Abstract
Exposure to perfluoroalkyl substances (PFASs) has in some studies been associated with reduced anogenital distance (AGD) in newborns as a sensitive indicator of prenatal anti-androgenic exposure. The aim of this study was to investigate the association between maternal PFAS exposure and offspring AGD in a population with wide ranges of PFAS exposures.
Participants were recruited in the Faroe Islands in 2007-2009, and information on AGD and PFAS exposure was obtained from 463 mother-infant pairs. Perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) were measured in maternal pregnancy serum. Data were analyzed using multiple linear regression analysis adjusted for birth weight, child age at examination, parity, and maternal education level.
Among boys, higher maternal serum concentrations of PFOA, PFOS, PFNA and PFDA were significantly associated with a longer AGD, both with the exposure entered as a continuous variable and as quartiles. Boys in the highest quartile of PFOA, PFOS, PFNA and PFDA exposure had an increase in AGD of 1.2 mm (95% CI 0.1;2.2), 1.3 mm (95% CI 0.3;2.3), 1.0 mm (95% CI 0.0:2.0) and 1.3 mm (95% CI 0.3;2.4), respectively, when compared to boys in the lowest quartile of exposure (p <0.05). No significant association was found between male AGD and PFHxS. No association was found for girls.
In conclusion, elevated maternal exposure to major PFASs was significantly associated with a longer AGD in boys. No significant associations were found among girls, thus suggesting a sex-dimorphic effect of PFAS exposure.
Keywords: Perfluorinated compounds, PFAS, Prenatal exposure, Anogenital Distance, Faroe Islands
1. Introduction
Perfluoroalkyl substances (PFASs) are a group of highly persistent synthetically manufactured chemicals used in fabrics and food packaging due to water-, stain-, and grease-resistant properties [1]. Human exposure to PFASs occurs through ingestion of contaminated food and drinking water, inhalation of indoor air and contact with other contaminated media [1–3]. Two of the previously most commonly used PFASs, perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been phased out by some of the major manufacturers [2]. Nonetheless, other PFAS, such as perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA), are still in production, and these PFASs have been studied to a lesser extent. PFASs cross the placental barrier and have been detected in human amniotic fluid and umbilical cord blood [3].
Among other important adverse effects, PFASs have potential endocrine-disrupting effects through affecting androgen receptor and estrogen receptor (ER) activity or altering the expression of estrogen-responsive genes [4, 5] leading to hormonal imbalance, and may thus play a central role in declining male reproductive health [6]. PFAS exposure has been associated with fetal growth, and a recent review found that most studies report association between maternal exposure to PFOA and PFOS and low birth weight [7, 8] but the mechanisms behind the influence of PFASs on fetal growth are largely unknown. Animal studies have reported that exposure to PFASs could impair the reproductive system during the developmental stage in male mice through an antiandrogen pathway[9, 10] and influence the expression of estrogen-responsive genes in animal studies[11], and PFAS-induced changes in sex hormone biosynthesis have been reported in vitro[12].
Anogenital distance (AGD), the distance from the anus to the genitalia, is routinely used in animal toxicology studies and is sensitive to anti-androgenic exposure. In rodents, AGD is a sensitive biomarker of androgen exposure during a critical embryonic window of testis development[13] and has been shown to reflect the amount of androgen to which a male fetus is exposed in early development where higher in utero androgen exposure resulted in a longer AGD [6, 14]. Two animal studies have found that exposure to PFASs was associated with shorter AGD in male rat fetuses and wild male minks[15, 16]. The suggested mechanisms by which PFASs affects AGD is by increasing the ratio between estrogens and androgens through transcriptional induction of the aromatase enzyme while also being a direct agonist to the estrogen receptor (ER) [17–19]. Some of these findings have been reproduced in humans, e.g., in regard to prenatal exposure to phthalates, which are anti-androgenic, and which have been found to reduce AGD in boys [20]. Still, the potential effect on AGD is still poorly understood.
Three birth cohort studies have examined the association between maternal PFAS exposure and AGD in the offspring, and the results have been somewhat unclear. A Danish study found higher PFOS, PFHxS, PFNA and PFDA concentrations to be associated with a shorter AGD in 3 month old girls, but a longer AGD in boys, suggesting a sex-dimorphic effect [21]. A Chinese study found that high maternal PFOS and PFDA exposure was associated with a shorter AGD in boys aged 1-3 days [22]. Lastly, a Canadian study found an association between high PFOA and longer AGD in boys but not in girls [23]. Thus, the limited evidence available shows somewhat unclear results and more studies are needed. We hypothesize that maternal exposure to PFASs has impact on the development of the reproductive tract and AGD in the offspring. Thus, the aim of this study was to investigate the association between maternal PFAS exposure and AGD in infants in a Faroese birth cohort, where ranges of PFAS exposure are wide.
2. Methods
2.1. Study population and data collection
The study is based on data from the Faroese Birth Cohort 5, a population-based prospective cohort of 490 children, recruited between October 2007 and April 2009 with a participation rate of 73%. Obstetric information was obtained from midwife records and included maternal age, parity, pre-pregnancy weight, height, education level, alcohol and smoking during pregnancy, partner smoking during pregnancy, and birth weight.
Two weeks after expected term date, a pediatric examination was performed during which AGD were measured. The AGD was measured with Vernier calipers according to a standardized method described by Salazar-Martinez [27]. The infants were laid on their back with hips flexed and light pressure was placed on the thighs until the hand of the examiner touched the subject’s abdomen.. In boys, AGD was determined as the distance from the center of the anus to the bottom of the scrotum (AGDAS) and, in girls, as the distance from the center of the anus to the posterior convergence of the fourchette (AGDAF). Measurement was repeated three times and a mean was calculated [27, 28]. All measurements were performed by the same trained pediatrician. During the examination, i.e., two weeks after expected term date, a sample of maternal blood was obtained for PFAS exposure assessment.
All protocols were approved by the Scientific Ethical Committee of the Faroe Islands and the institutional review board at Harvard T.H. Chan School of Public Health. Written informed consent was obtained from all mothers.
Exposure assessment
Maternal serum concentrations of PFASs (PFOA, PFOS, PFHxS, PFNA and PFDA) were measured by online solid-phase extraction followed by high-pressure liquid chromatography with tandem mass spectrometry [29, 30]. To ensure accuracy and reliability of data, each analytical series included quality control serum samples, calibration standards, as well as reagent and serum blanks. Imprecision within and between batches was less than 3.0 % and 5.2 %, respectively [31].
2.3. Statistics
Data were presented as counts and percentages for categorical variables, mean and standard deviation (SD) or medians and interquartile range (IQR) for continuous variables. Pearson’s correlation coefficient was used to evaluate correlation between the five measured PFASs. Data was assessed for normality by visual assessment using histograms and normal probability plots. One-way ANOVA-test was used for normally distributed covariates to test for differences in AGD in relation to the following covariates one at a time: maternal age, parity, maternal BMI, gestational age, birth weight, maternal education level, maternal alcohol use during pregnancy, maternal smoking during pregnancy, partner smoking during pregnancy, weight and age at examination adjusted to term-date. Differences in PFAS concentrations between the same covariates were tested with Kruskal-Wallis’ test for skewed distributions. PFAS distributions were skewed and divided into quartiles, while also entered as a continuous variable after logarithmic transformation to approach normality.
Stepwise multiple linear regression was used to examine the associations between PFASs and AGD. Confounders included were factors known a priori to be important predictors of birth outcomes or AGD [28, 29]. Weight at examination and infant’s age at examination were included in all analyses. The remaining covariates were gestational age (days), parity (0, 1, 2 and 3+), maternal smoking during pregnancy (yes/no), partner smoking during pregnancy (yes/no), maternal alcohol use during pregnancy (yes/no), maternal pre-pregnancy BMI (<20, 20–25, 25+ kg/m2), maternal education level (below high school/high school or above). The only covariates that changed the beta estimate by at least 10 % were parity and maternal education level, and both were therefore included in the final regression model. Associations were reported in terms of beta estimates with 95% confidence intervals (95% CI), and statistical significance was reached at a p-value of <0.05. All data analyses were performed using STATA/IC 16®.
3. Results
Of the cohort 490 mother-child pairs, a total of 463 pairs (94.4 % of the original cohort) had information on both PFAS exposure and AGD available and were thus included in the analyses; i.e., 27 mother-child pairs were excluded because of missing data. Mean maternal age at examination was 29.8 years and mean maternal pre-pregnancy BMI was 24.3kg/m2. Most women were multiparous (69 %) and of Scandinavian origin (97 %). A total of 73 women (16 %) reported smoking and 22 (5 %) reported alcohol consumption during pregnancy. The means of birth weight and AGD were 3787 g and 25.1 mm for boys and 3612 g and 13.4 mm for girls, respectively. For both sexes, birth weight, weight at examination, and age at examination were significantly associated with the AGD while maternal age, gestational age at birth, and maternal education level were significantly associated with AGD only among the girls (Table 1).
Table 1:
Maternal characteristics (n=463) according to mean AGD (standard deviation) stratified by sex (n=232 males; n=231 females
| ) | |||||
|---|---|---|---|---|---|
|
| |||||
| N (%) | Males n=232 |
Females n=231 |
|||
|
| |||||
| % | AGDAS mean (SD) |
% | AGDAF mean (SD) |
||
| All children | 463 | 25.1(3.1) | 13.4(2.1) | ||
|
| |||||
| Maternal age (years) | |||||
| <25 | 84 (18) | 15 | 24.9 (2.8) | 21 | 12.7 (1.8)* |
| 25-29.9 | 135 (29) | 30 | 25.5 (2.8) | 28 | 13.4 (1.8) |
| 30-35 | 175 (38) | 38 | 25.0 (3.1) | 37 | 13.2 (2.4) |
| >35 | 69 (15) | 16 | 25.1 (3.5) | 13 | 13.2 (1.8) |
|
| |||||
| Parity | |||||
| 0 | 141 (31) | 25 | 24.9 (2.9) | 36 | 13.2 (1.8) |
| 1 | 160 (34) | 37 | 25.0 (3.1) | 33 | 13.7 (2.6) |
| 2 | 102 (22) | 26 | 25.5 (2.7) | 18 | 13.0 (1.7) |
| 3+ | 59 (13) | 12 | 25.6 (3.7) | 13 | 13.6 (1.7) |
|
| |||||
| Maternal pre-pregnancy BMI | |||||
| <20 | 56 (12) | 12 | 24.7 (2.8) | 12 | 13.8 (2.1) |
| 20-25 | 248 (54) | 52 | 24.9 (3.2) | 55 | 13.3 (1.8) |
| >25 | 159 (34) | 36 | 25.7 (2.9) | 33 | 13.5(2.5) |
|
| |||||
| Gestational age at birth (weeks) | |||||
| <38 | 31 (7) | 7 | 25.4 (4.5) | 7 | 13.7 (1.3)* |
| 38-40 | 302 (65) | 58 | 25.2 (2.9) | 72 | 13.2 (1.9) |
| >40 | 130 (28) | 35 | 24.9 (3.0) | 21 | 14.1 (2.6) |
|
| |||||
| Birth weight (g) | |||||
| <2500 | 5 (1) | 1 | 21.0 (.)* | 2 | 14.3 (1.0)* |
| 2500-4500 | 430(93) | 92 | 25.1 (3.0) | 93 | 13.3 (1.8) |
| >4500 | 28 (6) | 7 | 26.7 (2.4) | 5 | 15.0 (4.7) |
|
| |||||
| Weight at examination (g) | |||||
| <3500 | 60 (13) | 11 | 23.2 (2.9)* | 15 | 12.4 (1.4)* |
| 3500-4500 | 300 (65) | 60 | 24.7 (2.7) | 69 | 13.5 (1.8) |
| >4500 | 103 (22) | 29 | 26.7 (3.0) | 16 | 14.1 (3.1) |
|
| |||||
| Age at examination (days) | |||||
| <14 | 100 (22) | 20 | 24.3 (2.3)* | 23 | 12.9 (1.5) |
| 14-23 | 255 (55) | 55 | 25.2 (3.2) | 55 | 13.4 (2.3) |
| >23 | 108 (23) | 25 | 25.8 (3.0) | 22 | 13.7 (1.9) |
|
| |||||
| Maternal education level | |||||
| Below high school | 70 (16) | 14 | 24.7 (2.6) | 18 | 13.2 (2.1) |
| High school or above | 378 (84) | 86 | 25.2 (3.1) | 82 | 13.4 (2.1) |
|
| |||||
| Maternal alcohol use during pregnancy | |||||
| Yes | 22 (5) | 5 | 25.7 (3.4) | 4 | 12.8 (1.3) |
| No | 441 (95) | 95 | 25.1 (3.0) | 96 | 13.4 (2.1) |
|
| |||||
| Maternal smoking during pregnancy | |||||
| Yes | 73 (16) | 15 | 25.2 (2.8) | 17 | 13.4 (2.2) |
| No | 389 (84) | 85 | 25.2 (3.1) | 83 | 13.4 (2.1) |
|
| |||||
| Partner smoking during pregnancy | |||||
| Yes | 152 (34) | 33 | 25.0 (3.4) | 34 | 13.3 (2.0) |
| No | 299 (66) | 67 | 25.3 (2.8) | 66 | 13.5 (2.2) |
|
| |||||
Note: : AGDAF, anofourchette distance; AGDAS, anoscrotal distance; ; SD, standard deviation
Missing values: parity: 1, maternal pre-pregnancy BMI: 1, maternal education level: 15, smoking during pregnancy: 1, partner smoking during pregnancy: 12
, marks a p-value <0.05 with one-way ANOVA test
All measured PFASs were found in quantifiable concentrations in serum from the 463 women (Supplementary table). PFOS had the highest average concentrations (8.3 μg/L), followed by PFOA (1.4 μg/L), PFNA (0.7 μg/L), PFDA (0.3 μg/L) and PFHxS (0.2 μg/L) and they were all mutually correlated (Pearson’s correlation coefficient r between 0.40 and 0.85, p<0.001). Older women had significantly lower concentrations of all five PFASs while women with higher parity had significantly lower concentrations of PFOA, PFOS and PFHxS. Women with elevated PFOA exposure were more often smokers and gave birth to children of lower birth weight and a lower weight at examination (Supplementary table). Significant association was observed between exposure to all PFASs and birth weight (data not shown).
In multiple linear regressions, after adjusting for weight and age at examination (adjusted to term date), parity and maternal education, higher maternal exposure to PFOA, PFOS, PFNA and PFDA was significantly associated with a longer AGDAS in boys, both with PFASs entered as continuous variable and as quartiles (Table 2). Boys in the highest quartile of exposure to PFOA, PFOS, PFNA and PFDA had an AGDAS increase of 1.2 mm (95 % CI 0.1;2.2), 1.3 mm (95% CI 0.3;2.3), 1.0 (0.0:2.0) and 1.3 mm (95% CI 0.3;2.4), respectively. No association was seen for PFHxS. No associations were found between maternal PFAS exposure and AGDAF in girls (Table 2).
Table 2:
Linear regression analysis (β-coefficient and 95 % confidence intervals) on maternal pregnancy PFASs in quartiles, continuous (transformed by the use of natural logarithm) and AGDAS and AGDAF.
| PFASs (ng/mL) | AGDAS (mm) 232 males |
AGDAF (mm) 231 females |
||
|---|---|---|---|---|
| β | 95 % CI | β | 95 % CI | |
| PFOA | ||||
| 1st | Reference | Reference | ||
| 2nd | 0.2 | −0.8;1.1 | 0.8 | −0.1;1.6 |
| 3rd | 1.2* | 0.1;2.2 | 0.3 | −0.6;1.2 |
| 4th | 1.1 | 0.0;2.3 | 0.2 | −0.7;1.1 |
| p-trend | 0.02 | 0.85 | ||
| Continuous ** | 0.8* | 0.1;1.5 | 0.0 | −0.6;0.6 |
| PFOS | ||||
| 1st | Reference | Reference | ||
| 2nd | 1.3* | 0.3;2.4 | 0.1 | −0.6;0.9 |
| 3rd | 1.3* | 0.2;2.3 | 0.4 | −0.5;1.1 |
| 4th | 1.3* | 0.3;2.3 | 0.0 | 0.8;0.9 |
| p-trend | 0.02 | 0.81 | ||
| Continuous** | 1.0* | 0.1;1.8 | 0.1 | −0.6;0.7 |
| PFHxS | ||||
| 1st | Reference | Reference | ||
| 2nd | −0.6 | −1.6;0.4 | 0.3 | −1.1;0.5 |
| 3rd | 0.1 | −0.9;1.2 | 0.00 | −0.8:0.8 |
| 4th | 0.5 | −0.6;1.6 | −0.5 | −1.3;0.3 |
| p-trend | 0.18 | 0.39 | ||
| Continuous** | 0.2 | −0.3;0.7 | −0.1 | −0.4;0.3 |
| PFNA | ||||
| 1st | Reference | Reference | ||
| 2nd | 0.2 | −0.8;1.3 | 0.2 | −0.7;1.0 |
| 3rd | 1.0* | 0.0;2.0 | 0.1 | −0.8;0.9 |
| 4th | 0.9 | −0.2;1.9 | −0.3 | −1.1;0.4 |
| p-trend | 0.03 | 0.3 | ||
| Continuous** | 1,1* | 0.1;2.0 | −0.1 | −0.8;0.5 |
| PFDA | ||||
| 1st | Reference | Reference | ||
| 2nd | 1.4* | 0.4;2.5 | 0.3 | −0.5;1.1 |
| 3rd | 1.0* | 0.0;2.1 | −0.1 | −0.9;0.7 |
| 4th | 1.3* | 0.3;2.4 | −0.2 | −1.0;0.6 |
| p-trend | 0.03 | 0.46 | ||
| Continuous** | 0.8* | 0.0;1,6 | −0.3 | −0.9;0.4 |
Note: Linear regression models are adjusted for parity, child age and weight at examination and mothers’ level of education. AGDAF, anofourchette distance; AGDAS, anoscrotal distance;;distance; PFASs, perfluoroalkyl substances; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonic acid; PFHxS, perflurohexane sulfonic acid; PFNA, perfluorononanoic acid; PFDA, perflurodecanoic acid. Missing values: parity: 1; maternal education level: 15
, marks a p-value <0.05
, logarithmically transformed
4. Discussion
In this birth cohort study, maternal PFAS exposure was significantly associated with a longer AGDAS in boys aged 14 days after term, whereas no association was found in girls.
To date, only three epidemiological studies have investigated the association between prenatal PFAS exposure and AGD, although showing somewhat uneven results (Table 3). Our findings of a longer AGD in boys exposed to PFAS is in agreement with a study from the Canadian that investigated the association between AGDAS, (anoscrotal distance), AGDAP, (anopenile distance), AGDAC, (anoclitoral distance), and AGDAF, (anofourchette distance), and PFOA, PFOS, and PFHxS in 403 children [23]. They found that elevated PFOA was significantly associated to a longer AGDAS, as was PFOS, albeit not significantly so. No association was observed with AGDAS or among girls and PFASs. The Canadian study population resembled ours regarding maternal age and parity but had a higher level of education and with children at a lower average birth weight (Table 3). The PFOA concentration was similar, but the Canadian study measured PFAS in first trimester as opposed to a median of 19 days post-partum in our cohort. Interestingly, a Danish study (n=547) from the Odense Child Cohort (OCC) [21] also found increased AGDAS in boys exposed to PFOS, PFNA and PFDA. Further, they reported a significant association between higher PFOS, PFHxS, PFNA and PFDA exposure and decreased AGDAC in girls (Table 3). The Faroese and Danish studies are comparable regarding maternal characteristics and serum PFAS concentrations although the Danish study measured PFAS in first trimester. The third study from the Shanghai-Minhang Birth Cohort, included 439 boys [22] and found significant associations between higher exposure to PFOS and PFDA and shorter AGDAS, though not AGDAP 1-3 days post-partum, i.e., findings inconsistent with our results. However, the PFAS exposure in the Chinese boys was much higher compared with our study (Table 3), and 84 % of the Chinese mothers were nulliparous which may have affected the differences in results. Besides the three studies above, to our knowledge, only two animal studies have investigated the association between PFAS exposure and AGD and with consistent associations. A reduction AGD in male rat fetuses after exposure to PFOS was observed[15] and PFOS, PFDA, PFUA, and total PFASs were related to shorter AGD in wild male minks[16]. Thus, our results are inconsistent with the findings in animals.
Table 3:
A comparison of studies on PFAS exposure and AGD in infants
| Study population | № of participants | PFAS concentrations in ng/mL | AGD measurements | Age at AGD-measurements | Confounders adjusted for | Results |
|||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| PFOA | PFOS | PFHxS | PFNA | PFDA | |||||||
| Odense Child Cohort | 316 boys 231 girls |
Gestational age of 5-12 weeks: |
AGDAS AGDAP AGDAF AGDAC |
3.5 months (range 2.1–6.8 months) | Age at examination, weight for age Z-score, pre-pregnancy BMI, parity, smoking | NA | AGDAC↓* AGDAS↑ʎ |
AGDAC↓*Ω AGDAS↓* |
AGDAC↓*Ω AGDAS↑* |
AGDAC↓ʎΩ AGDAS↑ʎΩ |
|
| PFOA | 1.7 | ||||||||||
| PFOS | 8.1 | ||||||||||
| PFHxS | 0.3 | ||||||||||
| PFNA | 0.7 | ||||||||||
| PFDA | 0.3 | ||||||||||
|
| |||||||||||
| Shanghai-Minhang Birth Cohort Study 1 | 439 boys 0 girls |
At gestational age of 12-16 weeks: |
AGDAS AGDAP |
1-3 days and 6 months | Age at examination, birth weight, parity, maternal education level, maternal age at delivery, gestational age, pre-pregnancy BMI | NA | AGDAS1-3d↓* AGDAS6mo↓* |
NA | NA | AGDAS1-3d↓* AGDAP1-3d↓* |
|
| PFOA | 20.1 | ||||||||||
| PFOS | 10.7 | ||||||||||
| PFHxS | 2.8 | ||||||||||
| PFNA | 1.8 | ||||||||||
| PFDA | 2.1 | ||||||||||
|
| |||||||||||
| Maternal-Infant Research on Environmental Chemicals Cohort | 198 boys 205 girls |
At gestational age of <15 week: |
AGDAS AGDAP AGDAF AGDAC |
Mean 3.41 days | Varied, but could include recruitment site, education, gestational age, weight-for-length Z-score, active smoking status and household income | AGDAS↑* | NA | NA | |||
| PFOA | 1.7 | ||||||||||
| PFOS | 4.5 | ||||||||||
| PFHxS | 1.1 | ||||||||||
|
| |||||||||||
| Faroese birth cohort, Cohort 5 | 232 girls 231 boys |
14 days after expected birth§: |
AGDAS AGDAF |
Mean 19.3 days | Age at examination, birth weight, parity, maternal education level | AGDAS↑*ʎΩ | AGDAS↑*ʎΩ | AGDAS↑ʎ | AGDAS↑*ʎΩ | AGDAS↑*ʎΩ | |
| PFOA | 1.4 | ||||||||||
| PFOS | 8.3 | ||||||||||
| PFHxS | 0.2 | ||||||||||
| PFNA | 0.7 | ||||||||||
| PFDA | 0.3 | ||||||||||
Note: NA, no association; AGDAF, anofourchette distance; AGDAS, anoscrotal distance AGDAP, anopenile distance; AGDAC, anoclitoral distance; PFASs, perfluoroalkyl substances; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonic acid; PFHxS, perflurohexane sulfonic acid; PFNA, perfluorononanoic acid; PFDA, perflurodecanoic acid.
: Did not investigate separate quartiles
: p <0.05 as continuous variable
: p <0.05 in higher quartiles (q3 and/or q4)
: p-trend <0.05
Mean 19.3 days after expected date of birth
In addition to AGD, a number of studies, both human and animal, have investigated the effects of PFAAs on reproductivity and development[32]. In humans increases in exposure to PFOA and PFOS has been associated with lower birth weight[7, 8], a finding supported by animal studies where increased PFOA, PFOS, PFNA, and PFDA is associated with lower birthweight[1]. This is in accordance with our study as we see that higher levels of all measured PFAS were significantly associated with lower birthweight.
The four epidemiological studies strongly indicate that PFASs exert a sex-dimorphic effect, likely through endocrine disruption. This disruption of PFASs has also been studied in vitro and in animals. Affected physiological pathways have been studied in vitro [11, 33–35]. One pathway is through PFOA and PFOS decreasing testosterone levels by inducing aromatase activity through transcriptional activation of CYP19, the aromatase enzyme and also induction of CYP11B2, coding for aldosterone synthase, which may disrupt several of the functions related to aldosterone. Further, studies have found some PFASs to bind directly to the ER with low affinity relative to endogenous estrogen [11, 33] and thereby exerting an estrogenic effect and PFOA and PFOS are found to enhance the effect of estradiol on estrogen-responsive genes [34]. In one animal study, blockage of androgen stimulation within a sensitive programming window in fetal development resulted in de-masculinization of male genitalia including a shorter AGD, increased risk of both hypospadias and cryptorchidism [14]. Generally, PFASs are suspected to cause endocrine disruption through an increase in the fetal estrogen/androgen ratio. Following such disruption, one would expect a shorter AGD in male offspring. . The suggested pathways which disrupt endocrine homeostasis found in vitro and animal studies, support our findings of a sex-dimorphic effect of PFAS.
In humans, changes in the AGD relate to the symptom complex of diseases in male reproductive system referred to as the testicular dysgenesis syndrome (TDS) [6], a syndrome causing decreased fertility through a complex of symptoms such as decreased sperm quality, and an increased risk of cryptorchidism and hypospadias [24, 36, 37]. The incidence of TDS is increasing and is hypothesized to be linked to environmental pollutants, such as PFASs, that disrupt normal endocrine signaling, thereby causing abnormal androgen action which alters the in utero development of the reproductive organs [38, 39]. Shorter AGD has been associated with, e.g., poorer semen quality [40, 41], genital malformations in men [42, 43], and an increased risk of testicular germ cell tumor development [44]. Besides, longer AGD has been suggested to be associated with higher sperm concentration, total sperm count, and total motile sperm count in adult men and with fatherhood and may predict normal male reproductive potential [36, 45, 46]. In females, the long-term implications of AGD on reproductive health have only been studied sporadically. In our study and the Canadian study [44], increased PFAS exposure was associated with longer AGD, which may seem to contradict the TDS hypothesis, but cannot be considered as harmless, as the long-term implications of longer AGDs are currently unknown.
Our study has several strengths. First, it is fairly large and comprised of a population-based birth cohort with a high participation rate [24–26], and thus the cohort is likely to be representative of the general population, thereby limiting selection bias. Further, the birth outcomes were objectively reported and AGD was measured three times by the same trained pediatrician to eliminate inter-examiner variance and reduce imprecision. However, some weaknesses need consideration. PFASs were measured approximately two weeks post-partum, i.e., several months after the hypothesized masculinization programming window occurring between 8- and 14 weeks of gestation [14]. Exposure misclassification may have occurred, as maternal serum concentrations of PFOS, PFOA, and PFNA average higher in the first trimester, as compared to the second and third trimester [47, 48]. However, due to the long half-life of PFASs, one can assume that PFAS concentrations, measured postpartum after the initiation of breastfeeding, were likely comparable or even lower than PFAS concentrations during the relevant stage of development [37]. The Faroese women were exposed to lower PFAS concentrations than the Chinese women, whereas the exposure levels were relatively similar to Danish and Canadian pregnant women (table 3). Additionally, unknown confounders associated with PFAS exposure and intra-uterine growth may exist. e.g. lifestyle, and exposure to other chemicals, including PCBs and methylmercury, that the Faroese population are highly exposed to [49]. Lastly, the number of endocrine disruption chemicals (EDCs) that the mothers have been exposed to is not limited to the five PFASs measured, and serum-PFAS concentrations could conceivably function as a surrogate for other EDCs that may be highly correlated. Of note, studies exist that associate other EDCs with shorter AGD, e.g. phthalates [6], which is opposite to the findings of the present study.
5. Conclusions
In conclusion, we found that maternal PFAS exposure was significantly associated with a longer AGDs in boys. No significant associations were found among girls, suggesting sex-dimorphic effects of PFASs. Whether the observed association with longer AGD and PFAS influences the reproductive health of males is unknown and needs to be investigated further.
Supplementary Material
Highlights:
Exposure to PFASs have a sex-dimorphic effect on anogenital distance in infants
Prenatal exposure PFASs results in a longer anogenital distance in infant boys
PFASs may have endocrine disruptive abilities
Acknowledgements
We thank the participating families for their willingness to participate in the studies. The efforts of pediatrician Oddmar Færø, nurse Nanna Kallsberg and midwife Annika Hoydal are highly appreciated.
Funding sources
The study was financially supported by the U.S. National Institute of Environmental Health Sciences (ES09797 and ES012199)
Footnotes
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Conflict of interest
The authors declare that they have no competing financial interests.
Declaration of interests
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests
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