Association of novel and legacy PFAS with reproductive hormones in women of child-bearing age

LIN Cai-yun, XIE Mei-hua, YIN Fei-fei, ZENG Liang-ying, ZHANG Jun, YANG Hua

1.Hainan Women and Children’s Medical Center, Haikou 570206, China

2.Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Medical University,Haikou 571199,China

3.Hainan Medical University-The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, Hainan Medical University,Haikou 571199,China

4.MOE-Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China

Keywords:

ABSTRACT Objective: To evaluate the legacy and novel per- and polyfluoroalkyl substances (PFAS)exposure in women of reproductive age, and to explore the effects on female reproductive hormones.Methods: We used cross-sectional data of 433 normal women of reproductive age from 2013 to 2015.Data of age, age at menarche, parity, BMI, education, and income were collected through questionnaires, serum samples were collected for reproductive hormones, and plasma samples for PFAS measurement by ultraperformance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS).Multiple linear regression and quantile g-computation were adopted to analyze the association between PFAS and reproductive hormones.Results:The detection rates of 6:2 Cl-PFESA, 8:2 Cl-PFESA and HFPO-DA in the plasma were 100%, 99.8% and 99.9%, respectively.The median concentrations of these chemicals were 2.27 ng/mL, 0.07 ng/mL and 0.03 ng/mL, respectively.Multiple linear regression analysis showed a positive association between branched PFOS [1m-PFOS (β=0.131, 95% CI:0.021, 0.242),br-PFOS (β=0.119, 95% CI: 0.005, 0.234)] and progesterone.In addition,mixed exposure model suggested that PFAS had a positive association with progesterone(β=0.549,95% CI: 0.323, 0.774).Conclusion: In this study, a mixed exposure model was used to evaluate the combined effects of PFAS mixtures, reflecting the association between multiple environmental PFAS exposure and reproductive hormones, and a higher concentration of novel PFAS was found in women of reproductive age, confirming that PFAS exposure may be related to reproductive hormone disorders in women of reproductive age.

1.Introduction

At present, the total fertility rate of Chinese women of childbearing age is showing a trend of continuous decline.The decline of birth rate is a key issue facing Chinese social development.To determine new intervention factors affecting reproductive health of people of childbearing age may reduce the downward risk of fertility rate to a large extent, which is of great social development and public health significance.Per- and polyfluoroalkyl substances (PFAS) are a class of organic compounds mainly composed of fluorocarbons and are widely used in textile, surfactants, food packaging, non-stick coating, fire extinguishing foam and other fields.Can persist in the environment, almost not biodegradable[1].PFAS exposure has been associated with a variety of adverse outcomes including reproductive and developmental toxicity[2].Animal and cellular experiments have shown that PFAS, as potential endocrine disruptors, may affect the function of reproductive hormones.The PFAS family includes: (1) perfluoroalkyl substances, mainly perfluoroalkyl acids;(2) polyfluoroalkyl substances such as fluoromeric monomers[3].Typical compounds of conventional PFAS include perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid, PFOA), which has been banned and restricted by the Stockholm Convention for its environmental persistence, bioaccumulation and multiple potential toxicity, leading to the development and application of alternatives such as perfluoropolyethers (PFPEs)[4].In recent years, a variety of PFPEs substitutes have been frequently detected in human body and drinking water, causing the environmental scientific community to pay attention to their safety[3,5-6].However, there are few studies on whether PFAS have a significant effect on the level of reproductive hormones in women preparing for pregnancy, thus affecting female fertility.Therefore, this paper discusses the correlation between the novel PFAs and reproductive hormones in women preparing for pregnancy.

2.Methods

2.1.Study population

This study adopted a cross-sectional survey and collected pregnant women of childbearing age from two hospitals in Shandong and Zhejiang from 2013 to 2015.Inclusion criteria: (1) Healthy women with family planning;(2) no medical or obstetrical diseases;(3) No hormone drugs were taken.Exclusion criteria: patients without complete clinical data.Overall, 433 women were included in the study.

2.2 Research Methods

2.2.1 Questionnaire SurveyUnified structured questionnaires were used to collect information,such as age, education level, family economic income, height and weight.The study was approved by the Ethics Association of Xinhua Hospital and put on record by the ethics committee of all participating hospitals.All participants signed informed consent.

2.2.2 Detection of PFAS

Fasting peripheral venous blood samples were collected, plasma was centrifuged and stored at -80 ℃ for future use.Agilent 1290 high performance liquid chromatograph and Agilent 6495C triple tandem quadrupole mass spectrometry were used to detect the concentrations of 23 PFAS in the plasma of the study subjects, including three novel alternatives to PFAS: 6:2 chlorinated Polyfluorinated Ether Sulfonate, 6:2 chlorinated polyfluorinated ether sulfonate, 6:2 Cl-PFESA), 8:2 chlorinated Polyfluorinated Ether Sulfonate, 8:2 Cl-PFESA) and hexafluoropropylene oxide dimer acid (HFPO-DA),8 conventional PFAS: PFOS, PFOA, perfluorohexanesulfonic acid (PFHxS), perfluorohexanesulfonic acid, Fluorodecanoic acid(PFHpS), Perfluoroononanoic acid (PFNA), perfluorodecanoic acid (PFDA), Perfluorodecanoic acid (Perfluorodecanoic acid,Fluorododecanoic acid (PFDoA), 8 fluorododecanoic acid (PFDOA),fluorododecanoic acid (PFDOA), Fluorododecanoic acid (PFDOA),fluorododecanoic acid (PFAS) isomers: n-PFOS, 1m PFOS, 3m PFOS, 4 m PFOS, 5 m PFOS, 6m PFOS, n-PFHxS and br-PFHxS,4 kinds of short-chain PFAS: Fluorobutanesulfonic acid (PFBA),perfluorobutanesulfonic acid (PFBS), perfluorobutanesulfonic acid,perfluorohexanoic acid, Fluoroheptanesulfonic acid (PFHpS) and perfluoroheptanesulfonic acid.The limits of detection (LOD) of PFAS were defined as the concentration when the signal-to-noise ratio (S/N) was equal to 3, ranging from 0.002 to 0.0083 ng/mL.

2.2.3 Determination of reproductive hormone

Venous blood was taken in the morning of the 2-5 d of menstruation of the subjects, and the blood samples were also used for PFAS detection.follicle-stimulating hormone (FSH), luteinizint hormone(LH), prolactin (LH), follicle-stimulating hormone (FSH),Luteinizint hormone (LH), and prolactin (LH) in serum were detected by Roche E170 automatic immune analyzer and supporting reagents.PRL), estradiol (E2), testosterone (total testosterone, TT)and progesterone (progesterone, P) levels.

2.3 Statistical Methods

SAS 9.4 software and R language 4.0.3 software were used to analyze the data.The mean ± standard deviation (±s) was used to represent the measurement data conforming to normal distribution,the median (interquartile distance) was used to represent the measurement data of non-normal distribution, and the quantity(percentage) was used to represent the categorical variable.Chisquare Chi-2 test and T-test were used to analyze the data, and multiple linear regression analysis and quantile g-computation model were used to analyze the correlation between single PFAS and mixed PFAS exposure and reproductive hormones.IfP＜0.05, the difference was statistically significant.

3.Result

3.1 Basic information of research objects

A total of 433 women of childbearing age were included in this study.The mean age of menarche was 29.6 years old, the mean age of menarche was 14.1 years old, the BMI was (22.7±3.4) kg/m2,and the level of reproductive hormones was in line with the overall standard for this age group.See Table 1.

Tab 1 General epidemiological characteristics of the study population (n=433)

3.2 PFAS content

According to the structure and use of PFAS, PFAS are divided into four categories: traditional PFAS, PFAS isomers, short-chain PFAS and PFAS substitutes.The results showed that 6:2 Cl-PFESA,n-PFHxS, n-PFOS, 1m PFOS, ∑3,4,5m-PFOS, 6m-PFOS, PFOA,PFOS, PFNA, PFDA, PFHxS and PFUdA were all detected, and the detection rate of PFAS substitutes was more than 98%.The detection rate of short - chain PFAS was more than 80%.The median concentration of PFOA was the highest (6.16 ng/mL), followed by total PFOS (4.11 ng/mL), n-PFOS (3.0 ng/mL) and 6:2 Cl-PFESA(2.27 ng/mL).The concentrations of other PFAS were lower, ranging from 0.01-0.61ng/mL.See Table 2.

Tab 2 PFAS concentration levels in the study population (ng/mL, n=433)

3.3 PFAS and reproductive hormone levels

After adjusting for confounding factors such as female age, age of menmene, birth time, BMI, study area, education and income level,natural logarithm conversion of PFAS and reproductive hormones was performed, multiple linear results showed that LH decreased by 0.075 (ln-IU/L) for each increase of natural logarithm unit concentration of 6:2 Cl-PFESA.95%CI(-0.139, -0.012); For each increase in the natural logarithmic unit concentration of 1m-PFOS and brPFOS, the progesterone changed by 0.131 (95%CI: 0.021,0.242) and 0.119 (95%CI: 0.005, 0.234), respectively, as shown in Table 3.In addition, quantile g-computation model also found a positive correlation between mixed PFAS exposure and progesterone levels [0.549 (95%CI: 0.322, 0.774)], as shown in Figure 1.However, no association was found between PFAS mixed exposure and other reproductive hormones.

Fig 1 Association between PFAS mixed exposure and progesterone (n=433)

4.Discussion

In this study, plasma concentrations of PFAS in women of reproductive age were investigated in Shandong and Zhejiang provinces.The results showed that the detection rates of all PFAS except brPFHxS were above 80%, suggesting that there was a high level of PFAS contamination in these two regions.In addition, a relatively high concentration of new PFAS substitutes was detected in this study, and the concentration was second only to PFOA and PFOS, suggesting that new PFAS substitutes are replacing traditional PFAS in human life and production, and entering the human body.This study found a positive correlation between PFAS and progesterone levels, suggesting that PFAS may have the potential to interfere with reproductive hormone homeostasis.

The results of this study showed that the concentration of the new substitute 6:2 Cl-PFESA in female plasma (2.27 ng/mL) in two regions was higher than that detected in Shenyang (1.75 ng/mL)[7], Shanxi (0.34 ng/mL)[8] and Wuhan (1.54 ng/mL)[8].The plasma 6:2 Cl-PFESA concentration was similar to that in Guangzhou(2.405 ng/mL)[9], but lower than that in Tianjin (5.48 ng/mL) [9],Huzhou (3.7 ng/mL)[10] and Zibo (5.48 ng/mL)[11].8:2 Cl-PFESA concentration in the plasma of women in this study was 0.07 ng/mL,It was higher than that of Shenyang (0.01 ng/mL)[11], Wuhan (0.01 ng/mL)[12], Guangzhou (0.001ng/mL)[9], Huzhou (0.0068 ng/mL)[9] and Zibo (0.06 ng/mL)[11].It was lower than that in Tianjin (0.15 ng/mL)[13].The concentration of HFPO-DA was 0.03 ng/mL, and no HFpo-Da was detected in Tianjin and Zibo.The reason why the concentration of substitutes is not exactly the same may be related to the different pollution degree, use time, people’s lifestyle, diet pattern and other factors of substitutes in different regions.

This study found a positive correlation between PFAS and progesterone levels, which was not completely consistent with the results of previous population studies.A Norwegian study recruited 178 healthy women aged 25-35 and found a negative correlation between PFOS and estradiol, and no association between PFAS and progesterone[14].Another study recruited 13,458 women aged 18-42 years using the C8 program and found that estradiol concentration decreased as PFOS concentration increased[15].Appropriate progesterone can promote endometrial differentiation, which is conducive to pregnancy, but increased progesterone level may cause ovulation disorder, reduce endometrial receptivity, cause and aggravate pregnancy complications, and lead to decreased female fertility[16].

At present, the mechanism of PFAS on reproductive hormones is not clear.It has been found that 5 mg/kg PFOA poisoning can stimulate the secretion of progesterone in C57Bl/6 mice[17], and high concentrations of PFOA and PFOS can slightly stimulate the secretion of estradiol and progesterone in H259R cells[18], but this effect has not been found in human concentrations[19].It is speculated that high concentration of PFAS may stimulate hormone secretion by promoting the expression of MC2R, CYP11B1,CYP11B2, and CYP19A1[19].

Considering the fact that human body is exposed to multiple pollutants at the same time, traditional PFAS, new alternatives to PFAS, PFAS isomers and short-chain PFAS were included in this study.The analysis method of mixed exposure model was adopted to reflect the association between multiple environmental PFAS exposure and reproductive hormones, and to accurately evaluate the combined action of PFAS mixture.It is of great significance for social economic development and public health to provide new guidance basis for the clinical protection of female fertility and new ideas for reducing the downward risk of fertility.However,considering that this study is a cross-sectional study, there are some limitations in the inference of causal association.Secondly, although confounding factors were taken into account in this study, the results may be affected to some extent because the samples come from different provinces and there are differences in nationality or lifestyle, such as eating habits.Finally, the sample size of this study is small, so it needs to be verified in a larger population sample in the future.

5.Conclusion

Disturbance of reproductive hormones (progesterone) may be associated with PFAS when exposure to novel PFAS is higher in women of childbearing age.