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The epidemiologic evidence linking prenatal and postnatal exposure to endocrine disrupting chemicals with male reproductive disorders: a systematic re ...

The epidemiologic evidence linking prenatal and postnatal exposure to endocrine disrupting... Human Reproduction Update, Vol.23, No.1 pp. 104–125, 2017 Advanced Access publication on September 21, 2016 doi:10.1093/humupd/dmw036 The epidemiologic evidence linking prenatal and postnatal exposure to endocrine disrupting chemicals with male reproductive disorders: a systematic review and meta-analysis 1,2, 1 3 Jens Peter Bonde *, Esben Meulengracht Flachs , Susie Rimborg , 1 4 Clara Helene Glazer , Aleksander Giwercman , 5 6 Cecilia Høst Ramlau-Hansen , Karin Sørig Hougaard , 1,7 1 Birgit Bjerre Høyer , Katia Keglberg Hærvig , 1 8 1 Sesilje Bondo Petersen , Lars Rylander , Ina Olmer Specht , 7 1,9 Gunnar Toft , and Elvira Vaclavik Bräuner 1 2 Department of Occupational and Environmental Medicine, Bispebjerg University Hospital, DK-2400 Copenhagen NV, Denmark Institute of Public Health, University of Copenhagen, DK-1016 Copenhagen K, Denmark The Royal Library/ University of Copenhagen Library, DK-2200 Copenhagen N, Denmark Molecular Reproductive Medicine, Department of Translational Medicine, Lund University, Lund, 5 6 Sweden Institute of Public Health, Aarhus University, DK-8000 Aarhus C, Denmark National Research Centre for the Working Environment, DK-2100 Copenhagen Ø, Denmark Department of Clinical Epidemiology, Aarhus University Hospital, DK-8200 Aarhus N, 8 9 Denmark Department of Occupational and Environmental Medicine, University of Lund, SE-221 85 Lund, Sweden Research Center for Prevention and Health (RCPH), University of Copenhagen, DK-2600 Glostrup, Denmark *Correspondence address. Department of Occupational and Environmental Medicine, Bispebjerg-Frederiksberg University Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Tel: +45-3043-8617; E-mail: jens.peter.ellekilde.bonde@regionh.dk Submitted on May 27, 2016; resubmitted on August 22, 2016; accepted on August 31, 2016 TABLE OF CONTENTS ........................................................................................................................... � Introduction � Methods Protocol and registration Information sources Eligibility criteria Search and study selection Data extraction Quality assessment Meta-analysis � Results The study base Global assessment: generic exposures and outcomes as one entity (Hypothesis 1) Specific exposures and outcomes as one entity (Hypothesis 2) Generic exposures and specific outcomes (Hypothesis 3) Specific exposures and specific outcomes (Hypothesis 4) Studies not eligible for meta-analysis © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permis- sions@oup.com Review on EDC and male reproduction 105 � Discussion Exposure assessment Exposure contrast within and across studies Outcome ascertainment The testicular dysgenesis syndrome hypothesis Studies addressing testicular cancer Studies addressing sperm count Methodological issues Cocktail effects Non-monotonic dose–response relationships Wider implications and conclusion BACKGROUND: More than 20 years ago, it was hypothesized that exposure to prenatal and early postnatal environmental xenobiotics with the potential to disrupt endogenous hormone signaling might be on the causal path to cryptorchidism, hypospadias, low sperm count and testicular cancer. Several consensus statements and narrative reviews in recent years have divided the scientific community and have elicited a call for systematic transparent reviews. We aimed to fill this gap in knowledge in the field of male reproductive disorders. OBJECTIVE AND RATIONALE: The aim of this study was to systematically synthesize published data on the risk of cryptorchidism, hypospadias, low sperm counts and testicular cancer following in utero or infant exposure to chemicals that have been included on the European Commission’s list of Category 1 endocrine disrupting chemicals defined as having documented adverse effects due to endocrine disruption in at least one intact organism. SEARCH METHODS: A systematic literature search for original peer reviewed papers was performed in the databases PubMed and Embase to identify epidemiological studies reporting associations between the outcomes of interest and exposures documented by bio- chemical analyses of biospecimens including maternal blood or urine, placenta or fat tissue as well as amnion fluid, cord blood or breast milk; this was followed by meta-analysis of quantitative data. OUTCOMES: The literature search resulted in 1314 references among which we identified 33 papers(28 study populations) fulfilling the eligibility criteria. These provided 85 risk estimates of links between persistent organic pollutants and rapidly metabolized compounds (phthalates and Bisphenol A) and male reproductive disorders. The overall odds ratio (OR) across all exposures and outcomes was 1.11 (95% CI 0.91–1.35). When assessing four specific chemical subgroups with sufficient data for meta-analysis for all outcomes, we found that exposure to one of the four compounds, p,p′-DDE, was related to an elevated risk: OR 1.35 (95% CI 1.04–1.74). The data did not indi- cate that this increased risk was driven by any specific disorder. WIDER IMPLICATIONS: The current epidemiological evidence is compatible with a small increased risk of male reproductive disorders following prenatal and postnatal exposure to some persistent environmental chemicals classified as endocrine disruptors but the evidence is limited. Future epidemiological studies may change the weight of the evidence in either direction. No evidence of distortion due to publi- cation bias was found, but exposure–response relationships are not evident. There are insufficient data on rapidly metabolized endocrine disruptors and on specific exposure–outcome relations. A particular data gap is evident with respect to delayed effects on semen quality and testicular cancer. Although high quality epidemiological studies are still sparse, future systematic and transparent reviews may provide pieces of evidence contributing to the narrative and weight of the evidence assessments in the field. Key words: cryptorchidism / endocrine disruption / hypospadias / infertility / prenatal exposure / sperm count / testicular cancer / xenobiotics gonadotrophins leading to disruption of normal development of the Introduction male fetal gonad during the late phase of the first trimester of pregnancy As early as 1979, Henderson et al. hypothesized that a relative excess (Sharpe and Skakkebaek, 1993). This so-called estrogen hypothesis has of estrogen, in particular at the time of testicle differentiation, is a major been scrutinized by a large number of studies addressing the risk of risk factor for testicular cancer (Henderson et al.,1979). Fourteen years male reproductive disorders in boys who, during pregnancy, were later, Sharpe and Skakkebaek proposed that not only testicular cancer exposed to high or low levels of estrogens because of twin pregnancy, but also other male reproductive disorders such as cryptorchidism, first parity, preeclampsia, hyperemesis or intended or incidental treat- hypospadias and low sperm counts may share a common fetal origin ment with synthetic hormones during first trimester. Three literature and that environmental exposure to chemicals with actions mimicking reviews provide no support for the estrogen hypothesis except that estrogens might play a pivotal role (Sharpe and Skakkebaek, 1993). The testicular cancer seems related to high prenatal estrogen exposure underlying mechanisms were thought to include increased negative (Sever et al.,1997; Storgaard et al., 2006; Martin et al.,2008). In par- feedback on the fetal pituitary resulting in reduced levels of ticular, it is notable that prenatal exposure to the highly potent synthetic 106 Bonde et al. estrogen diethylstilbestrol is only associated with low sperm count at and there is presently no consensus. In 2012, a Global Assessment of high doses (Gill et al.,1979; Leary et al.,1984). Subsequently a cap- the State-of-the-Science of Endocrine Disrupters commissioned by the acious amount of experimental research has indicated that adverse World Health Organization and United Nations Environment Program health effects, including male reproductive disorders, may be related to concluded that the direct evidence for endocrine disrupting effects with endocrine disruption of fetal development through xenobiotic inter- adverse health effects because of exposure to xenobiotic substances is action with steroid hormone receptors or through interference with limited in humans, but emphasized the need for continuous attention the synthesis, secretion, transport, metabolism or degradation of nat- (European workshop on the impact of endocrine disrupters on human ural hormones (European workshop on the impact of endocrine disrup- health and wildlife. Report of proceedings from a workshop held in ters on human health and wildlife. Report of proceedings from a Weybridge, UK, 2–4 December 1996; World Health Organisation workshop held in Weybridge, UK, 2–4 December 1996; World Health (WHO) and United Nations Environment Program (UNEP), 2012). As Organisation (WHO) and United Nations Environment Program several epidemiological studies addressing this issue have been pub- (UNEP), 2012). This broader endocrine disruption hypothesis with lished over the past 10 years, a systematic evaluation of the epidemio- focus on environmental pollutants has gained tremendous public and logical evidence on effects of environmental endocrine disrupting scientific attention in the USA and the European Union over the past xenobiotics including quantitative meta-analyses of data, where feasible, 15 years. Numerous narrative reviews and consensus statements have has now become timely (Beronius and Vandenberg, 2015). discussed supporting and contradicting evidence based on data of secu- Therefore, the objective of this review was to identify and evaluate lar trends of male reproductive health outcomes, wildlife impact and the epidemiological evidence linking adverse reproductive health disor- exposure levels in various populations, in addition to the large body of ders with in utero or early postnatal exposure to xenobiotic endocrine in vitro and in vivo experimental data (European workshop on the impact disrupting compounds. The key eligibility criteria are documentation of of endocrine disrupters on human health and wildlife. Report of pro- exposure contrast by chemical measurements of the compounds in ceedings from a workshop held in Weybridge, UK, 2–4December maternal blood or other biospecimens indicative of fetal or infant expos- 1996; Bergman et al.,2013; Cantonwine et al.,2014; Diamanti- ure. Specifically, we address the following four hypotheses on the risk of Kandarakis et al., 2009; Lamb et al.,2014; Nohynek et al.,2013; adverse male reproductive outcomes following prenatal and postnatal Sharpe, 2001; Sharpe and Irvine, 2004; Skakkebaek et al., 2001; environmental exposure to industrial chemicals classified as endocrine Takahashi et al.,2004; Toppari et al., 2010; Vandenberg et al.,2012; disrupting substances (McCarthy, 2011): (i) that all compounds regard- World Health Organisation (WHO) and International Programme on less of specific endocrine disrupting properties carry a risk of reproduct- Chemical Safety (IPCS), 2002; World Health Organisation (WHO) and ive disorders considered as one outcome entity; (ii) that specific United Nations Environment Program (UNEP), 2012). It is particularly compounds are heterogeneous with respect to reproductive disorders noteworthy that biomonitoring data from all over the world provide considered as one entity; (iii) that all compounds regardless of specific unequivocal documentation of human exposure to a range of xeno- endocrine disrupting properties carry a risk for specific outcomes; and biotic substances including persistent compounds (Govarts et al.,2012; (iv) that specific compounds carry a risk for specificoutcomes. Lenters et al., 2013; Gyalpo et al., 2016; Hung et al., 2016; Liu et al., 2016; Magulova and Priceputu, 2016) as well as rapidly metabolized Methods and excreted compounds that reach steady state concentrations in human blood and tissues because of continuous exposure (Arbuckle The review was conducted and reported in accordance with the MOOSE et al., 2016; Zhang et al.,2016). These xenobiotic compounds have guidelines for Meta-analyses and Systematic reviews of Observational been released into the environment intentionally via the widespread Studies (Stroup et al., 2000). application of pesticides or unintentionally by either degradation of industrial products and building materials or by leakage of packing mate- Protocol and registration rials into food items (Nam et al.,2010). New exposure pathways are A review protocol was registered at PROSPERO.org with registration described regularly. For instance, it has recently been reported that number CRD4201603742 prior to initiation of the review process, on 12 semi-volatile polychlorinated biphenyls (PCBs) represent an important April 2016 with amendments on 12 May 2016 (CRD420160374X). The inhalation exposure pathway for residents living in PCB contaminated amendments specified the four main hypotheses, provided details on sen- homes containing PCB in calking material, paints and sealant used >40 sitivity analyses and discarded the initial idea to analyze data according to years ago (Frederiksen et al.,2012; Meyer et al., 2013). Exposure by estrogenic or anti-androgenic activity of measured compounds which inhalation and possibly through the skin may more than double the PCB proved non-feasible. The protocol was updated before the review pro- body burden in individuals exposed at the residence or at school cess and data analysis were initiated. (Meyer et al.,2013). Considering the mounting evidence that numerous of these environmental xenobiotics exhibit endocrine activity in in vitro and in vivo assays, there is basis for substantial concern regarding the Information sources potential health effects in humans (Gabrielsen and Tanrikut, 2016; The databases PubMed and Embase were used as they cover the vast McLachlan, 2016; Trasande et al.,2016), even if alarming reports on, majority of relevant journals for the subject. for instance, declining sperm counts are circumstantial or not corrobo- rated (Bonde et al., 2011) and the best evidence we have is reassuring Eligibility criteria (Jorgensen et al.,2012). However, the epidemiological data on the adverse health effects are We conducted a systematic search of original peer-reviewed original sparse, particularly regarding links with prenatal and postnatal exposure, papers in English published between 1966 and 12 April 2016 to identify Review on EDC and male reproduction 107 journal articles providing quantitative data on the association between specimens (maternal blood or urine, placenta or fat tissue as well as xenobiotic endocrine disrupting chemicals and male reproductive disor- amnion fluid, cord blood or breast milk). In addition serum concen- ders in humans. The complete search specification is provided in the trations in adult life as proxies for fetal or early life exposure were online Supplementary Table 1. Eligibility criteria for inclusion in the sys- also included for testicular cancer. tematic review were as follows. (4) Outcome ascertainment by medical standardized examination or antecedent medical records or reporting to health registries. (1) Exposures: Chemicals that by the European Commission are classified (5) Risk estimates (rate risk [RR], OR, hazard ratio [HR]) for an outcome as Category 1 endocrine disruptors with high concern in terms of according to higher versus lower levels of prenatal and postnatal human or wildlife exposure (McCarthy, 2011). Category 1 compounds exposure defined by exposure contrasts within the given study. are defined as chemicals where at least one study has shown endocrine Studies reporting alternative measures of association such as differ- effects in an intact organism. The EU report lists 60 Category 1 sub- ence in mean values of exposure levels in cases and controls were stances (29 chemical groups) that are highly persistent or have high also included, and the authors were contacted to get risk estimates current production volume (McCarthy, 2011). In addition we include (Hosie et al., 2000; Damgaard et al., 2006; Main et al., 2007; the polyfluorinated chemicals, a group of ubiquitous biopersistent Choi et al., 2012; Fenichel et al., 2012; Komarowska et al., 2015; emerging endocrine disruptors (White et al., 2011) that are receiving Virtanen et al., 2012). Several authors responded positively but increasing attention. Table I lists the specific chemical substances with reanalysis of the original data could not be accomplished within the abbreviations that are referenced in the text and in Tables II–VII. given time-frame and the studies were therefore not included in the (2) Outcomes: Cryptorchidism (one or both testicles undescended) present review. ascertained at birth or during childhood, hypospadias, testicular can- Criteria for exclusion of studies were as follows: cer regardless of histological subgroup, and sperm count (number of spermatozoa per volume or mass unit of seminal fluid). (1) In vitro and in vivo experimental studies. (3) Exposure to specific chemicals in utero or in the first year of life (post- (2) Studies addressing mechanisms and other outcomes related to endo- natal exposure) documented by measurements in biological crine disruption, for example, effects on sexual hormone levels in tis- sues and measures of semen quality other than sperm concentration. (3) Studies based upon chemical analysis of exposure after puberty Table I List of specific chemical substances with except studies of testicular cancer. abbreviations. (4) Studies repeating risk estimates reported in previous publications for example, studies addressing risk according to gene polymorphisms Bisphenol A 4,4′-(Propan-2,2-diyl)diphenol for substances, where risk estimates for the entire population were mBP Mono-n-butyl-phthalate provided earlier. Chlordane cis-heptachlordane, cis-chlordane, ocychlordane, trans- (5) Ecological studies with exposure information at the population level mix nonachlordane, cis-nonachlordane rather than at the individual level. DBP Di-n-butyl-phthalate DDE 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene o,p′-DDT 1,1,1-Tricholoro-2-(p-chlororphenyl)-2-(o-chlorophenyl) Search and study selection ethane p,p′-DDT 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane We combined medical subject headings and generic terms for the expo- sures and outcomes (Supplementary Table 1) and obtained in total 1314 DEHP Di-(2-ethylhexyl)phthalate hits after removal of duplicates. Two authors (JPB and EVB) sifted titles Dieldrin Derivate of norbornadiene and and abstracts independently to assess eligibility and retrieved 110 papers hexachlorocyclopentadiene for full text reading. Among these, several reports failed to provide quan- HCB Hexachlorobenzene titative data on exposure levels and others were excluded for other rea- HCE Heptachlor epoxide sons detailed in Supplementary Table 2. Hand searches of the HCCH b-Hexachlorocyclohexane bibliographies of retrieved primary reports and reviews did not capture MEHP Mono-(2-ethylhexyl)phthalate additional papers. We selected 19 epidemiological studies (21 papers) that provided at 5cx-MEPP Di(2-ethylhexyl)phthalate [metabolite of DEHP] least one risk estimate for a male reproductive disorder according to at 7cx- Mono(4-ethyl-7-carboxylheptyl)phthalate [metabolite of least one measured xenobiotic classified as an endocrine disruptor. MMeHP DiNP] Moreover, nine studies (12 papers) that compared average exposure Mirex Dimerization derivate of hexachlorocyclopentadiene levels in cases and referents without providing risk estimates were n-NP n-Nonylphenol included. Thus, the final database comprised 33 papers included for t-OP t-Octylphenol qualitative systematic analysis and 21 papers included for quantitative PBB Polybrominated biphenyl meta-analysis. No attempt was made to retrieve papers from the unpublished literature. The steps in the literature search are displayed PBDE Polybrominated diphenyl ether in Fig. 1. PCB Polychlorinated biphenyl PCDD/Fs Polychlorinated dibenzo-p-dioxins/furans PFOS Perfluorooctane sulfonic acid PFOA Perfluorooctanoic acid Data extraction TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin Descriptive information (Tables II–V) was recorded from each publication using a standardized form constructed prior to the collection process. 108 Bonde et al. Table II Characteristics and risk estimates for 10 case-referent studies (18 risk estimates) addressing the risk of cryptorchidism following prenatal and postnatal exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure medium Exposure contrast Substance OR 95% CI CR Bias referents .......................................................................................................................................................................................................................................................... Longnecker et al. USA (12 centers) Collaborative Perinatal Project 219/552 Third trimester serum Highest versus lowest quintiles p,p′-DDE 1.30 0.70–2.24 9 0 (2002) Pierik et al. (2007) USA (12 centers) Collaborative Perinatal Project 219/564 Third trimester serum 75–90 percentile versus 0–10 b-HCCH 2.08 1.08–4.01 9 0 percentile HCE 0.82 0.41–1.65 HCB 0.85 0.52–1.40 McGlynn et al. USA (12 centers) Collaborative Perinatal Project 230/593 Third trimester serum Highest quartile versus lowest PCB (sum of 11 1.41 0.90–2.20 8 0 (2009a) congeners) Trabert et al. (2012) USA (12 centers) Collaborative Perinatal Project 217/557 Third trimester serum Highest quartile versus lowest trans-nonachlor 1.22 0.70–2.12 9 0 Oxychlordane 0.95 0.55–1.64 Bhatia et al. (2005) USA, San Francisco Child Health and Development 75/283 Maternal serum Highest versus lowest quartile p,p′- DDT 1.01 0.44–2.28 9 1 Study p,p′- DDE 1.34 0.51–3.48 Brucker-Davis et al. France, Nice Newborns at maternity clinics in 56/69 Colostrum Highest versus lowest. Three p,p′-DDE 2.16 0.94–4.98 7 1 (2008) Nice and Grasse. categories PCB (seven 2.74 1.15–6.53 congeners) mBP 2.13 0.66–6.83 Small et al. (2009) USA, Michigan Michigan Long-term PBB Study 9/464 Maternal serum Highest versus lowest (<LOD). PBB-153 (Includes 0.50 0.10–4.70 4 1 Three categories hypospadias) Vesterholm et al. Denmark and Finland Danish–Finnish prospective Birth 107/108 Cord blood Highest tertile versus lowest PFOS 0.83 0.39–1.79 9 0 (2014) Cohort Study PFOA 0.46 0.20–1.20 9 0 Jensen et al. (2015) Denmark Pregnancy screening registry, 270/300 Second trimester Highest tertile versus lowest 5cx-MEPP 0.90 0.57–1.41 9 1 Serum Institute amnion fluid 7cx-MMeHP 1.28 0.80–2.01 Toft et al. (2016) Denmark Pregnancy screening registry, 270/300 Second trimester Highest tertile versus lowest PFOS 1.01 0.66–1.53 9 1 Serum Institute amnion fluid CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. Review on EDC and male reproduction 109 Table III Characteristics of 10 case-referent studies (21 risk estimates) addressing the risk of hypospadias following prenatal and postnatal exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure Exposure contrast Substance OR 95% CI Meta- CR Bias Referents medium analysis .......................................................................................................................................................................................................................................................... Longnecker et al. (2002) USA (12 Collaborative Perinatal Project 199/552 Third trimester Highest versus lowest quintiles p,p′-DDE 1.2 0.60–2.40 Yes 8 0 centers) serum McGlynn et al. (2009a) USA (12 Collaborative Perinatal Project 201/593 Third trimester Highest versus lowest quartile PCB (sum of 11 1.69 1.06–2.68 Yes 8 1 centers) serum congeners) Trabert et al. (2012) USA (12 Collaborative Perinatal Project 197/557 Third trimester Highest quartile versus lowest trans-nonachlor 1.08 0.62–1.89 Yes 9 0 centers) serum Oxychlordane 1.24 0.69–2.22 Yes 9 Bhatia et al. (2005) USA, San Child Health and Development 66/283 Maternal serum Highest versus lowest quartile p,p′-DDT 0.79 0.33–1.89 Yes 9 0 Francisco Study p,p′-DDE 1.18 0.46–3.02 Yes 9 0 Small et al. (2009) USA, Michigan Long-term PBB Study 14/431 Maternal serum Highest versus lowest (<LOD). PBB-153 (includes 0.71 0.1–3.8 Yes 4 1 Michigan 3 categories cryptorchidism) Carmichael et al. (2010) USA, Pregnant women participating 20/28 Maternal serum OR for a 1–10 ng/g lipid PBDE-100 1.02 0.74–1.35 Yes 5 1 California in a screening program change in the analyte PCB-153 0.84 0.30–2.46 Yes 5 1 HCB 0.95 0.79–1.13 Yes 5 1 p,p′-DDT 1 0.99–1.01 No 5 1 p,p′-DDE 1 0.95–1.00 No 5 1 Giordano et al. (2010) Italy, Rome Children enrolled at two 37/21 Mid pregnancy Above versus below median p,p′-DDE 1.18 0.47–6.91 Yes 5 1 Hospitals serum HCB 5.5 1.25–24.31 Yes 5 1 PCB (four congeners) 1.89 0.51–6.93 Yes 5 1 Rignell-Hydbom et al. Sweden Southen Sweden maternity 237/237 Early pregnancy Highest quartile versus lowest PCB-153 0.60 0.30–1.19 Yes 10 0 (2012) cohort serum p,p′-DDE 1.68 0.92–3.08 Yes 10 0 HCB 1.59 0.86–2.93 Yes 10 0 Jensen et al. (2015) Denmark Pregnancy screening registry, 75/300 Second trimester Highest tertile versus lowest 5cx-MEPP 0.89 0.44–1.80 Yes 9 1 Serum Institute amnion fluid 7cx-MMeHP 1.69 0.78–3.67 Yes 9 1 Toft et al. (2016) Denmark Pregnancy screening registry, 75/300 Second trimester Highest tertile versus lowest PFOS 0.69 0.35–1.38 Yes 9 1 Serum Institute amnion fluid CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 110 Bonde et al. Table IV Characteristics of six case-referent studies (36 risk estimates) addressing the risk of testicular cancer following exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure medium Exposure contrast Substance RR 95% CI CR Bias referents .......................................................................................................................................................................................................................................................... Hardell et al. Sweden Patients referred to hospital 58/61 Blood, diagnosed men Above versus below PCB (sum of 38 1.10 0.50–2.60 9 1 (2003) departments in 5 Swedish median in referents congeners) cities HCB 1.70 0.80–3.60 9 1 p,p′-DDE 1.70 0.80–3.70 9 1 Chlordane 1.30 0.60–2.80 9 1 44/45 Blood, mothers at time of sons diagnosis Above versus below PCB (sum of 38 3.80 1.40–10.0 9 1 median in referents congeners) HCB 4.40 1.70–12.0 9 1 p,p′-DDE 1.30 0.50–3.00 9 1 Chlordane 1.90 0.70–5.00 9 1 Hardell et al. Sweden Patients referred to hospital 44/45 Blood, mothers at time of sons diagnosis Above versus below PBDE 47, 99, 153 2.50 1.02–6.00 9 1 (2006) departments in 5 Swedish median in referents cities Biggs et al. USA, The general population 246/630 Blood, men when diagnosed Above 85th percentile p,p′-DDT 1.17 0.68–2.00 9 1 (2008) Washing- versus below 50th o,p′-DDT 1.30 0.67–2.53 9 1 ton State percentile in referents Chlordane (six 0.93 0.51–1.68 9 1 substances) HCB 0.85 0.37–1.96 9 1 Mirex 0.87 0.50–1.53 9 1 Dieldrin 0.79 0.44–1.41 9 1 DDE 0.61 0.32–1.14 9 1 McGlynn USA US servicemen who had 739/915 Serum, adult males (bio-banked, (mean storage Highest quartile versus DDT 1.13 0.71–1.82 11 0 et al. (2008) donated serum between 1987 time 14.2 years)) lowest DDE 1.71 1.23–2.38 11 0 and 2002 Chlordane 1.51 1.09–2.10 11 0 Mirex 1.24 0.90–1.74 11 0 HCCH 0.90 0.65–1.24 11 0 McGlynn USA US servicemen who had 736/913 Serum, adult males (bio-banked, (mean storage Highest quartile versus PCB (sum of 11 0.61 0.43–0.86 11 0 et al. donated serum between 1987 time 14.2 years)) lowest congeners) (2009b) and 2002 Purdue et al. Norway Citizens with serum stored in 49/51 Serum, men (pre-diagnostic serum samples Highest tertile versus o,p′-DDT 1.40 0.40–4.50 7 1 (2009) the Norwegian cancer from between 1972 and 1978 in the Norwegian lowest p,p′-DDT 2.10 0.60–7.20 7 1 Registry Janus Serum Bank) p,p′-DDE 2.20 0.70–6.50 7 1 Chlordane 2.30 0.60–7.20 7 1 Total PCB 1.30 0.50–3.80 7 1 Dieldrin 2.10 0.50–9.50 7 1 HCB 2.90 0.50–15.20 7 1 β-HCCH 1.80 0.50–6.10 7 1 λ-HCCH 1.10 0.20–5.00 7 1 Mirex 1.20 0.40–3.00 7 1 Cohn et al. The Child Health and 15/45 Early postpartum maternal serum p,p′-DDT 0.70 0.26–1.64 8 1 (2010) Development Studies p,p′-DDE 0.19 0.04–0.62 8 Review on EDC and male reproduction 111 The standardized data extraction form is provided in Supplementary Table 3. Risk estimates with 95% CI were extracted for each measured compound and outcome. Sperm concentration (sperm count) was taken as the most relevant and robust measure of semen quality despite the substantial within- and between- individual variation (Schrader et al., 1988), while other indicators such as motility and morphology were ignored. When risk estimates were available for groups of chemicals, for example, PCB and PBDE congeners and phthalate metabolites, we used the summed risk estimate, if provided. Otherwise, we selected indicator congeners such as PCB-153 and PBDE-100 reflecting levels of all respect- ive congeners. When risk according to several levels of exposure was reported, the highest level versus the reference category was chosen. In one study, the highest level was greater than the 90th percentile (Pierik et al., 2004); therefore, we used the next highest exposure category in that study to allow for theoretical inverse relationships. If the relevant relative risks were not reported but data were available, the authors of this paper computed risk estimates and CIs. Quality assessment Reporting Each publication was evaluated for completeness of reporting of the fol- lowing 11 study characteristics modified after Bonzini et al., (2007): (i) study design, (ii) sampling frame and procedures, (iii) inclusion and exclu- sion criteria, (iv) population characteristics of exposed/unexposed or cases/referents, (v) response rates reported or given implicitly, (vi) meth- ods for exposure measurements (reference to method for chemical ana- lysis or detailed description), (vii) criteria for outcome ascertainment, (viii) external quality assurance program of biochemical analyses (certified laboratory and/or participating in analyses of spiked samples from other laboratories), (ix) detection level, recovery and precision (CV) provided on all issues, (x) statistical analysis, and (xi) reporting of exposure– response relationship (Supplementary Table 3). We evaluated whether each of these study characteristics were described or not and assigned a value of one if the criterion was fulfilled and zero if not. Giving equal weight to each of the 11 items, we consid- ered completeness of reporting as sufficient if the sum of the 0/1 scores for each paper was ≥8(Bonzini et al., 2007). Completeness of reporting is not a direct measure of quality but high completeness is needed for adequate evaluation of bias and confounding. Bias and confounding The decision to limit the review to observational epidemiological studies applying biological monitoring for exposure assessment is considered of major importance to counteract biased findings because of differential recall of exposure often introduced by self-reports or interview informa- tion. For each study, other sources of bias were evaluated against a pre- defined list of seven potential sources of bias and confounding adapted from validated checklists in order to fit the type of studies and research questions addressed in this review (Shamliyan et al., 2010); these are as follows: (1) Reporting of tested hypotheses: Many studies perform multiple comparisons because numerous chemical compounds have been measured. A high risk of bias towards inflated risk estimates is likely if risk estimates are selectively reported compared to objectives or available data. (2) Sample size justification (power calculations and/or addressing sam- ple size in discussion): Small numbers of cases and/or exposed may increase the risk of false negative reporting and thus cause bias towards the null. USA, Continuous contrasting o,p′-DDT 0.77 0.37–1.33 North 25th–75th interquartile California range in controls Giannandrea Italy, Hospital cases and controls 50/48 Serum, extrapolated backwards from male adult Above versus below LOD Sum of p,p′-DDE 3.34 1.09–10.2 7 1 et al. (2011) Rome undergoing evaluation for values (0.2 ng/mL) and HCB fertility status CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 112 Bonde et al. Table V Studies reporting associations between prenatal and postnatal exposure and cryptorchidism or hypospadias without risk estimates (not eligible for meta-analysis). a b Reference Location Study Study N Outcome Bio- Measured Results CR Bias population design population specimen xenobiotics .......................................................................................................................................................................................................................................................... Hosie et al. Germany Chilldren Cross- 18/30 Cryptochidism Fat 26 persistent Median values of 21 among 26 chemicals were higher in cases 31 (2000) undergoing sectional samples organochlorine and two compunds reached statistical significance (HCB and surgery at one pesticides and PCBs. HCE) hospital Damgaard Denmark/ Danish–Finnish Case- 62/68 Cryptochidism Breast milk 27 biopersistent Median values of 14 among 27 chemicals were higher in cases. 90 et al. (2006) Finland prospective Birth referent POPs No single compound significantly elevated. Monte Carlo Cohort Study analysis of median values indicated higher levels in cases when the eight compounds with the highest was analysed (P = 0.03) Main et al. Denmark/ Danish–Finnish Case- 95/185 Cryptochidism Placenta 14 PBDEs Placenta: no difference between cases and controls for 90 (2007) Finland prospective Birth referent placenta tissue, individual congeners or sum of 14 congeners Cohort Study samples, 62/ breast milk Breast milk: no difference between cases and controls for sum 68 milk of 14 PBDE congeners, while seven individual congenres were samples significantly higher in cases Virtanen et al. Denmark/ Danish–Finnish Case- 95/185 Cryptochidism Serum 17 TCDDs and No significant difference between cases and controls with 90 (2012) Finland prospective Birth referent placenta PCDD⁄ Fs) and 37 respect to sum of 37 PCBs, PCDD or dioxin equivalents Cohort Study samples, PCBs Shekharyadav Northern Children with Case- 80/120 Hypospadias Serum Three HCH isomers, Higher serum concentrations of all nine pesticides in cases, b- 51 et al. (2011) India hypospadias and referent aldrin, dieldrin, endo hexachlorohexane (HCH), g-HCH, and p,p′-dichlorodiphenyl age-matched I, endo II, p,p′-DDT, -dichloroethylene (p,p′-DDE) significantly higher control children p,p,-DDE Choi et al. Republic No data Cross- 80/80 Hypospadias Urine DEHP, DBP, MEHP, In urine five of eight substances higher in cases, in serum three 21 (2012) of Chorea sectional Serum MBP, PA, n-NP and substances higher, inconsistent with respect to DEHP t-OP (contamination?) Fenichel et al. Southern French studies on Case- 46/106 Cryptochidism Cord Bisphenol A Mean unconjugated Bisphenol A marginally higher in cases but 61 (2012) France cryptorchidism in referent blood not significant (P = 0.38) Nice and Grasse Komarowska Poland Children 0–4 Case- 98/57 Cryptochidism Serum Bisphenol A Free serum BPA level in cryptorchid boys and in the control 31 et al. (2015) years admitted to referent group was not statistically significant but the conjugated and paediatric total serumBPA level was elevated in cryptorchid boys department CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. Review on EDC and male reproduction 113 (3) Selection bias due to attrition in follow-up studies and non-response in case-referent studies: Attrition/non-response >20% or attrition/ non-response differing by >10% in exposed/cases and unexposed/ referents was considered as a potential cause of bias in an unpredict- able direction. (4) Information bias (outcome ascertainment): Outcomes identified by patient recall in questionnaires, by interview or medical examination not blinded towards exposure status is a likely cause of inflated risk estimates because of differential exposure misclassification. (5) Confounding: Failure to account by study design or analysis for major potentially confounding factors including maternal age, parity, prema- turity, social class and ethnicity was considered as high risk of bias in unpredictable directions. Other potentially confounding factors were considered on an outcome specific basis: Cryptorchidism is a strong risk factor for testis cancer and low sperm count, sexual abstinence and maternal smoking during pregnancy is related to sperm count and the sons age is related to risk of testis cancer. Weak and/or uncertain risk factors such as paternal age, maternal consumption of coffee and alcoholic beverages, infections, malformed uterus and poorly controlled diabetes were not considered. (6) Measuring of confounding factors: Potential bias in an unpredictable direction was considered if measures of confounding factors were invalid or inadequate. (7) Exposure contrast: Studies contrasting exposure levels by the median split rather than by at least exposure-tertiles were considered at risk of bias towards the null because of insufficient exposure contrast. We considered selective reporting related to multiple testing (1), selection bias (3), information bias related to outcome ascertainment (4) and failure to account for major confounding factors (5) as the most important threats to validity. Accordingly, we categorized a study as at higher risk of bias if two or more of these sources of bias were present. Risk of bias across studies was evaluated by means of funnel plots to assess risk of publication bias. Meta-analysis We applied a stepwise approach addressing four different scenarios for the association between chemical exposure and included outcomes with increasing level of specificity. Only cryptorchidism, hypospadias and tes- ticular cancer were included in Steps (1)–(4) below, as none of the four papers addressing sperm count provided risk estimates. The following associations were analysed: (1) The association between all types of endocrine disrupting chemicals with all outcomes assuming that effects occur regardless of specific mechanisms and that the four outcomes are manifestations of the same underlying condition (testicular dysgenesis syndrome; Skakkebaek, 2002). This analysis assumes that all types of examined individual compounds are proxies for mixtures of substances that car- ry the same risk for the adverse reproductive outcomes. This assumption is very unlikely to be true, but the analysis is informative when considering global statements on the impact of endocrine dis- ruptors that do not single out individual substances or mixtures of particular concern (Bergman et al., 2013). (2) The associations between specific chemicals and all outcomes. Hereby, we account for different endocrine disrupting actions and potencies exhibited by different compounds and address the tes- ticular dysgenesis hypothesis stating that all male reproductive out- comes at least partly are manifestations of the same underlying causal agents operating in early life (Skakkebaek et al., 2001; Table VI Studies reporting associations between prenatal exposure and sperm count without risk estimates (not eligible for meta-analysis). a b Reference Location Study population Study N population Biospecimen Measured Results CR Bias design xenobiotics .......................................................................................................................................................................................................................................................... Mocarelli Italy, Exposed men are sons of women living near the Mixed 39 exposed /58 Maternal serum in Serum Average sperm counts were almost 81 et al. (2011) Seveso Seveso chemical plant at the time of the accident design referents exposed men. Referents samples halved in the exposed group and the in 1976. Controls were blood donors whose had an assumed TCDD effect was most pronounced among mothers were not exposed exposure of 10 ppt breast fed men Vested et al. Denmark Pregnancy cohort, male offspring Follow-up 169 Maternal serum PFOS PFOA but not PFOS was associated 11 0 (2013) with lower adjusted sperm concentration (p-trend = 0.01) PFOA Vested et al. Denmark Pregnancy cohort, male offspring Follow-up 173 Maternal serum PCB (six Sum PCB and p,p′-DDE was not 11 0 (2014) congeners) associated with sperm count DDE Axelsson Sweden Mainly young men presenting for military health Follow-up 112 Maternal serum Three DEHP None of the six phthalate 71 et al. (2015) board but also a few from the general population metabolites metabolites were associated with (through announcements in schools) Three DiNP Sperm count metabolites CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 114 Bonde et al. Table VII ORs (95% CIs) of the summary estimate of analyses for associations between endocrine disrupting chemicals and cryptorchidism, hypospadias and testicular cancer. Strata with three or less risk estimates not eligible for meta- analysis. I only available for meta-analyses without bootstrapping (one risk estimate per study). Exposures Outcomes N studies N risk estimates OR 95% CI I (min-max per study) ............................................................................................................................................................................................. All exposures All outcomes 19 70 (1–10) 1.11 0.91–1.35 Cryptorchidism 6 16 (1–7) 1.03 0.72–1.47 Hypospadias 7 18 (1–4) 1.13 0.86–1.50 Testicular cancer 6 36 (2–10) 1.20 0.78–1.89 Bias less likely All outcomes 5 22 (2–7) 1.12 0.81–1.57 Large studies All outcomes 9 34 (1–7) 1.06 0.85–1.33 Excluding studies c. testis studies with adult exposure data All outcomes 13 34 (1–7) 1.07 0.87–1.32 + Biopersistence All outcomes 17 66 (1–10) 1.15 0.92–1.44 − Biopersistence All outcomes 3 4 (1–2) 1.12 0.71–1.77 DDE All outcomes 13 13 (1) 1.35 1.04–1.74 31.91 Cryptorchidism 3 3 (1) Hypospadias 4 4 (1) 1.38 0.93–2.04 0.00 Testicular cancer 6 6 (1) 1.19 0.60–2.36 78.05 DDT All outcomes 6 6 (1) 1.07 0.81–1.42 0.00 Cryptorchidism 1 1 (1) Hypospadias 1 1 (1) Testicular cancer 4 4 (1) 1.12 0.82–1.55 0.00 PCB All outcomes 9 9 (1) 1.26 0.82–1.96 62.41 Cryptorchidism 2 2 (1) Hypospadias 4 4 (1) 1.11 0.61–2.01 54.45 Testicular cancer 3 3 (1) HCB All outcomes 7 7 (1) 1.19 0.87–1.62 40.94 Cryptorchidism 1 1 (1) Hypospadias 3 3 (1) Testicular cancer 3 3 (1) Skakkebaek, 2003, 2004). One study reported multiple PCB and studies with high completeness of reporting and lower risk of bias and PBDE congener specific risk estimates but no summary estimate confounding as defined above; and (v) performed separate analyses of (Carmichael et al., 2010). For this study, we selected the PCB-153 large case-referent studies with at least 75 cases. and PBDE-100 as indicators of the total exposure to these com- We computed a common risk estimate across studies by weighing pounds and did not include the other congener specificestimates the risk ratio (RR) or equivalent (OR or HR) by the inverse variance (12 estimates). Another study reported the p,p′-DDE exposure computed from the mean of the provided confidence limits. This is justi- estimate as well as an estimate for the sum of HCB and p,p′-DDE fied becausethe outcomes arerare(prevalence <5%). Random effects but no separate estimate for HCB (too few cases). In this case, we estimates are presented regardless of tests for heterogeneity since all used the sum of the p,p′-DDE and HCB estimate (Giannandrea studies are conceptually heterogeneous. Nevertheless we also provide et al.,2011). I statistics as a formal measure of heterogeneity. Bootstrapping techni- (3) The associations between all exposures and specificout- ques were used in analyses where the same study yielded more than comes, assuming that endocrine disrupting chemicals in one risk estimate, to account for multiple risk estimates per person in general carry a risk for some but not other male reproductive studies where risk estimates were reported for several compounds or disorders. outcomes. Bootstrapping with 500 repeats was applied by sampling (4) Each of the three outcomes individually in relation to specific com- among all included risk estimates, ensuring that each study only contrib- pounds or group of compounds under the assumption of specific uted one risk estimate to each bootstrapping step. Mean values across mechanisms and specific outcomes, but without an a priori ranking of bootstrapped steps were used as overall measures of common risk esti- more or less likely specific hypotheses. These exploratory analyses mates and confidence limits. These analyses were repeated for each have a narrow scope because data are limited. We only included outcome to account for heterogeneity in the exposure–outcome exposure–outcome estimates in the meta-analysis if four or more relation. risk estimates were available. All statistical analyses were performed using R and a significance level of 0.05. We also used this software to create funnel plots of the standard In sensitivity analyses, we: (i) regrouped compounds into persistent or error by the logarithm of the RR separately for each of the exposures rapidly metabolised substances with half-lives in the range of hours; (ii) and for the higher quality studies, and inspected these plots for evidence excluded studies with blood sampling after the diagnosis of testicular can- of publication bias. cer; (iii) examined the effect of each study on the overall estimate by excluding the studies one at a time; (iv) performed separate analyses of Review on EDC and male reproduction 115 Records identified through database searching Records identified through hand Pubmed: 1,253; Embase: 220 searches (n = 0) Records after duplicates removed (n = 1,314) Records screened Records excluded (n =1,314) (n = 1,204) Ful-text articles assessed Full-text articles excluded, for eligibility with reasons (n = 110) (n = 77) Papersincluded in qualitative synthesis (n = 33) Papersincluded in quantitative synthesis (meta-analysis) (n = 21) Figure 1 Flow diagram showing results of a systematic literature search to identify peer review papers addressing risk of male reproductive disor- ders following prenatal and infancy exposure to environmental endocrine disrupting compounds. (Mocarelli et al., 2011; Axelsson et al., 2015; Vested et al., 2013, Results 2014). There were 12 papers, based upon nine different study popula- The study base tions, which compared exposure levels in cases and referents but did We identified 33 papers that were based upon 28 independent stud- not provide risk estimates. An overview of eligible studies and risk ies reported 89 associations between measured levels of endocrine estimates are given in Tables II–IV while the results of three studies disrupting chemicals and one or more of the outcomes: cryptorchid- (four papers) addressing sperm count and six studies (eight papers) ism (Hosie et al., 2000; Longnecker et al., 2002; Pierik et al., 2004, addressing cryptorchidism and hypospadias without risk estimates are 2007; Bhatia et al., 2005; Damgaard et al., 2006; Main et al., 2007; displayed in Tables V and VI. Most studies addressed persistent orga- Brucker-Davis et al., 2008; McGlynn et al., 2009a; Fenichel et al., nochlorines such as DDE, the metabolite of DDT (18 associations), 2012; Chevalier et al., 2015; Jensen et al., 2015; Komarowska et al., PCBs (12 associations), DDT and HCB (each 12 associations), while 2015; Small et al., 2009; Trabert et al., 2012; Vesterholm et al., 2014; studies on rapidly metabolized compounds such as phthalate ethers Toft et al., 2016; Virtanen et al., 2012); hypospadias (Longnecker and Bisphenol were sparse. The systematic literature search seems et al., 2002; Bhatia et al., 2005; Small et al., 2009; McGlynn et al., to have been efficient since no additional papers fulfilling the eligibility 2009a; Carmichael et al., 2010; Giordano et al., 2010; Choi et al., criteria were identified from hand searches of reference lists or 2012; Jensen et al., 2015; Rignell-Hydbom et al., 2012; Trabert et al., reviews. The majority of the studies were case-referent studies, many 2012; Toft et al., 2016); testicular cancer (Hardell et al., 2003, 2004; nested within large well-defined cohorts, while two studies of sperm Biggs et al., 2008; Cohn et al., 2010; Giannandrea et al., 2011; count were cohort studies (Vested et al., 2013, 2014) and two were McGlynn et al., 2008; 2009b; Purdue et al., 2009); and sperm count cross-sectional studies (Mocarelli et al., 2011; Axelsson et al., 2015). Eligibility Included Screening Identification 116 Bonde et al. The case-referent studies included a total of 2446 cases of which 671 with <75 cases, we found an attenuated summary estimate (OR cases were cryptorchidism, 570 cases were hypospadias and 1205 1.06, 95% CI 0.85–1.33) (Table VII, ‘Large studies’) but with no indi- cases were testicular cancer. In addition 324 men took part in studies cation of publication bias in the corresponding funnel plot (data not of semen quality. Of all of the studies, 19 were published within the shown). Leaving out five estimates originating from studies addressing past 10 years, of these 13 were carried out in the United States of xenobiotics with rapid metabolism and excretion (phthalates) pro- America or Scandinavia. The completeness of reporting assessed by duced similar results (Table VII‘+biopersistent/-biopersistent’). No 11 items were considered high (sum score ≥8 of 11 items) for over single study or exposure had a major impact on the summary risk 65% of the papers. But less than half of the studies were considered estimate. Studies addressing p,p-DDT, p,p′-DDE and HCB contribu- at low risk of bias and confounding. Only five case-referent studies ted the most. included >75 cases. Using the form in Supplementary Table 3, we extracted detailed information on each study and including our evalu- ation of completeness of reporting and potential biases (data available Specific exposures and outcomes as one on request). In the following sections, we first present the results of entity (Hypothesis 2) the quantitative meta-analysis and next we review papers that did Data were available for substance specific analyses of risk of any of the not report risk estimates. reproductive disorders for the most frequently examined compounds (p,p′-DDT, p,p′-DDE, PCB and HCB). Results are summarized in Table VII. The summary estimate was above unity of all exposures but Global assessment: generic exposures and only statistically significantly elevated for p,p′-DDE (Fig. 3). outcomes as one entity (Hypothesis 1) The meta-analysis, including all 70 eligible estimates addressing the Generic exposures and specific outcomes risk for cryptorchidism, hypospadias or testicular cancer and giving (Hypothesis 3) equal weight to each of the xenobiotic exposure indicators, showed an overall risk estimate marginally above unity with rather narrow Cryptorchidism 95% confidence limits including unity, OR 1.11 (95% CI 0.91–1.35) Ten case-referent studies based upon six study populations provided (Table VII), and the estimate was similar in studies less likely to be 16 risk estimates for cryptorchidism (Fig. 4). The most frequently biased, OR 1.12 (95% CI 0.81–1.57). A funnel plot revealed some studied exposures were persistent organochlorine pesticides and indication of publication bias as studies with few cases and large PCBs which were measured in maternal pregnancy or postpartum standard errors seemed overrepresented among studies with ele- blood samples, or cord blood or breast milk, and one study used vated risk estimates (Fig. 2). In a subsequent analysis excluding studies Figure 2 Funnel plot of all risk estimates from all studies included in the meta-analysis (n = 70) of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and male reproductive disorders. Risk estimates with identical colors are derived from same study. Review on EDC and male reproduction 117 amnion fluid to assess exposure to phthalates and perfluorooctane exposure and early infancy exposure (Biggs et al.,2008; Giannandrea sulfonic acid (PFOS). Among the 16 risk estimates, nine were above et al.,2011; McGlynn et al.,2008). Only a single study was based on unity and four exceeded a relative risk of 2. The meta-analytic sum- maternal serum samples (Cohn et al.,2010). Altogether, the eight mary estimate was close to unity, OR 1.03 (95% CI 0.72–1.47), case-referent studies provided 36 risk estimates with DDT being the Fig. 4. Funnel plots did not indicate publication bias (data not shown). most frequently examined compound (seven estimates). Of these, 26 estimates were above unity and nine exceeded a relative risk of 2. The summary estimate was slightly elevated but not statistically significantly Hypospadias different from one, OR 1.20 (95% CI 0.78–1.89). The funnel plot was Nine case-referent studies based upon seven study populations compatible with publication bias since small studies contrary to large addressed links between xenobiotics and hypospadias and all studies studies reported a non-proportional large number of elevated risk esti- except one measured xenobiotics in maternal serum (Fig. 5). Five of mates (data not shown). the studies also reported on cryptorchidism. Among 18 risk esti- mates, 11 were above unity and seven were below and one relative risk exceeded 2. The summary estimate was close to unity, OR 1.13 Sperm count (95% CI 0.86–1.50), Table VII. Funnel plots did not indicate publica- No risk estimates available. tion bias (data not shown). Testicular cancer Specific exposures and specific outcomes Eight case-referent studies based upon six study populations examined (Hypothesis 4) risk of testicular cancer according to environmental exposure to poten- tial endocrine disruptive chemicals (Fig. 6). Two early Swedish studies Available data for specific exposure and specific outcomes are lim- measured xenobiotics at the time of diagnosis in the men and in a sub- ited. Four or more risk estimates were only available for four expos- set of their mothers at the same time (Hardell et al.,2003, 2006), ure–outcome combinations. Results of the conventional meta- while a few later studies targeted the hypothesis of prenatal origins of analysis without bootstrapping (since risk estimates are independent) testicular cancer assuming that persistent organic pollutants in prediag- are shown in Table VII. The summary relative risk estimates were nostic serum samples of young men was a useful proxy of in utero slightly above unity but none were statistically significant. Figure 3 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between prenatal and postnatal exposure to DDE and cryptorchidism, hypospadias or testicular cancer (N = 13). 118 Bonde et al. Figure 4 Forest plot of the risk estimates from studies included in the meta-analysis of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and cryptorchidism (n = 16). Figure 5 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and hypospadias (n = 18). Studies not eligible for meta-analysis xenobiotics in fat samples, breast milk, placenta tissue, maternal serum Cryptorchidism or cord blood in cases and controls (Hosie et al., 2000; Damgaard Six case-referent studies based upon four independent study popula- et al., 2006; Main et al.,2007; Fenichel et al., 2012; Komarowska et al., tions did not provide risk estimates but compared concentrations of 2015; Virtanen et al., 2012), Table V. Altogether these papers report 104 exposure–outcome comparisons, ignoring that some studies Review on EDC and male reproduction 119 Figure 6 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between exposure to endocrine disrupting environmental chemicals and testicular cancer (n = 36). examined compounds in more than one type of biological medium. three out of eight substances were elevated in serum samples com- The findings with respect to biopersistent organochlorines results are pared to controls but these results were not entirely consistent with largely consistent with the findings in the quantitative meta-analysis, results based upon urine samples (Choi et al.,2012). The other study showing limited evidence of associations. This also applies to studies examined nine biopersistent pesticides and observed higher concentra- with high completeness of reporting and less likely risk of bias, that tions in serum from cases for them all (Shekharyadav et al.,2011). addressed PBDE, TCDD, PCDD and organochlorine pesticides (Table V). Bootstrapping analyses of the subset of the seven substances Sperm count with highest breast milk concentrations indicated higher median values To date only three studies (four papers) explicitly studying xenobiotic in cases but this may be a chance finding since the overall analysis of all prenatal exposure to potential endocrine disrupting compounds rela- compounds was non-significant (Damgaard et al.,2006). The study on tive to semen quality in adult men have been published (Table VI). brominated flame retardants found increased levels in breast milk sam- These studies are highly heterogeneous with respect to design and ples among cases, but this result was not confirmed in analyses based xenobiotics and a meta-analysis is not appropriate. Rather than pro- upon corresponding placenta tissue samples, which a priori were viding risk estimates for reduced sperm count these studies report thought to provide the most accurate measure of fetal exposure (Main measures of average sperm counts according to the level of xeno- et al.,2007). Findings in other studies mainly addressing rapidly meta- biotic. Sons of women living near a chemical plant exposed to dioxin bolized contaminants were also reassuring, but these studies were con- due to an accident in 1976 that contaminated the surroundings were sidered at high risk of bias (Table V). identified in a seminal study and serum dioxin concentration at the time of conception during the period 1977–1984 was estimated Hypospadias based upon bio-banked maternal serum samples. Sperm counts in Two studies, not providing best practice reporting and considered at the exposed group of sons were compared with counts from men higher risk of bias, did not provide risk estimates but compared con- with assumed background exposure. Average sperm counts were centrations of xenobiotics in blood and urine samples in cases and con- almost halved in the exposed group and the effect was most pro- trols (Shekharyadav et al., 2011; Choi et al., 2012; Table V). In the first nounced among men who had been breastfed during childhood study, compounds with short biological half-lives were examined and (Mocarelli et al., 2011). The sample included only 39 exposed men 120 Bonde et al. and was too small to allow for exposure-response analysis. In a lactation and not precisely at the time of differentiation of the male Danish study, sons of mothers with biobanked serum samples pro- gonads, that based upon animal studies in the rat, is assumed to vided semen samples when they were 19–21 years of age and sperm take place in gestational week 8–14 (Welsh et al., 2008; Macleod count was examined according to tertiles of maternal serum concen- et al.,2010). Considering the long half-lives of the persistent chemi- tration of a range of compounds. There was no indication that PCBs, cals with little expected fluctuation of tissue levels across a few the DDT metabolite DDE or PFOS were associated with reduced months, measurements around the time of birth seems a reason- sperm count, but high level PFOA exposure was significantly asso- able proxy for exposure levels during early pregnancy although dis- ciated with reduced sperm concentration (Vested et al., 2013, 2014). tribution kinetics during pregnancy may be an issue (Verner et al., Finally, a Swedish cross-sectional study using biobanked maternal ser- 2013). This is entirely different for compounds with a rapid metab- um samples did not show associations between sperm count and a olism and excretion such as phthalates and bisphenol A, which fluc- range of phthalate metabolites (Axelsson et al., 2015). tuate markedly within individuals as shown in studies with repeated sampling. Thus, in a study where three blood samples were taken during pregnancy the intra-class coefficient of variation were for most phthalate metabolites between 20% and 40% (Cantonwine Discussion et al., 2014). However, at the group level serum levels of metabo- This is the first systematic review with meta-analysis that has rigorously lites were remarkably stable. The direction of bias may be in either evaluated the epidemiological evidence on prenatal and postnatal direction in the individual study but tends, on average, to cause bias exposure to endocrine disrupting compounds and male reproductive towards null. One of the included studies measuring phthalate disorders. A total of 33 papers provided 89 risk estimates on which metabolites in amnion fluid in the second trimester found no associ- we found no strong support for a global effect as a whole or on any ation with cryptorchidism and hypospadias even though one DEHP specific outcome. However, one of four specific compounds with suffi- metabolite was related to higher fetal testosterone and lower cient data to allow for meta-analysis were related to a moderate insulin-like factor III (Jensen et al., 2015). increased risk of all outcomes taken together (p,p′-DDE). Although Assessment of fetal exposure using maternal blood or milk samples only limited data were available for specific exposure–outcome associa- assumes that the placenta is not an efficient barrier for chemical tions, these findings do not seem to be due to increased risk of any sin- transfer from the maternal to the fetal circulation and that fetal gle included disorder (cryptorchidism, hypospadias or testicular exposure is proportional to maternal exposure. It is therefore note- cancer). Thus findings provide some support to the hypothesis of worthy that knowledge regarding the transport of persistent xenobio- shared prenatal etiology of these outcomes, but they also point to het- tics over the placenta to the fetus has been known for decades erogeneity of compounds classified as endocrine disruptors with (Needham et al., 1999; Mazdai et al., 2003). More recent findings respect to potential effects on male reproduction. indicate that rapidly metabolized compounds also pass through the The strength of this review in comparison with narrative reviews placenta and these have been detected in cord blood and amnion and global assessments is primarily that it includes, to our knowledge, fluid (Jensen et al., 2015). all published epidemiological evidence fulfilling predefined criteria using a systematic and transparent search of the literature. Although the findings of this review provide some evidence for environmental endocrine disruption of male reproductive function, the limitations of Exposure contrast within and across studies observational epidemiology and the outcomes reported in the few The most prevalent persistent organochlorine contaminants were available high quality studies precludes strong concluding statements. banned in high income countries during the 1970s and subsequently their tissue levels have declined substantially. However, the studies included in this review encompass studies based upon samples bio- Exposure assessment banked >50 years ago such as the Collaborative Perinatal Study with Reliable exposure assessment is essential in environmental epidemi- relatively high serum concentrations (Longnecker et al., 2002; Pierik ology and measurements of the compounds of interest are consid- et al., 2007; McGlynn et al., 2008, 2009b; Trabert et al., 2012) as well ered as the gold standard. We therefore only included studies with as studies based upon sampling much later (Damgaard et al., 2006; actual measurements of the chemicals in tissues as exposure assess- Main et al., 2007; Virtanen et al., 2012). Unfortunately, data are too ment based on external determinants (such as job title) is crude sparse to allow for a formal meta-analysis based upon the variation in when it comes to endocrine disrupting chemicals. Analyses of blood exposure levels in reference populations throughout time; however, or tissue concentrations of chemicals were performed by gas chro- no individual studies with large exposure contrast found evidence for matography/mass spectrometry and most studies provided data on a relationship between exposure and response. recovery and between batch coefficients of variation. Although differ- Since this review is focused upon prenatal exposures, we ences in sample preparation, analytical technique and units may invali- excluded a wealth of cross-sectional studies linking occupational and date comparisons of absolute exposure levels across studies, this is environmental exposure in adulthood with semen quality (for a not expected to affect risk estimates that are derived from within- recent review and a large collaborate study summary, see Bonde study contrasts of exposure levels. et al., 2008; Bonde and Giwercman, 2014). In contrast to testicular Misclassification of exposure at the susceptible periods of devel- cancer, it is well established that spermatogenesis is susceptible to opment most likely results in bias towards the null. Most studies short-term effects of reproductive toxicants not least in the measured the contaminants late in pregnancy, at birth or during workplace. Review on EDC and male reproduction 121 in one study (Biggs et al., 2008). Although adult serum concentrations Outcome ascertainment of persistent chemicals may to some extent reflect prenatal and early The male reproductive disorders addressed in this review are distinct postnatal exposure, it is obvious that varying rates of metabolism as diseases with different pathologies, clinical characteristics and age of well as additional lifetime exposures during infancy, childhood and appearance. Cryptorchidism and hypospadias were either defined by adulthood also contribute to exposure levels. The resulting misclassi- specified criteria applied in systematic examinations of newborns in fication most is most likely is towards the null. Accordingly it is not prospective studies or from medical records. Although criteria for surprising that the overall risk estimate was attenuated in the analysis both disorders may differ substantially and contribute to apparent excluding studies on testis cancer with adult serum data only. secular trends and spatial shifts in prevalence rates, it is unlikely that differences in outcome ascertainment across studies disrupt the internal validity and the relative risk estimates. No studies used par- Studies addressing sperm count ticipant questionnaires or interviews to define outcomes and there- Although the effects of endocrine disrupting chemicals on male fertil- fore recall bias is not an issue in studies included in this review. ity has been the focus of heated debate (Bonde et al., 2011), only three human studies have to date linked prenatal and postnatal exposure to xenobiotics with sperm count in adults (Mocarelli et al., The testicular dysgenesis syndrome 2011; Axelsson et al., 2015; Vested et al., 2013). This is probably hypothesis explained by the difficult and costly logistics of population-based We analyzed the male reproductive disorders, with the exception of semen studies and the need for maternal biospecimens or cord blood low sperm count where no risk estimates were available, as one stored decades before follow-up takes place. In the study by entity with reference to the testicular dysgenesis syndrome hypoth- Mocarelli et al., the findings of halved sperm counts in men whose esis on shared etiology of the four male reproductive disorders mothers were exposed to higher levels of dioxin when pregnant are (Skakkebaek, 2002) and with reference to experimental evidence sug- intriguing but call for cautious interpretation. Exposure levels in gesting multiple mechanistic pathways of endocrine disruption women living in the contaminated area are overlapping the back- (Toppari, 2002). The strength of the testicular dysgenesis syndrome ground exposure, participants are few, referents are from a conveni- hypothesis and the applicability of this concept as explanation of the ence sample and significant results were limited to the subgroup of pathogenesis of the majority of its components is an important pre- men breast-fed as infants (Mocarelli et al., 2011). The pertinent ques- requisite for obtaining statistically significant associations when testing tion is whether sperm counts in the exposed group are low (in the hypotheses I and II, where cryptorchidism, hypospadias and testicular range of 45 mill/mL) or whether sperm counts in the reference cancer were merged together as end points in statistical analyses. group are high (in the range of 80 mill/mL). It is well established that However, the heterogeneity of the pathogenesis underlying those small cross-sectional semen studies are highly vulnerable to selection conditions and their relative weak or moderate mutual clinical asso- bias (Larsen et al., 1998). Unfortunately it is hardly possible to repli- ciations might indicate that only a minor proportion of these male cate the findings. Results from the sole study that was explicitly reproductive abnormalities fit into the testicular dysgenesis syndrome designed to test the hypothesis on delayed effects on sperm count concept (Thorup et al., 2010), which may explain the generally non- are reassuring with respect to organochlorines (Vested et al., 2014), significant or only weakly statistically significant findings of our but less so with respect to the much less studied but ubiquitous per- analyses. fluorinated hydrocarbons (Vested et al., 2013). The Swedish study addressing phthalate metabolites did not reveal adverse effects but exposure assessment of the rapidly metabolized compounds with Studies addressing testicular cancer high within-day and between-days variation within individuals is an Although focus was on developmental disorders induced by chemical issue. Nevertheless single spot urine samples may distinguish different exposure during critical time windows in early life (Lee and Jacobs, exposure levels at the population level if study populations are suffi- 2015), there only exists one small study using prenatal or postnatal ciently large (Hagmar et al., 2006; Preau et al., 2010; Ye et al., 2011). bio-specimens to assess testicular cancer (Cohn et al., 2010). We Obviously there is a need to design future studies with sufficient therefore included six studies on testicular cancer assessing exposure power to examine exposure–response associations. With the aging by measurement of chemicals in serum samples obtained after large European birth cohorts with stored maternal serum samples, puberty. Hereby, we assume that serum levels of persistent chemicals this will become feasible in a few years. in young men or their mothers are reasonable proxies for prenatal and postnatal exposure. There is however some evidence to support Methodological issues this strong assumption. First of all, the biological half-lives of many of these compounds are counted in decades (Hagmar et al., 2006). Many of the included studies examined several compounds for which Moreover, studies in a Faroese birth cohort demonstrated strong risk estimates are not independent. This was accounted for by the intra-individual correlations between persistent chemicals in cord use of bootstrapping statistical techniques by which analyses are iter- blood and serum concentrations at 7 and 14 years of age, respect- ated several hundred times randomly including only one risk estimate ively (Barr et al., 2006). Another study found high correlations of per- per population in each analysis and providing an average risk estimate sistent chemical levels in serum samples in adult men obtained up to giving equal weight to studies with few and many risk estimates. This 10 years apart (Hagmar et al., 2006). Finally, a history of breastfeed- method does not provide statistics for heterogeneity which is consid- ing was related to elevated serum levels in men up to 40 years later ered less of a problem since study populations and risk estimates 122 Bonde et al. obviously are heterogeneous. Therefore, we used random effects most often addressing effects of individual chemicals one by one. models rather than fixed effects models in all analyses. In spite of However, this criticism ignores to some extent that persistent organic strong heterogeneity across studies with respect to study popula- pollutants are highly correlated and epidemiological studies addres- tions, exposures and outcomes, it is remarkable that all summary risk sing one indicator chemical, for example PCB-153, are in fact reflect- estimates from substance and outcome specific supplementary ana- ing exposure to a mixture of numerous other chemicals. For lyses are of similar magnitude spanning a relative risk from 1.03 to example, in a study of persistent organic pollutants and fertility, it was 1.26 when ignoring one outlying observation. not possible to distinguish between the potential effects of PCBs and The number of studies addressing specific chemicals and outcomes the DDT metabolite DDE (Axmon et al., 2006). were few and did not permit robust meta-analysis. Therefore, this review is primarily informative with respect to the overall association Non-monotonic dose–response relationships and the hypothesis on shared etiology. A number of studies did not report risk estimates but used other measures of associations. Another concern is the possibility of non-monotonic dose–response However, those findings did not seem to deviate from the overall relationships which may explain apparently inconsistent findings picture. across studies because of different levels and ranges of exposures Since we only included studies that documented exposure by (Vandenberg et al., 2012). This is particularly a potential problem objective analysis of compounds in biological media, the often when extrapolating from high occupational exposure levels to low encountered risk of bias due to differential recall of exposure or out- environmental levels and in risk assessment with a need to extrapo- come was bypassed. Selection bias related to prior knowledge about late from high doses in in vitro and in vivo animal studies to many-fold levels of contaminants in biological tissues among patients or lower environmental levels that humans encounter. However, the researchers therefore also seems unlikely. Few, if any, know their possibility of non-monotonic dose–response relationship is less of a own tissue concentrations of the investigated contaminants to which problem in large population-based studies capturing a broad low- all persons are exposed. Moreover, the nested case-referent design level exposure range (Lee and Jacobs, 2015). None of the large stud- adopted by the majority of the included studies promotes compar- ies included in this review with substantial exposure contrast and ability of cases and referents with respect to social and behavioral comprehensive evaluation of risk according to exposure strata indi- factors that may be related to exposure levels and thus indirectly cate the existence of such non-monotonic exposure–response rela- cause bias. The two main reasons for rating some studies at a higher tionships (Longnecker et al., 1997, 2002). risk of confounding was either due to lack of or insufficient adjust- ment for potential confounding factors such as maternal age and pre- maturity in case of congenital malformations with unpredictable Wider implications and conclusion direction of bias or small study size that was not justified by power The widely stated view that ubiquitous endocrine disrupting chemi- calculations which may increase the risk of publication bias. In fact cals in our environment play a substantial role in the development of funnel plots indicated overrepresentation of higher risk estimates in male reproductive disorders through prenatal and perinatal mechan- small studies with larger standard errors. Selective reporting did not isms is to some extent challenged by this review. Although the cur- appear to be a problem. All studies seemed to provide data on all rent epidemiological evidence is compatible with a small increased measured substances regardless of association. Finally, only a few risk of male reproductive disorders following prenatal and postnatal studies corrected for weight gain during pregnancy which may cause exposure to some persistent environmental chemicals classified as false positive associations due to dilution of the distribution volume endocrine disruptors, the evidence is limited. In this light, estimates for biopersistent chemicals (Verner et al., 2013), an issue if the out- of the burden of disease and costs of exposure to endocrine chemi- come is related to lower birthweight or congenital malformations. cals (Trasande et al., 2016) seem highly speculative, at least with Nevertheless, the findings must be interpreted in the light of the respect to male reproductive disorders. Future epidemiological stud- complexity of this research field. Thus, the meta-analyses strongly ies may change the weight of the evidence in either direction and the violates basic assumptions in that populations, chemical substances need for appropriate risk assessment of chemicals based upon and outcomes differ across studies (Rothman and Greenland, 1998), experimental evidence should not be ignored. There are insufficient which necessarily must result in substantial heterogeneity disregarding data on rapidly metabolized endocrine disruptors and specific expos- results of any statistical test. The meta-analytic summary estimates ure–outcome relations. A particular data gap is evident with respect and their confidence limits are not quantitatively reliable measures of to delayed effects on semen quality and testicular cancer. specific exposure–outcome relations but merely serve to provide an overview of the available limited evidence. Supplementary data Supplementary data are available at http://humupd.oxfordjournals. Cocktail effects org/. Humans are simultaneously exposed to a plethora of hundreds of xenobiotics. Some in vitro and in vivo animal studies have demon- strated combined effects of endocrine disrupting chemicals at levels Acknowledgements at which the individual chemicals do not induce observable effects (Kortenkamp, 2007; Kortenkamp, 2014). Because of this ‘cocktail Research secretary Hanne Tulinius is thanked for typing an early ver- effect’ of mixtures, scientist have criticized epidemiological studies for sion of the manuscript. Review on EDC and male reproduction 123 contrasts in blood levels of persistent organochlorines. Environ Health Perspect Authors’ roles 2008;116:269–277. Bonzini M, Coggon D, Palmer KT. Risk of prematurity, low birthweight and pre- Jens Peter Bonde and Elvira Braüner conceived and organized the eclampsia in relation to working hours and physical activities: a systematic study, sifted titles and abstracts and independently included and review. Occup Environ Med 2007;64:228–243. excluded papers. Esben Meulengracht Flachs carried out the statistical Brucker-Davis F, Wagner-Mahler K, Delattre I, Ducot B, Ferrari P, Bongain A, analysis. Clara Helene Glazer, Karin Sørig Hougaard, Birgit Bjerre Kurzenne JY, Mas JC, Fenichel P. Cryptorchidism at birth in Nice area (France) is associated with higher prenatal exposure to PCBs and DDE, as assessed by col- Høyer, Katia Keglberg Hærvig, Sesilje Bondo Petersen and Ina Olmer ostrum concentrations. Hum Reprod 2008;23:1708–1718. Specht reviewed and rated the individual papers with respect to data Cantonwine DE, Cordero JF, Rivera-Gonzalez LO, Anzalota Del Toro LV, extraction, completeness of reporting, bias and confounding. All of Ferguson KK, Mukherjee B, Calafat AM, Crespo N, Jimenez-Velez B, Padilla IY the above-mentioned authors as well as Aleksander Giwercman, et al. Urinary phthalate metabolite concentrations among pregnant women in Cecilia Høst Ramlau-Hansen, Lars Rylander and Gunnar Toft contrib- Northern Puerto Rico: distribution, temporal variability, and predictors. Environ Int 2014;62:1–11. uted to the design and provided critical comments. Susie Rimborg did Carmichael SL, Herring AH, Sjodin A, Jones R, Needham L, Ma C, Ding K, Shaw the systematic literature search. Jens Peter Bonde drafted the manu- GM. Hypospadias and halogenated organic pollutant levels in maternal mid- script to which all authors contributed and approved the final version pregnancy serum samples. Chemosphere 2010;80:641–646. format. Chevalier N, Brucker-Davis F, Lahlou N, Coquillard P, Pugeat M, Pacini P, Panaia- Ferrari P, Wagner-Mahler K, Fenichel P. A negative correlation between insulin- like peptide 3 and bisphenol A in human cord blood suggests an effect of endo- crine disruptors on testicular descent during fetal development. Hum Reprod Funding 2015;30:447–453. 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Human Reproduction Update, Vol.23, No.1 pp. 104–125, 2017 Advanced Access publication on September 21, 2016 doi:10.1093/humupd/dmw036 The epidemiologic evidence linking prenatal and postnatal exposure to endocrine disrupting chemicals with male reproductive disorders: a systematic review and meta-analysis 1,2, 1 3 Jens Peter Bonde *, Esben Meulengracht Flachs , Susie Rimborg , 1 4 Clara Helene Glazer , Aleksander Giwercman , 5 6 Cecilia Høst Ramlau-Hansen , Karin Sørig Hougaard , 1,7 1 Birgit Bjerre Høyer , Katia Keglberg Hærvig , 1 8 1 Sesilje Bondo Petersen , Lars Rylander , Ina Olmer Specht , 7 1,9 Gunnar Toft , and Elvira Vaclavik Bräuner 1 2 Department of Occupational and Environmental Medicine, Bispebjerg University Hospital, DK-2400 Copenhagen NV, Denmark Institute of Public Health, University of Copenhagen, DK-1016 Copenhagen K, Denmark The Royal Library/ University of Copenhagen Library, DK-2200 Copenhagen N, Denmark Molecular Reproductive Medicine, Department of Translational Medicine, Lund University, Lund, 5 6 Sweden Institute of Public Health, Aarhus University, DK-8000 Aarhus C, Denmark National Research Centre for the Working Environment, DK-2100 Copenhagen Ø, Denmark Department of Clinical Epidemiology, Aarhus University Hospital, DK-8200 Aarhus N, 8 9 Denmark Department of Occupational and Environmental Medicine, University of Lund, SE-221 85 Lund, Sweden Research Center for Prevention and Health (RCPH), University of Copenhagen, DK-2600 Glostrup, Denmark *Correspondence address. Department of Occupational and Environmental Medicine, Bispebjerg-Frederiksberg University Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. Tel: +45-3043-8617; E-mail: jens.peter.ellekilde.bonde@regionh.dk Submitted on May 27, 2016; resubmitted on August 22, 2016; accepted on August 31, 2016 TABLE OF CONTENTS ........................................................................................................................... � Introduction � Methods Protocol and registration Information sources Eligibility criteria Search and study selection Data extraction Quality assessment Meta-analysis � Results The study base Global assessment: generic exposures and outcomes as one entity (Hypothesis 1) Specific exposures and outcomes as one entity (Hypothesis 2) Generic exposures and specific outcomes (Hypothesis 3) Specific exposures and specific outcomes (Hypothesis 4) Studies not eligible for meta-analysis © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permis- sions@oup.com Review on EDC and male reproduction 105 � Discussion Exposure assessment Exposure contrast within and across studies Outcome ascertainment The testicular dysgenesis syndrome hypothesis Studies addressing testicular cancer Studies addressing sperm count Methodological issues Cocktail effects Non-monotonic dose–response relationships Wider implications and conclusion BACKGROUND: More than 20 years ago, it was hypothesized that exposure to prenatal and early postnatal environmental xenobiotics with the potential to disrupt endogenous hormone signaling might be on the causal path to cryptorchidism, hypospadias, low sperm count and testicular cancer. Several consensus statements and narrative reviews in recent years have divided the scientific community and have elicited a call for systematic transparent reviews. We aimed to fill this gap in knowledge in the field of male reproductive disorders. OBJECTIVE AND RATIONALE: The aim of this study was to systematically synthesize published data on the risk of cryptorchidism, hypospadias, low sperm counts and testicular cancer following in utero or infant exposure to chemicals that have been included on the European Commission’s list of Category 1 endocrine disrupting chemicals defined as having documented adverse effects due to endocrine disruption in at least one intact organism. SEARCH METHODS: A systematic literature search for original peer reviewed papers was performed in the databases PubMed and Embase to identify epidemiological studies reporting associations between the outcomes of interest and exposures documented by bio- chemical analyses of biospecimens including maternal blood or urine, placenta or fat tissue as well as amnion fluid, cord blood or breast milk; this was followed by meta-analysis of quantitative data. OUTCOMES: The literature search resulted in 1314 references among which we identified 33 papers(28 study populations) fulfilling the eligibility criteria. These provided 85 risk estimates of links between persistent organic pollutants and rapidly metabolized compounds (phthalates and Bisphenol A) and male reproductive disorders. The overall odds ratio (OR) across all exposures and outcomes was 1.11 (95% CI 0.91–1.35). When assessing four specific chemical subgroups with sufficient data for meta-analysis for all outcomes, we found that exposure to one of the four compounds, p,p′-DDE, was related to an elevated risk: OR 1.35 (95% CI 1.04–1.74). The data did not indi- cate that this increased risk was driven by any specific disorder. WIDER IMPLICATIONS: The current epidemiological evidence is compatible with a small increased risk of male reproductive disorders following prenatal and postnatal exposure to some persistent environmental chemicals classified as endocrine disruptors but the evidence is limited. Future epidemiological studies may change the weight of the evidence in either direction. No evidence of distortion due to publi- cation bias was found, but exposure–response relationships are not evident. There are insufficient data on rapidly metabolized endocrine disruptors and on specific exposure–outcome relations. A particular data gap is evident with respect to delayed effects on semen quality and testicular cancer. Although high quality epidemiological studies are still sparse, future systematic and transparent reviews may provide pieces of evidence contributing to the narrative and weight of the evidence assessments in the field. Key words: cryptorchidism / endocrine disruption / hypospadias / infertility / prenatal exposure / sperm count / testicular cancer / xenobiotics gonadotrophins leading to disruption of normal development of the Introduction male fetal gonad during the late phase of the first trimester of pregnancy As early as 1979, Henderson et al. hypothesized that a relative excess (Sharpe and Skakkebaek, 1993). This so-called estrogen hypothesis has of estrogen, in particular at the time of testicle differentiation, is a major been scrutinized by a large number of studies addressing the risk of risk factor for testicular cancer (Henderson et al.,1979). Fourteen years male reproductive disorders in boys who, during pregnancy, were later, Sharpe and Skakkebaek proposed that not only testicular cancer exposed to high or low levels of estrogens because of twin pregnancy, but also other male reproductive disorders such as cryptorchidism, first parity, preeclampsia, hyperemesis or intended or incidental treat- hypospadias and low sperm counts may share a common fetal origin ment with synthetic hormones during first trimester. Three literature and that environmental exposure to chemicals with actions mimicking reviews provide no support for the estrogen hypothesis except that estrogens might play a pivotal role (Sharpe and Skakkebaek, 1993). The testicular cancer seems related to high prenatal estrogen exposure underlying mechanisms were thought to include increased negative (Sever et al.,1997; Storgaard et al., 2006; Martin et al.,2008). In par- feedback on the fetal pituitary resulting in reduced levels of ticular, it is notable that prenatal exposure to the highly potent synthetic 106 Bonde et al. estrogen diethylstilbestrol is only associated with low sperm count at and there is presently no consensus. In 2012, a Global Assessment of high doses (Gill et al.,1979; Leary et al.,1984). Subsequently a cap- the State-of-the-Science of Endocrine Disrupters commissioned by the acious amount of experimental research has indicated that adverse World Health Organization and United Nations Environment Program health effects, including male reproductive disorders, may be related to concluded that the direct evidence for endocrine disrupting effects with endocrine disruption of fetal development through xenobiotic inter- adverse health effects because of exposure to xenobiotic substances is action with steroid hormone receptors or through interference with limited in humans, but emphasized the need for continuous attention the synthesis, secretion, transport, metabolism or degradation of nat- (European workshop on the impact of endocrine disrupters on human ural hormones (European workshop on the impact of endocrine disrup- health and wildlife. Report of proceedings from a workshop held in ters on human health and wildlife. Report of proceedings from a Weybridge, UK, 2–4 December 1996; World Health Organisation workshop held in Weybridge, UK, 2–4 December 1996; World Health (WHO) and United Nations Environment Program (UNEP), 2012). As Organisation (WHO) and United Nations Environment Program several epidemiological studies addressing this issue have been pub- (UNEP), 2012). This broader endocrine disruption hypothesis with lished over the past 10 years, a systematic evaluation of the epidemio- focus on environmental pollutants has gained tremendous public and logical evidence on effects of environmental endocrine disrupting scientific attention in the USA and the European Union over the past xenobiotics including quantitative meta-analyses of data, where feasible, 15 years. Numerous narrative reviews and consensus statements have has now become timely (Beronius and Vandenberg, 2015). discussed supporting and contradicting evidence based on data of secu- Therefore, the objective of this review was to identify and evaluate lar trends of male reproductive health outcomes, wildlife impact and the epidemiological evidence linking adverse reproductive health disor- exposure levels in various populations, in addition to the large body of ders with in utero or early postnatal exposure to xenobiotic endocrine in vitro and in vivo experimental data (European workshop on the impact disrupting compounds. The key eligibility criteria are documentation of of endocrine disrupters on human health and wildlife. Report of pro- exposure contrast by chemical measurements of the compounds in ceedings from a workshop held in Weybridge, UK, 2–4December maternal blood or other biospecimens indicative of fetal or infant expos- 1996; Bergman et al.,2013; Cantonwine et al.,2014; Diamanti- ure. Specifically, we address the following four hypotheses on the risk of Kandarakis et al., 2009; Lamb et al.,2014; Nohynek et al.,2013; adverse male reproductive outcomes following prenatal and postnatal Sharpe, 2001; Sharpe and Irvine, 2004; Skakkebaek et al., 2001; environmental exposure to industrial chemicals classified as endocrine Takahashi et al.,2004; Toppari et al., 2010; Vandenberg et al.,2012; disrupting substances (McCarthy, 2011): (i) that all compounds regard- World Health Organisation (WHO) and International Programme on less of specific endocrine disrupting properties carry a risk of reproduct- Chemical Safety (IPCS), 2002; World Health Organisation (WHO) and ive disorders considered as one outcome entity; (ii) that specific United Nations Environment Program (UNEP), 2012). It is particularly compounds are heterogeneous with respect to reproductive disorders noteworthy that biomonitoring data from all over the world provide considered as one entity; (iii) that all compounds regardless of specific unequivocal documentation of human exposure to a range of xeno- endocrine disrupting properties carry a risk for specific outcomes; and biotic substances including persistent compounds (Govarts et al.,2012; (iv) that specific compounds carry a risk for specificoutcomes. Lenters et al., 2013; Gyalpo et al., 2016; Hung et al., 2016; Liu et al., 2016; Magulova and Priceputu, 2016) as well as rapidly metabolized Methods and excreted compounds that reach steady state concentrations in human blood and tissues because of continuous exposure (Arbuckle The review was conducted and reported in accordance with the MOOSE et al., 2016; Zhang et al.,2016). These xenobiotic compounds have guidelines for Meta-analyses and Systematic reviews of Observational been released into the environment intentionally via the widespread Studies (Stroup et al., 2000). application of pesticides or unintentionally by either degradation of industrial products and building materials or by leakage of packing mate- Protocol and registration rials into food items (Nam et al.,2010). New exposure pathways are A review protocol was registered at PROSPERO.org with registration described regularly. For instance, it has recently been reported that number CRD4201603742 prior to initiation of the review process, on 12 semi-volatile polychlorinated biphenyls (PCBs) represent an important April 2016 with amendments on 12 May 2016 (CRD420160374X). The inhalation exposure pathway for residents living in PCB contaminated amendments specified the four main hypotheses, provided details on sen- homes containing PCB in calking material, paints and sealant used >40 sitivity analyses and discarded the initial idea to analyze data according to years ago (Frederiksen et al.,2012; Meyer et al., 2013). Exposure by estrogenic or anti-androgenic activity of measured compounds which inhalation and possibly through the skin may more than double the PCB proved non-feasible. The protocol was updated before the review pro- body burden in individuals exposed at the residence or at school cess and data analysis were initiated. (Meyer et al.,2013). Considering the mounting evidence that numerous of these environmental xenobiotics exhibit endocrine activity in in vitro and in vivo assays, there is basis for substantial concern regarding the Information sources potential health effects in humans (Gabrielsen and Tanrikut, 2016; The databases PubMed and Embase were used as they cover the vast McLachlan, 2016; Trasande et al.,2016), even if alarming reports on, majority of relevant journals for the subject. for instance, declining sperm counts are circumstantial or not corrobo- rated (Bonde et al., 2011) and the best evidence we have is reassuring Eligibility criteria (Jorgensen et al.,2012). However, the epidemiological data on the adverse health effects are We conducted a systematic search of original peer-reviewed original sparse, particularly regarding links with prenatal and postnatal exposure, papers in English published between 1966 and 12 April 2016 to identify Review on EDC and male reproduction 107 journal articles providing quantitative data on the association between specimens (maternal blood or urine, placenta or fat tissue as well as xenobiotic endocrine disrupting chemicals and male reproductive disor- amnion fluid, cord blood or breast milk). In addition serum concen- ders in humans. The complete search specification is provided in the trations in adult life as proxies for fetal or early life exposure were online Supplementary Table 1. Eligibility criteria for inclusion in the sys- also included for testicular cancer. tematic review were as follows. (4) Outcome ascertainment by medical standardized examination or antecedent medical records or reporting to health registries. (1) Exposures: Chemicals that by the European Commission are classified (5) Risk estimates (rate risk [RR], OR, hazard ratio [HR]) for an outcome as Category 1 endocrine disruptors with high concern in terms of according to higher versus lower levels of prenatal and postnatal human or wildlife exposure (McCarthy, 2011). Category 1 compounds exposure defined by exposure contrasts within the given study. are defined as chemicals where at least one study has shown endocrine Studies reporting alternative measures of association such as differ- effects in an intact organism. The EU report lists 60 Category 1 sub- ence in mean values of exposure levels in cases and controls were stances (29 chemical groups) that are highly persistent or have high also included, and the authors were contacted to get risk estimates current production volume (McCarthy, 2011). In addition we include (Hosie et al., 2000; Damgaard et al., 2006; Main et al., 2007; the polyfluorinated chemicals, a group of ubiquitous biopersistent Choi et al., 2012; Fenichel et al., 2012; Komarowska et al., 2015; emerging endocrine disruptors (White et al., 2011) that are receiving Virtanen et al., 2012). Several authors responded positively but increasing attention. Table I lists the specific chemical substances with reanalysis of the original data could not be accomplished within the abbreviations that are referenced in the text and in Tables II–VII. given time-frame and the studies were therefore not included in the (2) Outcomes: Cryptorchidism (one or both testicles undescended) present review. ascertained at birth or during childhood, hypospadias, testicular can- Criteria for exclusion of studies were as follows: cer regardless of histological subgroup, and sperm count (number of spermatozoa per volume or mass unit of seminal fluid). (1) In vitro and in vivo experimental studies. (3) Exposure to specific chemicals in utero or in the first year of life (post- (2) Studies addressing mechanisms and other outcomes related to endo- natal exposure) documented by measurements in biological crine disruption, for example, effects on sexual hormone levels in tis- sues and measures of semen quality other than sperm concentration. (3) Studies based upon chemical analysis of exposure after puberty Table I List of specific chemical substances with except studies of testicular cancer. abbreviations. (4) Studies repeating risk estimates reported in previous publications for example, studies addressing risk according to gene polymorphisms Bisphenol A 4,4′-(Propan-2,2-diyl)diphenol for substances, where risk estimates for the entire population were mBP Mono-n-butyl-phthalate provided earlier. Chlordane cis-heptachlordane, cis-chlordane, ocychlordane, trans- (5) Ecological studies with exposure information at the population level mix nonachlordane, cis-nonachlordane rather than at the individual level. DBP Di-n-butyl-phthalate DDE 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene o,p′-DDT 1,1,1-Tricholoro-2-(p-chlororphenyl)-2-(o-chlorophenyl) Search and study selection ethane p,p′-DDT 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane We combined medical subject headings and generic terms for the expo- sures and outcomes (Supplementary Table 1) and obtained in total 1314 DEHP Di-(2-ethylhexyl)phthalate hits after removal of duplicates. Two authors (JPB and EVB) sifted titles Dieldrin Derivate of norbornadiene and and abstracts independently to assess eligibility and retrieved 110 papers hexachlorocyclopentadiene for full text reading. Among these, several reports failed to provide quan- HCB Hexachlorobenzene titative data on exposure levels and others were excluded for other rea- HCE Heptachlor epoxide sons detailed in Supplementary Table 2. Hand searches of the HCCH b-Hexachlorocyclohexane bibliographies of retrieved primary reports and reviews did not capture MEHP Mono-(2-ethylhexyl)phthalate additional papers. We selected 19 epidemiological studies (21 papers) that provided at 5cx-MEPP Di(2-ethylhexyl)phthalate [metabolite of DEHP] least one risk estimate for a male reproductive disorder according to at 7cx- Mono(4-ethyl-7-carboxylheptyl)phthalate [metabolite of least one measured xenobiotic classified as an endocrine disruptor. MMeHP DiNP] Moreover, nine studies (12 papers) that compared average exposure Mirex Dimerization derivate of hexachlorocyclopentadiene levels in cases and referents without providing risk estimates were n-NP n-Nonylphenol included. Thus, the final database comprised 33 papers included for t-OP t-Octylphenol qualitative systematic analysis and 21 papers included for quantitative PBB Polybrominated biphenyl meta-analysis. No attempt was made to retrieve papers from the unpublished literature. The steps in the literature search are displayed PBDE Polybrominated diphenyl ether in Fig. 1. PCB Polychlorinated biphenyl PCDD/Fs Polychlorinated dibenzo-p-dioxins/furans PFOS Perfluorooctane sulfonic acid PFOA Perfluorooctanoic acid Data extraction TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin Descriptive information (Tables II–V) was recorded from each publication using a standardized form constructed prior to the collection process. 108 Bonde et al. Table II Characteristics and risk estimates for 10 case-referent studies (18 risk estimates) addressing the risk of cryptorchidism following prenatal and postnatal exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure medium Exposure contrast Substance OR 95% CI CR Bias referents .......................................................................................................................................................................................................................................................... Longnecker et al. USA (12 centers) Collaborative Perinatal Project 219/552 Third trimester serum Highest versus lowest quintiles p,p′-DDE 1.30 0.70–2.24 9 0 (2002) Pierik et al. (2007) USA (12 centers) Collaborative Perinatal Project 219/564 Third trimester serum 75–90 percentile versus 0–10 b-HCCH 2.08 1.08–4.01 9 0 percentile HCE 0.82 0.41–1.65 HCB 0.85 0.52–1.40 McGlynn et al. USA (12 centers) Collaborative Perinatal Project 230/593 Third trimester serum Highest quartile versus lowest PCB (sum of 11 1.41 0.90–2.20 8 0 (2009a) congeners) Trabert et al. (2012) USA (12 centers) Collaborative Perinatal Project 217/557 Third trimester serum Highest quartile versus lowest trans-nonachlor 1.22 0.70–2.12 9 0 Oxychlordane 0.95 0.55–1.64 Bhatia et al. (2005) USA, San Francisco Child Health and Development 75/283 Maternal serum Highest versus lowest quartile p,p′- DDT 1.01 0.44–2.28 9 1 Study p,p′- DDE 1.34 0.51–3.48 Brucker-Davis et al. France, Nice Newborns at maternity clinics in 56/69 Colostrum Highest versus lowest. Three p,p′-DDE 2.16 0.94–4.98 7 1 (2008) Nice and Grasse. categories PCB (seven 2.74 1.15–6.53 congeners) mBP 2.13 0.66–6.83 Small et al. (2009) USA, Michigan Michigan Long-term PBB Study 9/464 Maternal serum Highest versus lowest (<LOD). PBB-153 (Includes 0.50 0.10–4.70 4 1 Three categories hypospadias) Vesterholm et al. Denmark and Finland Danish–Finnish prospective Birth 107/108 Cord blood Highest tertile versus lowest PFOS 0.83 0.39–1.79 9 0 (2014) Cohort Study PFOA 0.46 0.20–1.20 9 0 Jensen et al. (2015) Denmark Pregnancy screening registry, 270/300 Second trimester Highest tertile versus lowest 5cx-MEPP 0.90 0.57–1.41 9 1 Serum Institute amnion fluid 7cx-MMeHP 1.28 0.80–2.01 Toft et al. (2016) Denmark Pregnancy screening registry, 270/300 Second trimester Highest tertile versus lowest PFOS 1.01 0.66–1.53 9 1 Serum Institute amnion fluid CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. Review on EDC and male reproduction 109 Table III Characteristics of 10 case-referent studies (21 risk estimates) addressing the risk of hypospadias following prenatal and postnatal exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure Exposure contrast Substance OR 95% CI Meta- CR Bias Referents medium analysis .......................................................................................................................................................................................................................................................... Longnecker et al. (2002) USA (12 Collaborative Perinatal Project 199/552 Third trimester Highest versus lowest quintiles p,p′-DDE 1.2 0.60–2.40 Yes 8 0 centers) serum McGlynn et al. (2009a) USA (12 Collaborative Perinatal Project 201/593 Third trimester Highest versus lowest quartile PCB (sum of 11 1.69 1.06–2.68 Yes 8 1 centers) serum congeners) Trabert et al. (2012) USA (12 Collaborative Perinatal Project 197/557 Third trimester Highest quartile versus lowest trans-nonachlor 1.08 0.62–1.89 Yes 9 0 centers) serum Oxychlordane 1.24 0.69–2.22 Yes 9 Bhatia et al. (2005) USA, San Child Health and Development 66/283 Maternal serum Highest versus lowest quartile p,p′-DDT 0.79 0.33–1.89 Yes 9 0 Francisco Study p,p′-DDE 1.18 0.46–3.02 Yes 9 0 Small et al. (2009) USA, Michigan Long-term PBB Study 14/431 Maternal serum Highest versus lowest (<LOD). PBB-153 (includes 0.71 0.1–3.8 Yes 4 1 Michigan 3 categories cryptorchidism) Carmichael et al. (2010) USA, Pregnant women participating 20/28 Maternal serum OR for a 1–10 ng/g lipid PBDE-100 1.02 0.74–1.35 Yes 5 1 California in a screening program change in the analyte PCB-153 0.84 0.30–2.46 Yes 5 1 HCB 0.95 0.79–1.13 Yes 5 1 p,p′-DDT 1 0.99–1.01 No 5 1 p,p′-DDE 1 0.95–1.00 No 5 1 Giordano et al. (2010) Italy, Rome Children enrolled at two 37/21 Mid pregnancy Above versus below median p,p′-DDE 1.18 0.47–6.91 Yes 5 1 Hospitals serum HCB 5.5 1.25–24.31 Yes 5 1 PCB (four congeners) 1.89 0.51–6.93 Yes 5 1 Rignell-Hydbom et al. Sweden Southen Sweden maternity 237/237 Early pregnancy Highest quartile versus lowest PCB-153 0.60 0.30–1.19 Yes 10 0 (2012) cohort serum p,p′-DDE 1.68 0.92–3.08 Yes 10 0 HCB 1.59 0.86–2.93 Yes 10 0 Jensen et al. (2015) Denmark Pregnancy screening registry, 75/300 Second trimester Highest tertile versus lowest 5cx-MEPP 0.89 0.44–1.80 Yes 9 1 Serum Institute amnion fluid 7cx-MMeHP 1.69 0.78–3.67 Yes 9 1 Toft et al. (2016) Denmark Pregnancy screening registry, 75/300 Second trimester Highest tertile versus lowest PFOS 0.69 0.35–1.38 Yes 9 1 Serum Institute amnion fluid CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 110 Bonde et al. Table IV Characteristics of six case-referent studies (36 risk estimates) addressing the risk of testicular cancer following exposure to endocrine disrupting chemicals. a b Reference Location Study population N Cases/ Exposure medium Exposure contrast Substance RR 95% CI CR Bias referents .......................................................................................................................................................................................................................................................... Hardell et al. Sweden Patients referred to hospital 58/61 Blood, diagnosed men Above versus below PCB (sum of 38 1.10 0.50–2.60 9 1 (2003) departments in 5 Swedish median in referents congeners) cities HCB 1.70 0.80–3.60 9 1 p,p′-DDE 1.70 0.80–3.70 9 1 Chlordane 1.30 0.60–2.80 9 1 44/45 Blood, mothers at time of sons diagnosis Above versus below PCB (sum of 38 3.80 1.40–10.0 9 1 median in referents congeners) HCB 4.40 1.70–12.0 9 1 p,p′-DDE 1.30 0.50–3.00 9 1 Chlordane 1.90 0.70–5.00 9 1 Hardell et al. Sweden Patients referred to hospital 44/45 Blood, mothers at time of sons diagnosis Above versus below PBDE 47, 99, 153 2.50 1.02–6.00 9 1 (2006) departments in 5 Swedish median in referents cities Biggs et al. USA, The general population 246/630 Blood, men when diagnosed Above 85th percentile p,p′-DDT 1.17 0.68–2.00 9 1 (2008) Washing- versus below 50th o,p′-DDT 1.30 0.67–2.53 9 1 ton State percentile in referents Chlordane (six 0.93 0.51–1.68 9 1 substances) HCB 0.85 0.37–1.96 9 1 Mirex 0.87 0.50–1.53 9 1 Dieldrin 0.79 0.44–1.41 9 1 DDE 0.61 0.32–1.14 9 1 McGlynn USA US servicemen who had 739/915 Serum, adult males (bio-banked, (mean storage Highest quartile versus DDT 1.13 0.71–1.82 11 0 et al. (2008) donated serum between 1987 time 14.2 years)) lowest DDE 1.71 1.23–2.38 11 0 and 2002 Chlordane 1.51 1.09–2.10 11 0 Mirex 1.24 0.90–1.74 11 0 HCCH 0.90 0.65–1.24 11 0 McGlynn USA US servicemen who had 736/913 Serum, adult males (bio-banked, (mean storage Highest quartile versus PCB (sum of 11 0.61 0.43–0.86 11 0 et al. donated serum between 1987 time 14.2 years)) lowest congeners) (2009b) and 2002 Purdue et al. Norway Citizens with serum stored in 49/51 Serum, men (pre-diagnostic serum samples Highest tertile versus o,p′-DDT 1.40 0.40–4.50 7 1 (2009) the Norwegian cancer from between 1972 and 1978 in the Norwegian lowest p,p′-DDT 2.10 0.60–7.20 7 1 Registry Janus Serum Bank) p,p′-DDE 2.20 0.70–6.50 7 1 Chlordane 2.30 0.60–7.20 7 1 Total PCB 1.30 0.50–3.80 7 1 Dieldrin 2.10 0.50–9.50 7 1 HCB 2.90 0.50–15.20 7 1 β-HCCH 1.80 0.50–6.10 7 1 λ-HCCH 1.10 0.20–5.00 7 1 Mirex 1.20 0.40–3.00 7 1 Cohn et al. The Child Health and 15/45 Early postpartum maternal serum p,p′-DDT 0.70 0.26–1.64 8 1 (2010) Development Studies p,p′-DDE 0.19 0.04–0.62 8 Review on EDC and male reproduction 111 The standardized data extraction form is provided in Supplementary Table 3. Risk estimates with 95% CI were extracted for each measured compound and outcome. Sperm concentration (sperm count) was taken as the most relevant and robust measure of semen quality despite the substantial within- and between- individual variation (Schrader et al., 1988), while other indicators such as motility and morphology were ignored. When risk estimates were available for groups of chemicals, for example, PCB and PBDE congeners and phthalate metabolites, we used the summed risk estimate, if provided. Otherwise, we selected indicator congeners such as PCB-153 and PBDE-100 reflecting levels of all respect- ive congeners. When risk according to several levels of exposure was reported, the highest level versus the reference category was chosen. In one study, the highest level was greater than the 90th percentile (Pierik et al., 2004); therefore, we used the next highest exposure category in that study to allow for theoretical inverse relationships. If the relevant relative risks were not reported but data were available, the authors of this paper computed risk estimates and CIs. Quality assessment Reporting Each publication was evaluated for completeness of reporting of the fol- lowing 11 study characteristics modified after Bonzini et al., (2007): (i) study design, (ii) sampling frame and procedures, (iii) inclusion and exclu- sion criteria, (iv) population characteristics of exposed/unexposed or cases/referents, (v) response rates reported or given implicitly, (vi) meth- ods for exposure measurements (reference to method for chemical ana- lysis or detailed description), (vii) criteria for outcome ascertainment, (viii) external quality assurance program of biochemical analyses (certified laboratory and/or participating in analyses of spiked samples from other laboratories), (ix) detection level, recovery and precision (CV) provided on all issues, (x) statistical analysis, and (xi) reporting of exposure– response relationship (Supplementary Table 3). We evaluated whether each of these study characteristics were described or not and assigned a value of one if the criterion was fulfilled and zero if not. Giving equal weight to each of the 11 items, we consid- ered completeness of reporting as sufficient if the sum of the 0/1 scores for each paper was ≥8(Bonzini et al., 2007). Completeness of reporting is not a direct measure of quality but high completeness is needed for adequate evaluation of bias and confounding. Bias and confounding The decision to limit the review to observational epidemiological studies applying biological monitoring for exposure assessment is considered of major importance to counteract biased findings because of differential recall of exposure often introduced by self-reports or interview informa- tion. For each study, other sources of bias were evaluated against a pre- defined list of seven potential sources of bias and confounding adapted from validated checklists in order to fit the type of studies and research questions addressed in this review (Shamliyan et al., 2010); these are as follows: (1) Reporting of tested hypotheses: Many studies perform multiple comparisons because numerous chemical compounds have been measured. A high risk of bias towards inflated risk estimates is likely if risk estimates are selectively reported compared to objectives or available data. (2) Sample size justification (power calculations and/or addressing sam- ple size in discussion): Small numbers of cases and/or exposed may increase the risk of false negative reporting and thus cause bias towards the null. USA, Continuous contrasting o,p′-DDT 0.77 0.37–1.33 North 25th–75th interquartile California range in controls Giannandrea Italy, Hospital cases and controls 50/48 Serum, extrapolated backwards from male adult Above versus below LOD Sum of p,p′-DDE 3.34 1.09–10.2 7 1 et al. (2011) Rome undergoing evaluation for values (0.2 ng/mL) and HCB fertility status CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 112 Bonde et al. Table V Studies reporting associations between prenatal and postnatal exposure and cryptorchidism or hypospadias without risk estimates (not eligible for meta-analysis). a b Reference Location Study Study N Outcome Bio- Measured Results CR Bias population design population specimen xenobiotics .......................................................................................................................................................................................................................................................... Hosie et al. Germany Chilldren Cross- 18/30 Cryptochidism Fat 26 persistent Median values of 21 among 26 chemicals were higher in cases 31 (2000) undergoing sectional samples organochlorine and two compunds reached statistical significance (HCB and surgery at one pesticides and PCBs. HCE) hospital Damgaard Denmark/ Danish–Finnish Case- 62/68 Cryptochidism Breast milk 27 biopersistent Median values of 14 among 27 chemicals were higher in cases. 90 et al. (2006) Finland prospective Birth referent POPs No single compound significantly elevated. Monte Carlo Cohort Study analysis of median values indicated higher levels in cases when the eight compounds with the highest was analysed (P = 0.03) Main et al. Denmark/ Danish–Finnish Case- 95/185 Cryptochidism Placenta 14 PBDEs Placenta: no difference between cases and controls for 90 (2007) Finland prospective Birth referent placenta tissue, individual congeners or sum of 14 congeners Cohort Study samples, 62/ breast milk Breast milk: no difference between cases and controls for sum 68 milk of 14 PBDE congeners, while seven individual congenres were samples significantly higher in cases Virtanen et al. Denmark/ Danish–Finnish Case- 95/185 Cryptochidism Serum 17 TCDDs and No significant difference between cases and controls with 90 (2012) Finland prospective Birth referent placenta PCDD⁄ Fs) and 37 respect to sum of 37 PCBs, PCDD or dioxin equivalents Cohort Study samples, PCBs Shekharyadav Northern Children with Case- 80/120 Hypospadias Serum Three HCH isomers, Higher serum concentrations of all nine pesticides in cases, b- 51 et al. (2011) India hypospadias and referent aldrin, dieldrin, endo hexachlorohexane (HCH), g-HCH, and p,p′-dichlorodiphenyl age-matched I, endo II, p,p′-DDT, -dichloroethylene (p,p′-DDE) significantly higher control children p,p,-DDE Choi et al. Republic No data Cross- 80/80 Hypospadias Urine DEHP, DBP, MEHP, In urine five of eight substances higher in cases, in serum three 21 (2012) of Chorea sectional Serum MBP, PA, n-NP and substances higher, inconsistent with respect to DEHP t-OP (contamination?) Fenichel et al. Southern French studies on Case- 46/106 Cryptochidism Cord Bisphenol A Mean unconjugated Bisphenol A marginally higher in cases but 61 (2012) France cryptorchidism in referent blood not significant (P = 0.38) Nice and Grasse Komarowska Poland Children 0–4 Case- 98/57 Cryptochidism Serum Bisphenol A Free serum BPA level in cryptorchid boys and in the control 31 et al. (2015) years admitted to referent group was not statistically significant but the conjugated and paediatric total serumBPA level was elevated in cryptorchid boys department CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. Review on EDC and male reproduction 113 (3) Selection bias due to attrition in follow-up studies and non-response in case-referent studies: Attrition/non-response >20% or attrition/ non-response differing by >10% in exposed/cases and unexposed/ referents was considered as a potential cause of bias in an unpredict- able direction. (4) Information bias (outcome ascertainment): Outcomes identified by patient recall in questionnaires, by interview or medical examination not blinded towards exposure status is a likely cause of inflated risk estimates because of differential exposure misclassification. (5) Confounding: Failure to account by study design or analysis for major potentially confounding factors including maternal age, parity, prema- turity, social class and ethnicity was considered as high risk of bias in unpredictable directions. Other potentially confounding factors were considered on an outcome specific basis: Cryptorchidism is a strong risk factor for testis cancer and low sperm count, sexual abstinence and maternal smoking during pregnancy is related to sperm count and the sons age is related to risk of testis cancer. Weak and/or uncertain risk factors such as paternal age, maternal consumption of coffee and alcoholic beverages, infections, malformed uterus and poorly controlled diabetes were not considered. (6) Measuring of confounding factors: Potential bias in an unpredictable direction was considered if measures of confounding factors were invalid or inadequate. (7) Exposure contrast: Studies contrasting exposure levels by the median split rather than by at least exposure-tertiles were considered at risk of bias towards the null because of insufficient exposure contrast. We considered selective reporting related to multiple testing (1), selection bias (3), information bias related to outcome ascertainment (4) and failure to account for major confounding factors (5) as the most important threats to validity. Accordingly, we categorized a study as at higher risk of bias if two or more of these sources of bias were present. Risk of bias across studies was evaluated by means of funnel plots to assess risk of publication bias. Meta-analysis We applied a stepwise approach addressing four different scenarios for the association between chemical exposure and included outcomes with increasing level of specificity. Only cryptorchidism, hypospadias and tes- ticular cancer were included in Steps (1)–(4) below, as none of the four papers addressing sperm count provided risk estimates. The following associations were analysed: (1) The association between all types of endocrine disrupting chemicals with all outcomes assuming that effects occur regardless of specific mechanisms and that the four outcomes are manifestations of the same underlying condition (testicular dysgenesis syndrome; Skakkebaek, 2002). This analysis assumes that all types of examined individual compounds are proxies for mixtures of substances that car- ry the same risk for the adverse reproductive outcomes. This assumption is very unlikely to be true, but the analysis is informative when considering global statements on the impact of endocrine dis- ruptors that do not single out individual substances or mixtures of particular concern (Bergman et al., 2013). (2) The associations between specific chemicals and all outcomes. Hereby, we account for different endocrine disrupting actions and potencies exhibited by different compounds and address the tes- ticular dysgenesis hypothesis stating that all male reproductive out- comes at least partly are manifestations of the same underlying causal agents operating in early life (Skakkebaek et al., 2001; Table VI Studies reporting associations between prenatal exposure and sperm count without risk estimates (not eligible for meta-analysis). a b Reference Location Study population Study N population Biospecimen Measured Results CR Bias design xenobiotics .......................................................................................................................................................................................................................................................... Mocarelli Italy, Exposed men are sons of women living near the Mixed 39 exposed /58 Maternal serum in Serum Average sperm counts were almost 81 et al. (2011) Seveso Seveso chemical plant at the time of the accident design referents exposed men. Referents samples halved in the exposed group and the in 1976. Controls were blood donors whose had an assumed TCDD effect was most pronounced among mothers were not exposed exposure of 10 ppt breast fed men Vested et al. Denmark Pregnancy cohort, male offspring Follow-up 169 Maternal serum PFOS PFOA but not PFOS was associated 11 0 (2013) with lower adjusted sperm concentration (p-trend = 0.01) PFOA Vested et al. Denmark Pregnancy cohort, male offspring Follow-up 173 Maternal serum PCB (six Sum PCB and p,p′-DDE was not 11 0 (2014) congeners) associated with sperm count DDE Axelsson Sweden Mainly young men presenting for military health Follow-up 112 Maternal serum Three DEHP None of the six phthalate 71 et al. (2015) board but also a few from the general population metabolites metabolites were associated with (through announcements in schools) Three DiNP Sperm count metabolites CR: completeness of reporting on a scale from 0 (low completeness) to 11 (high completeness). Bias and confounding: 1 = higher risk of bias, 0 = lower risk of bias. 114 Bonde et al. Table VII ORs (95% CIs) of the summary estimate of analyses for associations between endocrine disrupting chemicals and cryptorchidism, hypospadias and testicular cancer. Strata with three or less risk estimates not eligible for meta- analysis. I only available for meta-analyses without bootstrapping (one risk estimate per study). Exposures Outcomes N studies N risk estimates OR 95% CI I (min-max per study) ............................................................................................................................................................................................. All exposures All outcomes 19 70 (1–10) 1.11 0.91–1.35 Cryptorchidism 6 16 (1–7) 1.03 0.72–1.47 Hypospadias 7 18 (1–4) 1.13 0.86–1.50 Testicular cancer 6 36 (2–10) 1.20 0.78–1.89 Bias less likely All outcomes 5 22 (2–7) 1.12 0.81–1.57 Large studies All outcomes 9 34 (1–7) 1.06 0.85–1.33 Excluding studies c. testis studies with adult exposure data All outcomes 13 34 (1–7) 1.07 0.87–1.32 + Biopersistence All outcomes 17 66 (1–10) 1.15 0.92–1.44 − Biopersistence All outcomes 3 4 (1–2) 1.12 0.71–1.77 DDE All outcomes 13 13 (1) 1.35 1.04–1.74 31.91 Cryptorchidism 3 3 (1) Hypospadias 4 4 (1) 1.38 0.93–2.04 0.00 Testicular cancer 6 6 (1) 1.19 0.60–2.36 78.05 DDT All outcomes 6 6 (1) 1.07 0.81–1.42 0.00 Cryptorchidism 1 1 (1) Hypospadias 1 1 (1) Testicular cancer 4 4 (1) 1.12 0.82–1.55 0.00 PCB All outcomes 9 9 (1) 1.26 0.82–1.96 62.41 Cryptorchidism 2 2 (1) Hypospadias 4 4 (1) 1.11 0.61–2.01 54.45 Testicular cancer 3 3 (1) HCB All outcomes 7 7 (1) 1.19 0.87–1.62 40.94 Cryptorchidism 1 1 (1) Hypospadias 3 3 (1) Testicular cancer 3 3 (1) Skakkebaek, 2003, 2004). One study reported multiple PCB and studies with high completeness of reporting and lower risk of bias and PBDE congener specific risk estimates but no summary estimate confounding as defined above; and (v) performed separate analyses of (Carmichael et al., 2010). For this study, we selected the PCB-153 large case-referent studies with at least 75 cases. and PBDE-100 as indicators of the total exposure to these com- We computed a common risk estimate across studies by weighing pounds and did not include the other congener specificestimates the risk ratio (RR) or equivalent (OR or HR) by the inverse variance (12 estimates). Another study reported the p,p′-DDE exposure computed from the mean of the provided confidence limits. This is justi- estimate as well as an estimate for the sum of HCB and p,p′-DDE fied becausethe outcomes arerare(prevalence <5%). Random effects but no separate estimate for HCB (too few cases). In this case, we estimates are presented regardless of tests for heterogeneity since all used the sum of the p,p′-DDE and HCB estimate (Giannandrea studies are conceptually heterogeneous. Nevertheless we also provide et al.,2011). I statistics as a formal measure of heterogeneity. Bootstrapping techni- (3) The associations between all exposures and specificout- ques were used in analyses where the same study yielded more than comes, assuming that endocrine disrupting chemicals in one risk estimate, to account for multiple risk estimates per person in general carry a risk for some but not other male reproductive studies where risk estimates were reported for several compounds or disorders. outcomes. Bootstrapping with 500 repeats was applied by sampling (4) Each of the three outcomes individually in relation to specific com- among all included risk estimates, ensuring that each study only contrib- pounds or group of compounds under the assumption of specific uted one risk estimate to each bootstrapping step. Mean values across mechanisms and specific outcomes, but without an a priori ranking of bootstrapped steps were used as overall measures of common risk esti- more or less likely specific hypotheses. These exploratory analyses mates and confidence limits. These analyses were repeated for each have a narrow scope because data are limited. We only included outcome to account for heterogeneity in the exposure–outcome exposure–outcome estimates in the meta-analysis if four or more relation. risk estimates were available. All statistical analyses were performed using R and a significance level of 0.05. We also used this software to create funnel plots of the standard In sensitivity analyses, we: (i) regrouped compounds into persistent or error by the logarithm of the RR separately for each of the exposures rapidly metabolised substances with half-lives in the range of hours; (ii) and for the higher quality studies, and inspected these plots for evidence excluded studies with blood sampling after the diagnosis of testicular can- of publication bias. cer; (iii) examined the effect of each study on the overall estimate by excluding the studies one at a time; (iv) performed separate analyses of Review on EDC and male reproduction 115 Records identified through database searching Records identified through hand Pubmed: 1,253; Embase: 220 searches (n = 0) Records after duplicates removed (n = 1,314) Records screened Records excluded (n =1,314) (n = 1,204) Ful-text articles assessed Full-text articles excluded, for eligibility with reasons (n = 110) (n = 77) Papersincluded in qualitative synthesis (n = 33) Papersincluded in quantitative synthesis (meta-analysis) (n = 21) Figure 1 Flow diagram showing results of a systematic literature search to identify peer review papers addressing risk of male reproductive disor- ders following prenatal and infancy exposure to environmental endocrine disrupting compounds. (Mocarelli et al., 2011; Axelsson et al., 2015; Vested et al., 2013, Results 2014). There were 12 papers, based upon nine different study popula- The study base tions, which compared exposure levels in cases and referents but did We identified 33 papers that were based upon 28 independent stud- not provide risk estimates. An overview of eligible studies and risk ies reported 89 associations between measured levels of endocrine estimates are given in Tables II–IV while the results of three studies disrupting chemicals and one or more of the outcomes: cryptorchid- (four papers) addressing sperm count and six studies (eight papers) ism (Hosie et al., 2000; Longnecker et al., 2002; Pierik et al., 2004, addressing cryptorchidism and hypospadias without risk estimates are 2007; Bhatia et al., 2005; Damgaard et al., 2006; Main et al., 2007; displayed in Tables V and VI. Most studies addressed persistent orga- Brucker-Davis et al., 2008; McGlynn et al., 2009a; Fenichel et al., nochlorines such as DDE, the metabolite of DDT (18 associations), 2012; Chevalier et al., 2015; Jensen et al., 2015; Komarowska et al., PCBs (12 associations), DDT and HCB (each 12 associations), while 2015; Small et al., 2009; Trabert et al., 2012; Vesterholm et al., 2014; studies on rapidly metabolized compounds such as phthalate ethers Toft et al., 2016; Virtanen et al., 2012); hypospadias (Longnecker and Bisphenol were sparse. The systematic literature search seems et al., 2002; Bhatia et al., 2005; Small et al., 2009; McGlynn et al., to have been efficient since no additional papers fulfilling the eligibility 2009a; Carmichael et al., 2010; Giordano et al., 2010; Choi et al., criteria were identified from hand searches of reference lists or 2012; Jensen et al., 2015; Rignell-Hydbom et al., 2012; Trabert et al., reviews. The majority of the studies were case-referent studies, many 2012; Toft et al., 2016); testicular cancer (Hardell et al., 2003, 2004; nested within large well-defined cohorts, while two studies of sperm Biggs et al., 2008; Cohn et al., 2010; Giannandrea et al., 2011; count were cohort studies (Vested et al., 2013, 2014) and two were McGlynn et al., 2008; 2009b; Purdue et al., 2009); and sperm count cross-sectional studies (Mocarelli et al., 2011; Axelsson et al., 2015). Eligibility Included Screening Identification 116 Bonde et al. The case-referent studies included a total of 2446 cases of which 671 with <75 cases, we found an attenuated summary estimate (OR cases were cryptorchidism, 570 cases were hypospadias and 1205 1.06, 95% CI 0.85–1.33) (Table VII, ‘Large studies’) but with no indi- cases were testicular cancer. In addition 324 men took part in studies cation of publication bias in the corresponding funnel plot (data not of semen quality. Of all of the studies, 19 were published within the shown). Leaving out five estimates originating from studies addressing past 10 years, of these 13 were carried out in the United States of xenobiotics with rapid metabolism and excretion (phthalates) pro- America or Scandinavia. The completeness of reporting assessed by duced similar results (Table VII‘+biopersistent/-biopersistent’). No 11 items were considered high (sum score ≥8 of 11 items) for over single study or exposure had a major impact on the summary risk 65% of the papers. But less than half of the studies were considered estimate. Studies addressing p,p-DDT, p,p′-DDE and HCB contribu- at low risk of bias and confounding. Only five case-referent studies ted the most. included >75 cases. Using the form in Supplementary Table 3, we extracted detailed information on each study and including our evalu- ation of completeness of reporting and potential biases (data available Specific exposures and outcomes as one on request). In the following sections, we first present the results of entity (Hypothesis 2) the quantitative meta-analysis and next we review papers that did Data were available for substance specific analyses of risk of any of the not report risk estimates. reproductive disorders for the most frequently examined compounds (p,p′-DDT, p,p′-DDE, PCB and HCB). Results are summarized in Table VII. The summary estimate was above unity of all exposures but Global assessment: generic exposures and only statistically significantly elevated for p,p′-DDE (Fig. 3). outcomes as one entity (Hypothesis 1) The meta-analysis, including all 70 eligible estimates addressing the Generic exposures and specific outcomes risk for cryptorchidism, hypospadias or testicular cancer and giving (Hypothesis 3) equal weight to each of the xenobiotic exposure indicators, showed an overall risk estimate marginally above unity with rather narrow Cryptorchidism 95% confidence limits including unity, OR 1.11 (95% CI 0.91–1.35) Ten case-referent studies based upon six study populations provided (Table VII), and the estimate was similar in studies less likely to be 16 risk estimates for cryptorchidism (Fig. 4). The most frequently biased, OR 1.12 (95% CI 0.81–1.57). A funnel plot revealed some studied exposures were persistent organochlorine pesticides and indication of publication bias as studies with few cases and large PCBs which were measured in maternal pregnancy or postpartum standard errors seemed overrepresented among studies with ele- blood samples, or cord blood or breast milk, and one study used vated risk estimates (Fig. 2). In a subsequent analysis excluding studies Figure 2 Funnel plot of all risk estimates from all studies included in the meta-analysis (n = 70) of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and male reproductive disorders. Risk estimates with identical colors are derived from same study. Review on EDC and male reproduction 117 amnion fluid to assess exposure to phthalates and perfluorooctane exposure and early infancy exposure (Biggs et al.,2008; Giannandrea sulfonic acid (PFOS). Among the 16 risk estimates, nine were above et al.,2011; McGlynn et al.,2008). Only a single study was based on unity and four exceeded a relative risk of 2. The meta-analytic sum- maternal serum samples (Cohn et al.,2010). Altogether, the eight mary estimate was close to unity, OR 1.03 (95% CI 0.72–1.47), case-referent studies provided 36 risk estimates with DDT being the Fig. 4. Funnel plots did not indicate publication bias (data not shown). most frequently examined compound (seven estimates). Of these, 26 estimates were above unity and nine exceeded a relative risk of 2. The summary estimate was slightly elevated but not statistically significantly Hypospadias different from one, OR 1.20 (95% CI 0.78–1.89). The funnel plot was Nine case-referent studies based upon seven study populations compatible with publication bias since small studies contrary to large addressed links between xenobiotics and hypospadias and all studies studies reported a non-proportional large number of elevated risk esti- except one measured xenobiotics in maternal serum (Fig. 5). Five of mates (data not shown). the studies also reported on cryptorchidism. Among 18 risk esti- mates, 11 were above unity and seven were below and one relative risk exceeded 2. The summary estimate was close to unity, OR 1.13 Sperm count (95% CI 0.86–1.50), Table VII. Funnel plots did not indicate publica- No risk estimates available. tion bias (data not shown). Testicular cancer Specific exposures and specific outcomes Eight case-referent studies based upon six study populations examined (Hypothesis 4) risk of testicular cancer according to environmental exposure to poten- tial endocrine disruptive chemicals (Fig. 6). Two early Swedish studies Available data for specific exposure and specific outcomes are lim- measured xenobiotics at the time of diagnosis in the men and in a sub- ited. Four or more risk estimates were only available for four expos- set of their mothers at the same time (Hardell et al.,2003, 2006), ure–outcome combinations. Results of the conventional meta- while a few later studies targeted the hypothesis of prenatal origins of analysis without bootstrapping (since risk estimates are independent) testicular cancer assuming that persistent organic pollutants in prediag- are shown in Table VII. The summary relative risk estimates were nostic serum samples of young men was a useful proxy of in utero slightly above unity but none were statistically significant. Figure 3 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between prenatal and postnatal exposure to DDE and cryptorchidism, hypospadias or testicular cancer (N = 13). 118 Bonde et al. Figure 4 Forest plot of the risk estimates from studies included in the meta-analysis of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and cryptorchidism (n = 16). Figure 5 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between prenatal and postnatal exposure to endocrine disrupting environmental chemicals and hypospadias (n = 18). Studies not eligible for meta-analysis xenobiotics in fat samples, breast milk, placenta tissue, maternal serum Cryptorchidism or cord blood in cases and controls (Hosie et al., 2000; Damgaard Six case-referent studies based upon four independent study popula- et al., 2006; Main et al.,2007; Fenichel et al., 2012; Komarowska et al., tions did not provide risk estimates but compared concentrations of 2015; Virtanen et al., 2012), Table V. Altogether these papers report 104 exposure–outcome comparisons, ignoring that some studies Review on EDC and male reproduction 119 Figure 6 Forest plot of risk estimates (95% CI) from studies included in the boot strap meta-analysis of the association between exposure to endocrine disrupting environmental chemicals and testicular cancer (n = 36). examined compounds in more than one type of biological medium. three out of eight substances were elevated in serum samples com- The findings with respect to biopersistent organochlorines results are pared to controls but these results were not entirely consistent with largely consistent with the findings in the quantitative meta-analysis, results based upon urine samples (Choi et al.,2012). The other study showing limited evidence of associations. This also applies to studies examined nine biopersistent pesticides and observed higher concentra- with high completeness of reporting and less likely risk of bias, that tions in serum from cases for them all (Shekharyadav et al.,2011). addressed PBDE, TCDD, PCDD and organochlorine pesticides (Table V). Bootstrapping analyses of the subset of the seven substances Sperm count with highest breast milk concentrations indicated higher median values To date only three studies (four papers) explicitly studying xenobiotic in cases but this may be a chance finding since the overall analysis of all prenatal exposure to potential endocrine disrupting compounds rela- compounds was non-significant (Damgaard et al.,2006). The study on tive to semen quality in adult men have been published (Table VI). brominated flame retardants found increased levels in breast milk sam- These studies are highly heterogeneous with respect to design and ples among cases, but this result was not confirmed in analyses based xenobiotics and a meta-analysis is not appropriate. Rather than pro- upon corresponding placenta tissue samples, which a priori were viding risk estimates for reduced sperm count these studies report thought to provide the most accurate measure of fetal exposure (Main measures of average sperm counts according to the level of xeno- et al.,2007). Findings in other studies mainly addressing rapidly meta- biotic. Sons of women living near a chemical plant exposed to dioxin bolized contaminants were also reassuring, but these studies were con- due to an accident in 1976 that contaminated the surroundings were sidered at high risk of bias (Table V). identified in a seminal study and serum dioxin concentration at the time of conception during the period 1977–1984 was estimated Hypospadias based upon bio-banked maternal serum samples. Sperm counts in Two studies, not providing best practice reporting and considered at the exposed group of sons were compared with counts from men higher risk of bias, did not provide risk estimates but compared con- with assumed background exposure. Average sperm counts were centrations of xenobiotics in blood and urine samples in cases and con- almost halved in the exposed group and the effect was most pro- trols (Shekharyadav et al., 2011; Choi et al., 2012; Table V). In the first nounced among men who had been breastfed during childhood study, compounds with short biological half-lives were examined and (Mocarelli et al., 2011). The sample included only 39 exposed men 120 Bonde et al. and was too small to allow for exposure-response analysis. In a lactation and not precisely at the time of differentiation of the male Danish study, sons of mothers with biobanked serum samples pro- gonads, that based upon animal studies in the rat, is assumed to vided semen samples when they were 19–21 years of age and sperm take place in gestational week 8–14 (Welsh et al., 2008; Macleod count was examined according to tertiles of maternal serum concen- et al.,2010). Considering the long half-lives of the persistent chemi- tration of a range of compounds. There was no indication that PCBs, cals with little expected fluctuation of tissue levels across a few the DDT metabolite DDE or PFOS were associated with reduced months, measurements around the time of birth seems a reason- sperm count, but high level PFOA exposure was significantly asso- able proxy for exposure levels during early pregnancy although dis- ciated with reduced sperm concentration (Vested et al., 2013, 2014). tribution kinetics during pregnancy may be an issue (Verner et al., Finally, a Swedish cross-sectional study using biobanked maternal ser- 2013). This is entirely different for compounds with a rapid metab- um samples did not show associations between sperm count and a olism and excretion such as phthalates and bisphenol A, which fluc- range of phthalate metabolites (Axelsson et al., 2015). tuate markedly within individuals as shown in studies with repeated sampling. Thus, in a study where three blood samples were taken during pregnancy the intra-class coefficient of variation were for most phthalate metabolites between 20% and 40% (Cantonwine Discussion et al., 2014). However, at the group level serum levels of metabo- This is the first systematic review with meta-analysis that has rigorously lites were remarkably stable. The direction of bias may be in either evaluated the epidemiological evidence on prenatal and postnatal direction in the individual study but tends, on average, to cause bias exposure to endocrine disrupting compounds and male reproductive towards null. One of the included studies measuring phthalate disorders. A total of 33 papers provided 89 risk estimates on which metabolites in amnion fluid in the second trimester found no associ- we found no strong support for a global effect as a whole or on any ation with cryptorchidism and hypospadias even though one DEHP specific outcome. However, one of four specific compounds with suffi- metabolite was related to higher fetal testosterone and lower cient data to allow for meta-analysis were related to a moderate insulin-like factor III (Jensen et al., 2015). increased risk of all outcomes taken together (p,p′-DDE). Although Assessment of fetal exposure using maternal blood or milk samples only limited data were available for specific exposure–outcome associa- assumes that the placenta is not an efficient barrier for chemical tions, these findings do not seem to be due to increased risk of any sin- transfer from the maternal to the fetal circulation and that fetal gle included disorder (cryptorchidism, hypospadias or testicular exposure is proportional to maternal exposure. It is therefore note- cancer). Thus findings provide some support to the hypothesis of worthy that knowledge regarding the transport of persistent xenobio- shared prenatal etiology of these outcomes, but they also point to het- tics over the placenta to the fetus has been known for decades erogeneity of compounds classified as endocrine disruptors with (Needham et al., 1999; Mazdai et al., 2003). More recent findings respect to potential effects on male reproduction. indicate that rapidly metabolized compounds also pass through the The strength of this review in comparison with narrative reviews placenta and these have been detected in cord blood and amnion and global assessments is primarily that it includes, to our knowledge, fluid (Jensen et al., 2015). all published epidemiological evidence fulfilling predefined criteria using a systematic and transparent search of the literature. Although the findings of this review provide some evidence for environmental endocrine disruption of male reproductive function, the limitations of Exposure contrast within and across studies observational epidemiology and the outcomes reported in the few The most prevalent persistent organochlorine contaminants were available high quality studies precludes strong concluding statements. banned in high income countries during the 1970s and subsequently their tissue levels have declined substantially. However, the studies included in this review encompass studies based upon samples bio- Exposure assessment banked >50 years ago such as the Collaborative Perinatal Study with Reliable exposure assessment is essential in environmental epidemi- relatively high serum concentrations (Longnecker et al., 2002; Pierik ology and measurements of the compounds of interest are consid- et al., 2007; McGlynn et al., 2008, 2009b; Trabert et al., 2012) as well ered as the gold standard. We therefore only included studies with as studies based upon sampling much later (Damgaard et al., 2006; actual measurements of the chemicals in tissues as exposure assess- Main et al., 2007; Virtanen et al., 2012). Unfortunately, data are too ment based on external determinants (such as job title) is crude sparse to allow for a formal meta-analysis based upon the variation in when it comes to endocrine disrupting chemicals. Analyses of blood exposure levels in reference populations throughout time; however, or tissue concentrations of chemicals were performed by gas chro- no individual studies with large exposure contrast found evidence for matography/mass spectrometry and most studies provided data on a relationship between exposure and response. recovery and between batch coefficients of variation. Although differ- Since this review is focused upon prenatal exposures, we ences in sample preparation, analytical technique and units may invali- excluded a wealth of cross-sectional studies linking occupational and date comparisons of absolute exposure levels across studies, this is environmental exposure in adulthood with semen quality (for a not expected to affect risk estimates that are derived from within- recent review and a large collaborate study summary, see Bonde study contrasts of exposure levels. et al., 2008; Bonde and Giwercman, 2014). In contrast to testicular Misclassification of exposure at the susceptible periods of devel- cancer, it is well established that spermatogenesis is susceptible to opment most likely results in bias towards the null. Most studies short-term effects of reproductive toxicants not least in the measured the contaminants late in pregnancy, at birth or during workplace. Review on EDC and male reproduction 121 in one study (Biggs et al., 2008). Although adult serum concentrations Outcome ascertainment of persistent chemicals may to some extent reflect prenatal and early The male reproductive disorders addressed in this review are distinct postnatal exposure, it is obvious that varying rates of metabolism as diseases with different pathologies, clinical characteristics and age of well as additional lifetime exposures during infancy, childhood and appearance. Cryptorchidism and hypospadias were either defined by adulthood also contribute to exposure levels. The resulting misclassi- specified criteria applied in systematic examinations of newborns in fication most is most likely is towards the null. Accordingly it is not prospective studies or from medical records. Although criteria for surprising that the overall risk estimate was attenuated in the analysis both disorders may differ substantially and contribute to apparent excluding studies on testis cancer with adult serum data only. secular trends and spatial shifts in prevalence rates, it is unlikely that differences in outcome ascertainment across studies disrupt the internal validity and the relative risk estimates. No studies used par- Studies addressing sperm count ticipant questionnaires or interviews to define outcomes and there- Although the effects of endocrine disrupting chemicals on male fertil- fore recall bias is not an issue in studies included in this review. ity has been the focus of heated debate (Bonde et al., 2011), only three human studies have to date linked prenatal and postnatal exposure to xenobiotics with sperm count in adults (Mocarelli et al., The testicular dysgenesis syndrome 2011; Axelsson et al., 2015; Vested et al., 2013). This is probably hypothesis explained by the difficult and costly logistics of population-based We analyzed the male reproductive disorders, with the exception of semen studies and the need for maternal biospecimens or cord blood low sperm count where no risk estimates were available, as one stored decades before follow-up takes place. In the study by entity with reference to the testicular dysgenesis syndrome hypoth- Mocarelli et al., the findings of halved sperm counts in men whose esis on shared etiology of the four male reproductive disorders mothers were exposed to higher levels of dioxin when pregnant are (Skakkebaek, 2002) and with reference to experimental evidence sug- intriguing but call for cautious interpretation. Exposure levels in gesting multiple mechanistic pathways of endocrine disruption women living in the contaminated area are overlapping the back- (Toppari, 2002). The strength of the testicular dysgenesis syndrome ground exposure, participants are few, referents are from a conveni- hypothesis and the applicability of this concept as explanation of the ence sample and significant results were limited to the subgroup of pathogenesis of the majority of its components is an important pre- men breast-fed as infants (Mocarelli et al., 2011). The pertinent ques- requisite for obtaining statistically significant associations when testing tion is whether sperm counts in the exposed group are low (in the hypotheses I and II, where cryptorchidism, hypospadias and testicular range of 45 mill/mL) or whether sperm counts in the reference cancer were merged together as end points in statistical analyses. group are high (in the range of 80 mill/mL). It is well established that However, the heterogeneity of the pathogenesis underlying those small cross-sectional semen studies are highly vulnerable to selection conditions and their relative weak or moderate mutual clinical asso- bias (Larsen et al., 1998). Unfortunately it is hardly possible to repli- ciations might indicate that only a minor proportion of these male cate the findings. Results from the sole study that was explicitly reproductive abnormalities fit into the testicular dysgenesis syndrome designed to test the hypothesis on delayed effects on sperm count concept (Thorup et al., 2010), which may explain the generally non- are reassuring with respect to organochlorines (Vested et al., 2014), significant or only weakly statistically significant findings of our but less so with respect to the much less studied but ubiquitous per- analyses. fluorinated hydrocarbons (Vested et al., 2013). The Swedish study addressing phthalate metabolites did not reveal adverse effects but exposure assessment of the rapidly metabolized compounds with Studies addressing testicular cancer high within-day and between-days variation within individuals is an Although focus was on developmental disorders induced by chemical issue. Nevertheless single spot urine samples may distinguish different exposure during critical time windows in early life (Lee and Jacobs, exposure levels at the population level if study populations are suffi- 2015), there only exists one small study using prenatal or postnatal ciently large (Hagmar et al., 2006; Preau et al., 2010; Ye et al., 2011). bio-specimens to assess testicular cancer (Cohn et al., 2010). We Obviously there is a need to design future studies with sufficient therefore included six studies on testicular cancer assessing exposure power to examine exposure–response associations. With the aging by measurement of chemicals in serum samples obtained after large European birth cohorts with stored maternal serum samples, puberty. Hereby, we assume that serum levels of persistent chemicals this will become feasible in a few years. in young men or their mothers are reasonable proxies for prenatal and postnatal exposure. There is however some evidence to support Methodological issues this strong assumption. First of all, the biological half-lives of many of these compounds are counted in decades (Hagmar et al., 2006). Many of the included studies examined several compounds for which Moreover, studies in a Faroese birth cohort demonstrated strong risk estimates are not independent. This was accounted for by the intra-individual correlations between persistent chemicals in cord use of bootstrapping statistical techniques by which analyses are iter- blood and serum concentrations at 7 and 14 years of age, respect- ated several hundred times randomly including only one risk estimate ively (Barr et al., 2006). Another study found high correlations of per- per population in each analysis and providing an average risk estimate sistent chemical levels in serum samples in adult men obtained up to giving equal weight to studies with few and many risk estimates. This 10 years apart (Hagmar et al., 2006). Finally, a history of breastfeed- method does not provide statistics for heterogeneity which is consid- ing was related to elevated serum levels in men up to 40 years later ered less of a problem since study populations and risk estimates 122 Bonde et al. obviously are heterogeneous. Therefore, we used random effects most often addressing effects of individual chemicals one by one. models rather than fixed effects models in all analyses. In spite of However, this criticism ignores to some extent that persistent organic strong heterogeneity across studies with respect to study popula- pollutants are highly correlated and epidemiological studies addres- tions, exposures and outcomes, it is remarkable that all summary risk sing one indicator chemical, for example PCB-153, are in fact reflect- estimates from substance and outcome specific supplementary ana- ing exposure to a mixture of numerous other chemicals. For lyses are of similar magnitude spanning a relative risk from 1.03 to example, in a study of persistent organic pollutants and fertility, it was 1.26 when ignoring one outlying observation. not possible to distinguish between the potential effects of PCBs and The number of studies addressing specific chemicals and outcomes the DDT metabolite DDE (Axmon et al., 2006). were few and did not permit robust meta-analysis. Therefore, this review is primarily informative with respect to the overall association Non-monotonic dose–response relationships and the hypothesis on shared etiology. A number of studies did not report risk estimates but used other measures of associations. Another concern is the possibility of non-monotonic dose–response However, those findings did not seem to deviate from the overall relationships which may explain apparently inconsistent findings picture. across studies because of different levels and ranges of exposures Since we only included studies that documented exposure by (Vandenberg et al., 2012). This is particularly a potential problem objective analysis of compounds in biological media, the often when extrapolating from high occupational exposure levels to low encountered risk of bias due to differential recall of exposure or out- environmental levels and in risk assessment with a need to extrapo- come was bypassed. Selection bias related to prior knowledge about late from high doses in in vitro and in vivo animal studies to many-fold levels of contaminants in biological tissues among patients or lower environmental levels that humans encounter. However, the researchers therefore also seems unlikely. Few, if any, know their possibility of non-monotonic dose–response relationship is less of a own tissue concentrations of the investigated contaminants to which problem in large population-based studies capturing a broad low- all persons are exposed. Moreover, the nested case-referent design level exposure range (Lee and Jacobs, 2015). None of the large stud- adopted by the majority of the included studies promotes compar- ies included in this review with substantial exposure contrast and ability of cases and referents with respect to social and behavioral comprehensive evaluation of risk according to exposure strata indi- factors that may be related to exposure levels and thus indirectly cate the existence of such non-monotonic exposure–response rela- cause bias. The two main reasons for rating some studies at a higher tionships (Longnecker et al., 1997, 2002). risk of confounding was either due to lack of or insufficient adjust- ment for potential confounding factors such as maternal age and pre- maturity in case of congenital malformations with unpredictable Wider implications and conclusion direction of bias or small study size that was not justified by power The widely stated view that ubiquitous endocrine disrupting chemi- calculations which may increase the risk of publication bias. In fact cals in our environment play a substantial role in the development of funnel plots indicated overrepresentation of higher risk estimates in male reproductive disorders through prenatal and perinatal mechan- small studies with larger standard errors. Selective reporting did not isms is to some extent challenged by this review. Although the cur- appear to be a problem. All studies seemed to provide data on all rent epidemiological evidence is compatible with a small increased measured substances regardless of association. Finally, only a few risk of male reproductive disorders following prenatal and postnatal studies corrected for weight gain during pregnancy which may cause exposure to some persistent environmental chemicals classified as false positive associations due to dilution of the distribution volume endocrine disruptors, the evidence is limited. In this light, estimates for biopersistent chemicals (Verner et al., 2013), an issue if the out- of the burden of disease and costs of exposure to endocrine chemi- come is related to lower birthweight or congenital malformations. cals (Trasande et al., 2016) seem highly speculative, at least with Nevertheless, the findings must be interpreted in the light of the respect to male reproductive disorders. Future epidemiological stud- complexity of this research field. Thus, the meta-analyses strongly ies may change the weight of the evidence in either direction and the violates basic assumptions in that populations, chemical substances need for appropriate risk assessment of chemicals based upon and outcomes differ across studies (Rothman and Greenland, 1998), experimental evidence should not be ignored. There are insufficient which necessarily must result in substantial heterogeneity disregarding data on rapidly metabolized endocrine disruptors and specific expos- results of any statistical test. The meta-analytic summary estimates ure–outcome relations. A particular data gap is evident with respect and their confidence limits are not quantitatively reliable measures of to delayed effects on semen quality and testicular cancer. specific exposure–outcome relations but merely serve to provide an overview of the available limited evidence. Supplementary data Supplementary data are available at http://humupd.oxfordjournals. Cocktail effects org/. Humans are simultaneously exposed to a plethora of hundreds of xenobiotics. Some in vitro and in vivo animal studies have demon- strated combined effects of endocrine disrupting chemicals at levels Acknowledgements at which the individual chemicals do not induce observable effects (Kortenkamp, 2007; Kortenkamp, 2014). Because of this ‘cocktail Research secretary Hanne Tulinius is thanked for typing an early ver- effect’ of mixtures, scientist have criticized epidemiological studies for sion of the manuscript. Review on EDC and male reproduction 123 contrasts in blood levels of persistent organochlorines. Environ Health Perspect Authors’ roles 2008;116:269–277. Bonzini M, Coggon D, Palmer KT. Risk of prematurity, low birthweight and pre- Jens Peter Bonde and Elvira Braüner conceived and organized the eclampsia in relation to working hours and physical activities: a systematic study, sifted titles and abstracts and independently included and review. Occup Environ Med 2007;64:228–243. excluded papers. Esben Meulengracht Flachs carried out the statistical Brucker-Davis F, Wagner-Mahler K, Delattre I, Ducot B, Ferrari P, Bongain A, analysis. Clara Helene Glazer, Karin Sørig Hougaard, Birgit Bjerre Kurzenne JY, Mas JC, Fenichel P. Cryptorchidism at birth in Nice area (France) is associated with higher prenatal exposure to PCBs and DDE, as assessed by col- Høyer, Katia Keglberg Hærvig, Sesilje Bondo Petersen and Ina Olmer ostrum concentrations. Hum Reprod 2008;23:1708–1718. Specht reviewed and rated the individual papers with respect to data Cantonwine DE, Cordero JF, Rivera-Gonzalez LO, Anzalota Del Toro LV, extraction, completeness of reporting, bias and confounding. All of Ferguson KK, Mukherjee B, Calafat AM, Crespo N, Jimenez-Velez B, Padilla IY the above-mentioned authors as well as Aleksander Giwercman, et al. Urinary phthalate metabolite concentrations among pregnant women in Cecilia Høst Ramlau-Hansen, Lars Rylander and Gunnar Toft contrib- Northern Puerto Rico: distribution, temporal variability, and predictors. Environ Int 2014;62:1–11. uted to the design and provided critical comments. Susie Rimborg did Carmichael SL, Herring AH, Sjodin A, Jones R, Needham L, Ma C, Ding K, Shaw the systematic literature search. Jens Peter Bonde drafted the manu- GM. Hypospadias and halogenated organic pollutant levels in maternal mid- script to which all authors contributed and approved the final version pregnancy serum samples. Chemosphere 2010;80:641–646. format. Chevalier N, Brucker-Davis F, Lahlou N, Coquillard P, Pugeat M, Pacini P, Panaia- Ferrari P, Wagner-Mahler K, Fenichel P. A negative correlation between insulin- like peptide 3 and bisphenol A in human cord blood suggests an effect of endo- crine disruptors on testicular descent during fetal development. Hum Reprod Funding 2015;30:447–453. 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Human Reproduction UpdatePubmed Central

Published: Dec 14, 2016

References