access to the data in the study and take responsibility for the integrity and the accuracy of the data analysis. All authors critically reviewed the manuscript and approved the submitted version. All authors contributed to the article and approved the submitted version.