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Exposures to Atmospheric PM10 and PM10-2.5 Affect Male Semen Quality: Results of MARHCS Study Niya Zhou, Changtan Jiang, Qing Chen, Huan Yang, Xiaogang Wang, Peng Zou, Lei Sun, Jiaojiao Liu, Ling Li, Lianbing Li, Linping Huang, Hongqiang Chen, Lin Ao, Ziyuan Zhou, Jinyi Liu, Zhihong Cui, and Jia Cao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05206 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Environmental Science & Technology

Cui, Zhihong; Key Lab of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Institute of Toxicology, College of Preventive Medicine, Third Military Medical University Cao, Jia; Key Lab of Medical Protection for Electromagnetic Radiation, Ministry of Education of China, Institute of Toxicology, College of Preventive Medicine, Third Military Medical University

ACS Paragon Plus Environment


Exposures to Atmospheric PM10 and PM10-2.5 Affect Male Semen

1 2

Quality: Results of MARHCS Study

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Niya Zhou1,†, Changtan Jiang2,†, Qing Chen1,†, Huan Yang1, Xiaogang

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Wang1, Peng Zou1, Lei Sun1, Jiaojiao Liu2, Ling Li2, Lianbing Li3,

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Linping Huang1, Hongqiang Chen1, Lin Ao1, Ziyuan Zhou4, Jinyi Liu1,

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Zhihong Cui1*, and Jia Cao1*

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† Authors equally contributed

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1

Key Lab of Medical Protection for Electromagnetic Radiation, Ministry of

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Education of China, Institute of Toxicology, College of Preventive Medicine, Third

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Military Medical University, Chongqing, China.

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2

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China.

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3

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and Family Planning Commission (Chongqing Population and Family Planning

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Science and Technology Research Institute), Chongqing, China.

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4

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Military Medical University, Chongqing, China.

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*Correspondence address: Institute of Toxicology, College of Preventive Medicine,

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Third Military Medical University, Chongqing 400038, PR China.

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Tel: +86-23-6875-2289 (J.C.)/+86-23-6875-2291 (Z.C.);

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Fax: +86-23-6875-2276(J.C.)/+86-23-6875-2276(Z.C.);

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E-mail: [email protected](J.C.)/[email protected] (Z.C.)

Ecological and Environmental Monitoring Center of Chongqing, Chongqing,

Key Laboratory of Birth Defects and Reproductive Health of the National Health

Department of Environmental Health, College of Preventive Medicine, Third

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Abstract

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Studies have shown that the effects of ambient particulate matter (PM) may be related

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to particle’s size. However, results on the relationships between different PM and

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reproductive health are controversial. To explore the impacts of various PM fractions

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on male reproductive health, a total of 796 eligible subjects recruited in 2013 baseline

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investigation. In addition, there were 656 (82.4%) and 568 (71.3%) subjects

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participated follow-up surveys in 2014 and 2015, respectively. We used multivariable

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regression analysis and mixed-effect model to investigate the associations between air

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pollutants PM10, PM10-2.5 and PM2.5 exposures and semen quality, sperm DNA

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fragmentation and serum reproductive hormones of subjects. In the preliminary

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regression analysis, PM10, PM10-2.5 and PM2.5 exposure all associated with sperm

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concentration, morphology, sperm high DNA stainability (HDS), serum estradiol and

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testosterone levels. However in mixed models, we only found that PM10 exposure

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were negatively associated with sperm normal morphology (95% CI: -14.13, -24.47)

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but positively associated with sperm progressive motility (95% CI: 23.00, 8.49), and

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PM10-2.5 exposure was inversely associated with sperm concentration (95% CI:

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-9.06, -27.31) after multiplicity adjustment. Our results provide the evidence that air

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PM10 and PM10-2.5 exposures, not PM2.5, are risk factors of semen quality.

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2



44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 3


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Introduction

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The health risk of ambient particulate matter (PM) pollution have been of great

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concern to the public and researchers.1-6 Associations between air PM pollution and

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male reproductive health also have been reported by lots of studies.7-13

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Inhalable particulate matter (aero dynamic diameter ≤ 10 μ m, PM10) and fine

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particulate matter (aero dynamic diameter ≤ 2.5μm, PM2.5) are most well studied

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particles. Researchers have demonstrated that the PM10 and PM2.5 can

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carry toxic chemicals like polycyclic aromatic hydrocarbons (PAHs) that are harmful

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to reproductive health.14-17 In one of our published report, we employed urinary PAH

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metabolites as biomarkers of PAH exposure to monitoring environmental pollution,

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and we found environmental level of PAH exposure is associated with increased

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sperm DNA damage.18

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Airborne PM is a mixture of solid and liquid particles and varies in size. There is

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growing number of studies shown that the harmful effects of particulate matter are

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related to particle’s size, different particles are not equally toxic.19 Over the recent

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decades, epidemiological investigations suggested that coarse particulate matter (aero

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dynamic diameter between 10 and 2.5μm, PM10-2.5) may also exerts detrimental

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effects on health.2,20-26 A recent study explored association between gestational

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exposure of PM10-2.5 and birth weight and suggested that PM10-2.5 have adverse

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effects on the birth weight.27 But the studies still limited for the potential effects of

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coarse fraction (PM10-2.5), which may or may not be associated with reproductive

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health. 4



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Previous studies on the reproductive health effects of PM pollution have focused

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on PM10 and PM2.5, but the results were largely inconsistent. Hammoud et al. found

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a negative correlation between PM 2.5 values and sperm motility recorded 2 and 3

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months later.8 Radwan et al. found that air pollutants PM10 and PM2.5 were

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positively associated with abnormal morphology, and negatively associated with the

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level of testosterone.28 A newly study conducted in China and found PM2.5 and PM10

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exposures during the 0 to 90 days period of semen examination were negatively

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associated with sperm concentration.12 On the contrary, there are two studies shown

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no significant associations between PM and semen quality. Hansen et al.

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demonstrated that PM2.5 exposure was not associated with sperm outcomes.7 Sokol et

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al. studied the relationship between air pollutant levels and semen quality and found

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that exposure to environmental ozone instead of PM10 was associated with semen

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quality.29 The reason for these conflicting results may be due to racial or geographic

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differences and the differences in methods and study designs.

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In order to address the gaps in the literature, we established The Male

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Reproductive Health in Chongqing College Students (MARHCS) cohort. The aim of

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this study was to investigate the effects of air pollution exposure on male reproductive

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health.30 We examined the associations between long-term exposure to various

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particulate matter size fractions (PM10, PM10-2.5 and PM2.5) and semen quality and

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serum hormone levels in a prospective cohort of 796 young adult males. A

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longitudinal data with two extended follow-ups in our large cohort would be made the

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results more definitive. 5


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Methods

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Study population

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Due to the needs of fast development and the increasing number of students, many

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Chinese universities are facing a problem of campus space shortage. In order to solve

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the problem, local governments usually assigned a region for universities to build a

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new campus. Generally, these new campuses have distinct environment form old one.

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To Chongqing city, for instance, the old campus located in Shapingba district, the

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central area of Chongqing City, and suffered from severe air pollution. The new one

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however, located in a new district with better surrounding environment and far from

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the urban area. For the first two years of school, students usually stay in new campus

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and they will be relocation to the old campus when achieving higher grades.

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Therefore, we established The Male Reproductive Health in Chongqing College

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Students (MARHCS) cohort to investigate the effects of air pollution exposure change

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on male reproductive health, as described in details in previous publication.30

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This study was approved by the Ethics Committees of Third Military Medical

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University. All participants were informed of the purpose of the study and signed an

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informed consent form if they agreed to take part in this project. From June 8th 2013

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to June 21th 2013, the baseline was established within the male college students in the

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new campus of the university town. A total of 872 volunteers participated in this study

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and 796 eligible subjects finished all investigations. In the follow-up Ⅰ(June 5th

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2014 to June 16th 2014), subjects were recruited only from men who eligible in 2013 6



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baseline investigation. There were 130 subjects dropped out due to personal reasons.

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A total of 666 subjects attended the followed-up procedure, among which 10 failed to

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provide semen sample. Therefore, 656 (82.4%) of the 796 eligible subjects were

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followed up and finished all the procedures. In follow-up Ⅱ (May 23th 2015 to June

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7th 2015), subjects were recruited only from men who finished the baseline

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investigation and follow-up Ⅰ. Finally, there were 88 subjects dropped out due to

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personal reasons and 568 (71.3%) of the 796 eligible subjects completed the

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follow-upⅡ survey (Figure 1).

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Air pollution data and exposure assessment

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Air pollution data were obtained from the Institute of Environmental Science and

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Technology of Chongqing. Outdoor PM10 and PM2.5 concentrations were measured

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from 2 ambient air quality monitoring stations that belonged to the national air

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pollutant detection network. One monitoring station, Huxi is just located in the new

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campus of Chongqing University and the other (Gaojiahuayuan) is located beside the

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old campus (＜500 meters) of Chongqing university. The data for PM10 and PM2.5

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represented a 24h average and were recorded daily. From January 1, 2013 to

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December 31, 2015, a total of 1095 days of air pollution data were recorded. We

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obtained data on PM10-2.5 by subtracting the daily PM2.5 concentration from the

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daily PM10 concentration at each location.31 Data on daily ambient average

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temperature, humidity and wind speed in these two study sites were obtained from the

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Chongqing Climate Center.

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In addition, to analyze the association between the estimated air pollutants and 7


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semen quality outcomes, we measured PM concentrations at Huxi and Gaojiahuayuan

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monitoring stations during three investigation periods (June 8th 2013 to June 21th

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2013, June 5th 2014 to June 16th 2014 and May 23th 2015 to June 7th 2015). We

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employed average exposure data of the pollutants within an exposure period of 90

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days before undertaking semen sampling from each subject. Specifically the PM

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exposure in 90 days before semen collection, no daily PM exposure information was

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missing (100%).

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Measurement of semen/hormones outcomes

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The methods for semen collection and analyses have been described in detail

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previously.30 Briefly, the semen analyses were performed according to the

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recommendations of World Health Organization methodology.32 Participant was

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contributing a semen sample by masturbation after 2 to 7 days of abstinence. We

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assessed conventional semen parameters of semen volume, sperm concentration, total

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sperm number, progressive motility and normal morphology. Semen volume was

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measured by weighing; sperm concentration and motility were measured by

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computer-aided sperm analysis (SCA CASA System; Microptic S.L., Barcelona,

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Spain). For sperm morphology assessment, air-dried smear were fixed in 96% ethanol,

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then Diff-Quik stained and analyzed according to the WHO criteria. In order to reduce

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the variation of assessment of sperm characteristics, all the analyses of semen quality

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were performed by one technician who was well trained in semen analysis and

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participated in the Continuous Quality Control System under the supervision of the

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Chongqing Science and Technology Commission. 8



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The

detection

of

serum

reproductive

hormones

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levels

(estradiol,

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follicle-stimulating hormone, luteinizing hormone, prolactin, progesterone, and

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testosterone) were conducted at the clinical laboratory of Southwest Hospital

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(Chongqing, China) by using the Immunology Analyzer (Unicel Dxi 800, Beckman

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Coulter Inc., Brea, CA) and commercial kits. Details are available in previous

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publication.33

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Sperm chromatin structure assay

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Sperm DNA integrity was tested by sperm chromatin structural assay (SCSA), as

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described previously.34 A total of 2000 sperm cells had been calculated for each

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sample which analyzed by flow cytometry (Beckman FC 500 MCL/MPL, USA). The

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DNA fragmentation index (DFI) represents the percentage of spermatozoa within the

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sample with fragmented or damaged DNA was calculated from the ratio between the

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red and total (red plus green) fluorescence intensity. The high DNA stainability

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(HDS) was the percentage of spermatozoa with high DNA stainability, which

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represents the percentage of sperm within the sample with incomplete chromatin

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condensation. For the flow cytometer set-up and calibration, a normal human

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ejaculate sample was used as an internal standard reference. Aliquots reference

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samples were run in duplicate with an intra-assay average quality control coefficient

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of variation (CV) of 11.5%.

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Statistical analysis

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The basic characteristics of the study population were described using untransformed

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data. Variables were represented median (25th to 75th percentile) or n (percentage). 9


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Crude trends between air pollution exposure concentrations during 90 days before

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investigation were evaluated by Spearman’s rank correlations test.

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Firstly, we analyzed the associations between air PM pollutions and the sperm

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parameters and serum hormone levels during the 2013 baseline investigation by using

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multivariate linear regression analysis. For the parameters with skewed distribution,

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we performed transformations on these variables to better approximate the normality

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assumption of the model. They were natural-log transformed before the multi-variate

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analyses and the results were then back-transformed after their analyses to acquire the

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percentage of change and the 95% confidential interval (CI). Selection of covariates

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for the regression model was based on their importance in the literature and biological

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plausibility. A coefficient was thought to be a potential confounder if the regression

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coefficient changed by ＞10% when it was included one by one in the models.

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Finally, age, body mass index (BMI), abstinence time, tobacco smoking and alcohol

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consumption were included in the models as potential confounders.

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Secondly, the mixed-effects model was used to jointly investigate the association

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between PM and the semen parameters and reproductive hormones, with integration

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of the three years data and modeling of the longitudinal relationships among the

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baseline and the two follow-ups. To determine the robustness of our findings to

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cut-point selection, we examined PM exposures categorizations (1st: < 90 µg /m3, 2nd:

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90-100 µg /m3 and 3rd: > 100 µg /m3 for PM10, 1st: < 35 µg /m3, 2nd: 35-40 µg /m3 and

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3rd: > 40 µg /m3 for PM2.5- 10, and 1st: < 50 µg /m3, 2nd: 50-60 µg /m3 and 3rd: > 60

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µg /m3 for PM2.5, respectively) in mixed model. Multiplicity adjustment by a 10



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Bonferroni correction was conducted for the regression analyses by multiplying the

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crude P-value by the number of analyses. The statistical analyses were carried out by

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SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) and SAS version 9.1 (SAS Institute

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Inc., Cary, NC, USA).

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Results

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Ambient PM exposure and semen/hormone outcomes for the subjects

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The descriptive characteristics are summarized in Table 1. In baseline investigation of

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2013, there are a total of 796 eligible subjects whom finished all examinations. 656

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(82.4%) and 568 (71.3%) of the 796 eligible subjects were finished all the procedures

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in followed-up Ⅰ and followed-up Ⅱ , respectively. All of the participants were

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undergraduate students with average ages of 20.0, 21.0, and 22.0 years and mean

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BMIs of 20.9, 21.2, and 21.8, respectively. The predominant race of the subjects is

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Han (83.0%, 90.3%, and 88.9% for 2013, 2014 and 2015). In addition, there are

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characteristics regarding health behaviors like smoking, drinking, cola and fried food

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consumption are described in detail in previous publication.35

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Table 1 shows the PM exposure data for the 90-day period before sampling. The

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median ambient PM10 concentration was 86.0µg/m3, 99.5µg/m3 and 87.0µg/m3 in

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2013, 2014 and 2015, respectively. The median concentrations were 37.0µg/m3,

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29.0µg/m3 and 41.0µg/m3 for PM10-2.5 and 53.0µg/m3, 65.0µg/m3 and 44.0µg/m3 for

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PM2.5, respectively. For meteorological data, the average temperature for 90 days

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preceding the collection of semen samples were 26.0℃, 22.5℃ and 24.5℃ in 2013,

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2014 and 2015. The relative humidity was 75.0%, 78.0% and 73.0% and wind speed 11


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was 8.0Km/h, 7.0Km/h and 8.0Km/h in 2013, 2014 and 2015, respectively.

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For the distribution of three 90-day individual average exposures for PM10,

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PM10-2.5 and PM2.5, mean ambient PM10 concentration was 92.0µg/m3 (range:

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69.3-104.1), mean ambient PM10-2.5 concentration was 37.2µg/m3 (range: 23.9-44.9)

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and mean ambient PM2.5 concentration was 54.8µg/m3 (range: 42.0-68.7) (data not

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shown).

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Correlations between PM concentrations and male reproductive parameters

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As shown in Figure 2, from 2013 to 2015, for the 90-day period before investigation,

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the air pollution concentrations of PM10-2.5 and PM10 were highly positively

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correlated (r = 0.905, 0.809, 0.867, P

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