Study population

We conducted a cross-sectional study based on a primary health program that aimed to promote reproductive health of couples in less developed areas by providing free medical counseling as well as free semen quality assays for couples of childbearing age. The study was conducted from July to August, 2012 in Sandu County (Autonomous County of Shui nationality), Guizhou Province, China. Inclusion criteria included males, 18–55 years and having at least one child. The men who (1) had been diagnosed with disease of urogenital system including testis, epididymis and prostate, (2) had severe heart, liver, lung, kidney, endocrine diseases or (3) any history of hospital diagnosed mental illness were excluded from the study.

A total of 774 eligible men, corresponding to a participation rate of 75%, agreed to participate. Among them, 507 men provided both semen samples and urine samples for BPA assay. We further excluded 7 men who did not provide any in-person information. Finally, 500 men (64.6% of eligible males) were included in the study. None of the participants or their wives were received any medical interventions, and 66.36%, 19.72%, 7.89% and 6.03% conceived the first child within 6 months, 6–12 months, 12–24 months and greater than 24 months, respectively.

In-person interview and bio-sample collections

The participants were invited to local clinics and interviewed with structured questionnaires. We obtained information on demographics, lifestyle factors (smoking and alcohol intake), history of exposure to pesticides, occupational exposure to high temperature, and reproductive history. Individuals who reported smoking at least one cigarette per day for the last six months were considered as smokers. Individuals who reported drinking alcoholic beverage (wine, beer, or liquor) at least once a week for the last six months were considered as drinkers. History of exposure to pesticides was defined as contacting any pesticides at least half an hour each day for at least 7 days each year. Individuals who reported to be workers of casting, smelting or bread baking were considered as occupational exposure to high temperature. Body height and weight were measured with barefoot and clad in light underwear, following the recommendations of the Committee on Nutritional Anthropometry of National Health and Nutrition Examination Survey. Body mass index (BMI) was calculated as weight (kg) divided by squared height (m2).

A single-spot urine sample was collected from each subject in a polypropylene cup (BPA free) when they visited the clinic for interview. The urine was frozen without preservatives and stored at −20 °C in local clinics within 14 days before they were shipped on dry ice by plane to the laboratory at the Shanghai Institute of Planned Parenthood Research (Shanghai, China) and stored at −80 °C for further analysis. A single semen sample was also, at the same time, collected by masturbation into 25-ml sterile polystyrene jars (BPA free) from each participant.

Semen analysis

The semen samples were analyzed within 1 hour following ejaculation. Semen analyses were conducted after liquefaction at the clinics by WLJY-9000 CASA system (Beijing Weili New Century Science & Tech Dev. Co. Ltd., Beijing, China)27. Main technical parameters are as follows: image acquisition frame: low and middle sperm concentration collected at 20 Hz, while high sperm concentration at 7 Hz; acquisition interval: 3 ms; maximum sperm motile velocity: 200 μm/s; area range of spermatozoa head detected at 7–60 μm2. Features of system software: adjusting greyscale threshold, collecting spermatozoa and excluding nonsperm granules.

The same professional technician performed semen analysis for all specimens according to World Health Organization (WHO) guidelines and instruction of the instrument manufacturer. Briefly, to measure both sperm concentration and motility, 5 μL of semen from each sample was placed into a pre-warmed (37 °C) 10 um-depth MAcro. counting chamber provided by the instrument manufacturer. A minimum of 200 sperm cells from at least four different fields were analyzed from each specimen. Sperm concentration, total count, forward mobility, and nine parameters of sperm movement characteristics were measured to profile men’s sperm quality. Total sperm count (106) was calculated by multiplying sperm concentration (106/ml) by semen sample volume (ml). Motile sperm was defined as WHO grade “a” sperm (rapidly progressive with a velocity ≥ 25 μm/s at 37 °C) plus “b” grade sperm (slow/sluggish progressive with a velocity ≥ 5 m/s but <25 μm/s). Sperm movement characteristics are composed of three indicators on sperm motion velocity (Curvilinear velocity (VCL), μm/s; Straight-line velocity (VSL), μm/s; Average path velocity (VAP), μm/s), three on velocity ratio (Linearity (LIN) equals to VSL/VCL; Straightness (STR) equals to VSL/VAP; Wobble (WOB) equals to VAP/VCL), and three on sperm swing characteristics (Amplitude of lateral head displacement (ALH), μm; Mean angular displacement (MAD), degree; Beat-cross frequency (BCF), Hz)28.

Urinary BPA measurements

The urine samples were analyzed at the collaborative laboratory of East China University of Science and Technology (Shanghai, China). Urinary concentrations of total BPA (free plus conjugated species) were measured by modified high-performance liquid chromatography (HPLC) as described previously3. In brief, urine samples were treated with 100 μL phosphorous acid buffer of 0.01 mol/L (PH = 5.0) and 20 μL β-glucuronidase (Sigma) for hydrolyzation, and were subsequently extracted twice using ether (HPLC grade, Dikma). We collected and evaporated the supernatants using nitrogen gas. The residue was dissolved in 60% acetonitrile (HPLC grade, Dikma) and analyzed using HPLC equipment on the following parameters: column, Inertsil ODS-3, 4.6 mm × 250 mm, 5 mm; mobile phase A and B, acetonitrile/water (40:60, v/v), equivalent grade; flow: 1.0 mL/min; FLD, excitation wavelength 275 nm, emission wavelength 300 nm. Water used in the assay was from Millipore Super-Q Plus water purification system (Bedford, MA).

Glass-made analytic instruments were used throughout entire analytic procedure to avoid possible contamination. Besides, blank samples, which used purified water instead of urine, were conducted randomly during each HPLC analysis to confirm an absence of BPA contamination. The BPA fraction was confirmed by the standard BPA (HPLC grade, Shanghai Yuanxing Company) with the same HPLC base. The relative standard deviation (RSD) of replicate analysis of samples was less than 5.26%. Absolute recoveries of BPA ranged from 83.7% to 98.7%.

The limit of detection (LOD) of BPA in this study was 0.12 μg/L, which was comparable to those reported at 0.1–0.4 μg/L in some studies29,30,31. BPA levels below the LOD were assigned a value of LOD divided by the square root of 2, based on a conventionally accepted practice32. Adjustment for creatinine was performed to account for urine volume, which was applied in all analyses.

Statistical analysis

Descriptive statistics of demographic characteristics, urinary BPA concentration and semen parameters were tabulated. Due to skewed distributions, sperm concentration and total count were natural logarithm (ln) transformed. Linear regression models were used to estimate associations of sperm parameters with BPA exposure. BPA exposure was first dichotomized as undetected and detected groups and differences of sperm parameters between the two groups were estimated. Those with detected BPA were further categorized into 3 groups according to tertiles and associations of sperm parameters with different BPA terties were examined with subjects with undetected BPA as reference group. The associations between BPA and sperm parameters were examined by introducing BPA categories as a discrete variable in the linear regression models. To test the dose-response pattern of the association, we repeated the analysis by introducing BPA categories as a continuous variable (0–3).

We adjusted for covariates, including age (<25, 25–29, 30–34, 35–39 and ≥40 years), education (≤primary school, junior high school and ≥senior high school), ethnicity (Shui, Bouyei, Miao, and other nationalities), smoking (Yes, No), alcohol intake (Yes, No), abstinence period (<2, 2–7 and >7days), BMI (<18.5, 18.5–24.9 and ≥25 kg/m2), history of exposure to pesticides (Yes, No), and occupational exposure to high temperature (Yes, No).

We re-examined the relationships between BPA exposure and sperm parameters using unadjusted BPA concentrations of all participants (shown in Supplemental Material, Table S1). The relatively large study sample recruited from communities made it almost inevitable to cause discrepancies in time of urine collection and abstinence periods. Thus, sub-group analyses by time of urine collection and abstinence periods were considered a priori. In order to reduce the misclassification of BPA level resulting from a large discrepancy in time of urine collection, we restricted analyses to the subjects whose urine samples were collected between 9 to 11 am (showed in Supplemental Material, Table S2). We also restricted analyses to the participants whose semen samples were obtained within an abstinence period of 2 to 7 days, which would reduce the misclassification of semen measures with duration of abstinence.

All data handling and statistical analyses were performed in SAS version 9.4 (SAS Institute, Inc., Cary, North Carolina).

Ethics

The study was reviewed and approved by the ethics committee board of Shanghai Institute of Planned Parenthood Research (IRB00008297). All participants gave written informed consent before engaging in the study. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.