【小夏的论文笔记】来自中国出生队列的孕妇尿对羟基苯甲酸酯水平和子代体格

Maternal urinary paraben levels and offspring size at birth from a Chinese birth cohort
中国出生队列孕妇尿中对羟基苯甲酸酯水平和子代体格

原文链接：http://www.sciencedirect.com/sci ... i/S0045653516318744


亮点：第一项评估中国孕妇对羟基苯甲酸酯暴露的研究；中国孕妇大多数尿样中检测到对羟基苯甲酸酯；检验了孕妇尿中对羟基苯甲酸酯与婴儿出生体重的关系；孕妇尿中对羟基苯甲酸甲脂和男婴出生身长呈正相关。

这项研究从中国湖北省的纵向健康婴儿队列（HBC）中随机选择1006对母婴，收集分娩前孕妇尿样测量对羟基苯甲酸酯浓度。通过广义线性模型分析孕妇尿中对羟基苯甲酸酯水平和子代体格之间的关系。结果发现尿样中对羟基苯甲酸甲脂，对羟基苯甲酸乙酯，对羟基苯甲酸丙酯浓度的调整几何均数分别为5.41, 0.11，0.94 ng/mL。孕妇尿中对羟基苯甲酸酯浓度与男孩出生体重呈正相关，女孩则成负相关，差异无统计学意义。孕妇尿中对羟基苯甲酸甲脂水平和男婴出生身长呈正相关，女孩中没有发现具有统计学意义的关联。

这篇文章其实主要是一个阴性结果，研究没有发现中国孕妇对羟基苯甲酸酯水平和出生婴儿体格的强相关。不过其实可以推想一下，过高的防腐剂暴露理论上是很有可能对胎儿造成不良影响的，不过一种可能是这种影响并没有体现在婴儿出生时的体格方面，另外也有可能是研究对象的对羟基苯甲酸酯暴露其实并没有达到引起不良效应的阈值。当然后者也是公共卫生研究的一个自然限制，即不伤害的伦理原则，我们不能像对待实验室里的老鼠一样人为地控制暴露量那样，去给研究对象施加负面干预，我们只能观察自然发生的现象，去发现其中的规律并给出合理的解释。


【Highlights】

•This is the first study to assess paraben exposure in Chinese pregnant women.
•Parabens were detected in the majority of urine samples in Chinese pregnant women.
•The association of maternal urinary parabens with infants' birth size was examined.
•Maternal urinary levels of MeP were positively associated with birth length in boys.

【Abstract】

Background

Parabens are suspected to impair fetal growth because of their endocrine disrupting effects. Epidemiological studies regarding the effects of prenatal exposure to parabens on birth outcomes are limited.

Objectives

Our aim was to evaluate the association between prenatal paraben exposure and size of infants at birth.

Methods

Within the longitudinal Healthy Baby Cohort (HBC) in Hubei Province, China, we randomly selected 1006 mother-infant pairs recruited in Wuhan City in 2014. Concentrations of parabens were measured in maternal urine collected before delivery. General linear models were used to analyze the associations of maternal parabens exposure levels with birth weight and birth length.

Results

The specific gravity adjusted geometric means for urinary concentrations of methyl paraben (MeP), ethyl paraben (EtP), and propyl paraben (PrP) were 5.41, 0.11, and 0.94 ng/mL, respectively. Maternal urinary concentrations of parabens tended to be positively associated with birth weight in boys, while opposite trends were found in girls, though these associations were not significant. Higher maternal urinary levels of MeP were positively associated with birth length in boys (β = 0.30, 95% CI: 0.01, 0.58 for the medium tertile, and β = 0.30, 95% CI: 0.01, 0.58 for the highest tertile compared to the lowest tertile); however, no significant associations with birth length were observed in girls.

Conclusions

Maternal urinary levels of MeP were positively associated with length at birth in boys. Besides, we did not find strong associations of the current exposure levels of parabens in Chinese pregnant women with size of infants at birth.

【Keywords】

Parabens; Birth size; Maternal urine; Birth cohort

引言讲述了

1.对羟基苯甲酸酯在生活中的用途，以及尿液中对羟基苯甲酸酯原型或代谢物的水平可以作为衡量对羟基苯甲酸酯暴露的有效生物标志物。

2.对内分泌可能的干扰作用和动物研究发现的危害。

3.简述了过去的三项结果完全不同的研究，以及本次研究的简介。


1. Introduction
Parabens, also named as esters of parahydroxybenzoic acid, are widely used as antimicrobial preservatives in the cosmetic and pharmaceutical industry, and several parabens are often used in combination (Golden et al., 2005 ;  Calafat et al., 2010). The chemicals can be found in multiple products, including personal care products (such as cosmetics, lotions, and hair care productions), foods and beverages (Andersen, 2008 ;  Crinnion, 2010). Exposure to parabens in the general population may occur through ingestion, inhalation, or dermal absorption (Mortensen et al., 2014). As the chemicals with short half-lives of less than 24 h in the human body, parabens are rapidly metabolized and are mainly eliminated through urine (Janjua et al., 2008). The levels of total form of the parent compounds and some new metabolites found in urine can be valid biomarkers for recent paraben exposure (Ye et al., 2006 ;  Moos et al., 2016).

Although parabens have been extensively used as preservatives for a long time, several concerns have been raised over the past twenty years about the safety of parabens due to their potential endocrine disrupting effects (SCCP, 2005; Darbre and Harvey, 2008 ;  Diamanti-Kandarakis et al., 2009). Prenatal exposure is of particular concern due to the potential health effects on the vulnerable embryos, in which exposure may cause lifetime adverse health impacts. Some animal studies have showed that maternal exposure to several parabens impaired growth, hormone balance, neurodevelopment, learning ability and behavior of the offspring (Kang et al., 2002; Boberg et al., 2008 ;  Kawaguchi et al., 2009).

Human data are sparse regarding the effects of prenatal exposure to parabens on fetal growth. A study conducted in 191 French male newborns reported no associations between maternal urinary levels of parabens and offspring size (Philippat et al., 2012). However, after enlarging the sample size to 520 male newborns, they came to the conclusion that maternal urinary levels of methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) tended to be positively associated with weight at birth (Philippat et al., 2014). On the contrary, another study conducted among only 34 newborns from immigrant population in Brooklyn, New York, demonstrated that PrP in cord blood plasma was associated with decreased length at birth (Geer et al., 2017).

Given the absence of data concerning the levels of paraben exposure in Chinese pregnant women, in this study, we analyzed urinary concentrations of five commonly used varieties of parabens, including MeP, EtP, PrP, BuP, and benzyl paraben (BzP) in 1006 pregnant women in Wuhan city, China to fill the gap of the biological monitoring data. As many previous studies suggested that endocrine-disrupting chemicals can have adverse effects at low doses (Melnick et al., 2002 ;  Vandenberg et al., 2012), we also aimed to find out whether the current exposure levels of parabens could affect fetal growth by evaluating their relationships with weight and length of infants at birth.

2. Materials and methods
2.1. Study population

The Healthy Baby Cohort (HBC) study, a longitudinal prospective birth cohort in Hubei Province, China, is committed to investigate the associations between environmental exposures and children's health. Details of the cohort study have been described previously (Yang et al., 2016). The population in this present study was selected from participants in Wuhan city, which enrolled 11,311 pregnant women from September 2012 through October 2014.

The present study restricted to a subset of women with paraben measurements in urine samples collected before delivery (n = 1016). These urine samples were randomly chosen from the participants who were enrolled in Wuhan during January to October 2014. Ineligible women who gave birth to infant with birth defects (n = 7), or had missing medical record data (n = 3) were excluded, leaving 1006 mother-infant pairs for analyses. None of the participants reported smoking or drinking during pregnancy. The women included in the present study did not significantly differ in the basic characteristics or the characteristics of their infants (sex, birth weight, gestational age) with the parent cohort.

All participants in this study provided written informed consent before enrollment. The research protocol received the approvals of the ethics committee of Tongji Medical College, Huazhong University of Science and Technology, and the study hospital.

2.2. Birth outcomes and covariates

At the time of delivery, routine anthropometric measurements including birth weight (g) and length (cm) were measured by trained nurses with standardized procedures. Information concerning history of gestation (parity), maternal age, education level, weight at delivery, and birth outcomes (infant's birth date, sex, gestational age at birth, birth weight, and birth length) were obtained from electronic medical records. Gestational age (in days) was calculated based on the date of last menstrual period (LMP) or assessed by ultrasound data if it differed from the LMP-based estimation by over 7 d due to the concerns over the reliability of the self-reported LMP estimation. Questionnaire information regarding maternal demographic characteristics and lifestyle factors (smoking, drinking, etc.) was collected by a face to face interview after delivery by specially trained nurses in the hospital. The pre-pregnancy body mass index (BMI) was calculated by self-reported pre-pregnancy weight and height.

2.3. Urine sample collection and paraben exposure assessments

The urine samples were collected immediately after the pregnant women admitted to the hospital for delivery (within 3 d before delivery), and divided into aliquots storing in the 5-mL polypropylene cryovials at −20 °C until further analysis.

Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to analyze the urinary paraben concentrations, as previously reported by Wang et al. (2013) with some modifications. In brief, 1 mL urine sample was added with 25 μL isotope-labeled internal standard solution which contained 13C6-MeP, 13C6-EtP, 13C6-n-PrP, and 13C6-n-BuP (10 ng each), and then the mixture was incubated with 10 μL of β-glucuronidase/sulfatase at 37 °C overnight. The digested samples were further extracted for three times with 3 mL methyl tert-butyl ether (MTBE). The total supernatant organic layer was collected and concentrated to near-dryness at room temperature under a gentle stream of nitrogen gas. Finally, a 500-μL acetonitrile-water (6:4) was added, vortexed, and filtered into a vial for UPLC-MS/MS analysis.

Chromatographic separation and detection of target analytes were accomplished using Waters Acquity UPLC system (Waters Corporation, Maple Street Milford, MA, USA), interfaced with a Waters TQD triple quadrupole tandem mass spectrometer, negative-ion electrospray ionization mass spectrometry (ESI-MS/MS) and multiple reaction monitoring mode (Waters Corporation). Five μL of the extract was injected onto an analytical column (Betasil C18, 100 × 2.1 mm column; Thermo Electron Corporation, Waltham, MA), which was connected serially to a guard column (Betasil C18, 20 × 2.1 mm column; Thermo Electron Corporation, Waltham, MA, USA). The mobile phase comprised methanol and ultrapure water. All samples were coded anonymously during the measurement. Each batch of analytical run included calibration standards, thirty samples, procedural blanks, matrix spiked samples, and duplicates. Recoveries of all target compounds corrected by isotope-labeled internal ranged from 85% to 109%. The mean recovery of 13C6-labeled internal standard was 73% for 13C6-MeP, 89% for 13C6-EtP, 87% for 13C6-n-PrP and 80% for 13C6-n-BuP, respectively. The analysis of selected duplicate samples showed that the between-assay and within-assay coefficient of variation were both < 15%. Field blanks were also conducted to evaluate the potential contamination. Quantification of parabens was calculated based on the ratios between areas of the target analytes and their respective internal standards, which were 13C6-MeP for MeP, 13C6-EtP for EtP, 13C6-n-PrP for PrP, and 13C6-n-BuP for BuP and BzP. Instrumental calibration ranged in concentrations of target chemicals from 0.2 to 500 ng/mL, and the regression coefficients (r) were above 0.995 for all target analytes. The limits of detection (LODs) were 0.02 ng/mL for EtP and BzP, 0.03 ng/mL for MeP, and 0.06 ng/mL for PrP and BuP.

Specific gravity (SG) of the urine samples were measured at room temperature using a hand-held digital refractometer (Atago Co., Ltd., Tokyo, Japan) and the concentration of urinary parabens were corrected by the measured SG to adjust for urine dilution through the following formula: Pc = P [(1.012–1)/(SG–1)], where Pc is the SG-adjusted urinary concentration (ng/mL), P is the measured urinary concentration (ng/mL), and SG is the specific gravity of the urine sample. The value of 1.012 was the median SG for the urine samples of this study population.

2.4. Statistical analysis

Geometric means and specific percentiles, including unadjusted and SG adjusted concentration, were calculated to describe the distributions of urinary parabens among pregnant women in this study. The sum of paraben (SumP) concentration was calculated by adding the molar concentrations of MeP, EtP, PrP, BuP and BzP. The SG adjusted concentrations of parabens were stratified into tertiles to categorize participants into low, medium and high exposure groups in order to explore nonlinear relationship between paraben exposure levels and size of infants at birth. In addition, values for paraben concentrations below the limit of detection (LOD) were replaced with LOD divided by the square root of 2 (Hornung and Reed, 1990), and natural log (ln) transformation of paraben concentrations were proceeded to produce normal distributions. Pearson correlation coefficients were calculated between different types of parabens using ln-transformed concentrations.

General linear models were used to compare the differences in the SG adjusted ln-transformed concentrations of parabens between categories of pre-pregnancy BMI (normal: 18.5–23.9; underweight: < 18.5; overweight: ≥ 24 kg/m2), pregnancy weight gain (< 16 and ≥ 16 kg, median as the cut-point), maternal height (< 1.60 and ≥ 1.60 m, median as the cut-point), paternal height (< 1.73 and ≥ 1.73 m, median as the cut-point), maternal age (< 25; 25–29; 30–34; ≥ 35 years), parity [0 (nulliparous) and ≥ 1 (multiparous)], education level (high school and below; more than high school), and infant sex (male and female).

Associations between SG adjusted concentrations of maternal urinary parabens and continuous birth weight, along with birth length were estimated using separate general linear models. The analysis of association with birth weight was further restricted to the term birth infants. Test for trend was conducted by modeling the tertile-specific median biomarker levels as continuous variable and evaluated the statistical significance of this predictor. We took biological and statistical reasons into consideration to include the covariates in the final models. Potential confounders such as gestational age, pre-pregnancy BMI, weight gain during pregnancy, maternal age, maternal education level, maternal and paternal height (for models which estimated the association between birth length and paraben exposure), and infant's sex (except in models conducting stratified analysis by sex) were entered into the final multi-variable model with p-value < 0.2 for the statistical consideration. The cut off at p-value was broadened from 0.1 to 0.2 for the variables entering into the multivariable model in order to avoid omitting some important confounders, as some previous studies suggested ( Holm Tveit et al., 2009 ;  Cao et al., 2016). Parity was included in final models even with p-value > 0.2, in consideration of the biological reason ( Shah, 2010). Considering that parabens are hormonally active, we further examined the associations between urinary paraben exposure levels and size at birth stratified by infant sex. Potential interactions between infant sex and paraben exposure in models with birth weight and length as outcomes were tested by inserting infant sex × ln-transformed SG adjusted urinary paraben concentrations into the models.

Statistical analyses were performed using Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., Cary, NC, USA). All tests were two sided and statistical significance was defined as a p value < 0.05.

资料与方法：
1.中国湖北省的纵向前瞻性出生队列——健康宝宝队列（HBC）
2.介绍了测量出生相关信息的方法和测量尿样方法（色谱）。
3.统计方法采用了计算得出的对羟基苯甲酸酯浓度，分为三层将参与者分为低中高暴露组；广义线性模型比较不同的怀孕前BMI分组、孕期体重增加量分组、母亲身高分组、父亲身高分组、母亲年龄分组、parity分组（根据后面的分组应该是“产次”的意思）、教育程度分组和婴儿性别分组之间根据妊娠前尿比重校正的尿样对羟基苯甲酸酯浓度的差异；separate general linear model估计校正的尿样对羟基苯甲酸酯浓度与出生体重之间的关系，混杂因素考虑了胎龄，怀孕前BMI，怀孕期间体重增加，孕产妇年龄，孕产妇教育水平，孕产妇、父母身高、和婴儿的性别，并纳入最终的多变量模型；考虑到对羟基苯甲酸酯具有激素活性，进一步分析了暴露水平与按性别分层的出生体格之间的关系；并检验了各混杂因素与对羟基苯甲酸酯浓度的潜在交互作用。


"In addition, values for paraben concentrations below the limit of detection (LOD) were replaced with LOD divided by the square root of 2 (Hornung and Reed, 1990)”
这里学习到了传说中低于检出限的值取检出限除以根号2的出处，拿小本本记下来，以后论文用得上：
Richard W. Hornung, and Laurence D. Reed. "Estimation of Average Concentration in the Presence of Nondetectable Values." Applied Occupational & Enviromental Hygiene 5.1(1990):46-51.

其实相比前面方法的介绍，后面的数据反倒并不是我们这种泛读所需要重点关注的。不过这篇文章的讨论里面看到了一个有意思的结果：
高教育水平与PrP（丙酯）的尿浓度增加有关，可能是是由于高教育水平女性使用了更多的化妆品等个人护理用品。


为了节约考试周的时间，保证每天临床课程刷书时间的前提下，坚持每天读一篇的节奏，之后的论文笔记只贴出摘要、引言和方法部分。至于结果和讨论，如果发现了有意思的结论也会在帖子里简要分享一下。