Antenatal exposure to DES: lessons learned…future concerns

DES-exposed offspring : certain complications have no time limit and continued follow-up is necessary, 2007


The short- and long-term effects of the widespread use of diethylstilbestrol (DES) over 3 decades have become a distant memory for many clinicians. Others are too young to remember the flurry of activity in the early 1970s on the part of many medical centers to identify the offspring of women who were prescribed DES during their pregnancies.

This medication was given in an attempt to prevent multiple pregnancy-related problems such as miscarriage, premature birth, and abnormal bleeding.

The recognition of the association of DES with an increased incidence of cervical and vaginal cancers in very young women led the Food and Drug Administration to ban its use during pregnancy in 1971.

Other pregnancy-related problems for the daughters and genitourinary tract changes in the sons did not become apparent until years later.

Ongoing follow-up of these offspring has raised concerns for their future as well as their mothers’ future. Clinicians need to be up-to-date with current knowledge regarding risks for cancer and other health-related issues.

Abstract (Third-Generation Effects)

Animal studies have shown tumor growth in older third-generation mice (human equivalent to age 70).

Multigenerational studies in humans are currently underway.

Several small studies of teenage third-generation females have not shown the same type of changes as in their mothers. Sons of DES daughters are at increased risk for hypospadias.


  • Antenatal exposure to DES: lessons learned…future concerns, Obstetrical and gynecological survey, NCBI PubMed PMID: 17634156, 2007 Aug.
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DES and Cryptorchidism

EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, 2015


It was subsequently determined that exposed offspring of both sexes had increased risk for multiple reproductive disorders, certain cancers, cryptorchidism (boys), and other diseases, although the risk for sons is more controversial.

New data are emerging to implicate increased disease risk in grandchildren.


  • EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, Endocrine Reviews, Volume 36, Issue 6, Pages E1–E150,, 01 December 2015.

Informatively empty clusters with application to multigenerational studies

Recently, Kioumourtzoglou examined the impact of in-utero DES exposure among nurses on attention-deficit/hyperactivity disorder (ADHD) in their children

2019 Summary

Exposures with multigenerational effects have profound implications for public health, affecting increasingly more people as the exposed population reproduces. Multigenerational studies, however, are susceptible to informative cluster size, occurring when the number of children to a mother (the cluster size) is related to their outcomes, given covariates. A natural question then arises: what if some women bear no children at all? The impact of these potentially informative empty clusters is currently unknown.

This article first evaluates the performance of standard methods for informative cluster size when cluster size is permitted to be zero. We find that if the informative cluster size mechanism induces empty clusters, standard methods lead to biased estimates of target parameters. Joint models of outcome and size are capable of valid conditional inference as long as empty clusters are explicitly included in the analysis, but in practice empty clusters regularly go unacknowledged. In contrast, estimating equation approaches necessarily omit empty clusters and therefore yield biased estimates of marginal effects.

To resolve this, we propose a joint marginalized approach that readily incorporates empty clusters and even in their absence permits more intuitive interpretations of population-averaged effects than do current methods. Competing methods are compared via simulation and in a study of the impact of in-utero exposure to diethylstilbestrol on the risk of attention-deficit/hyperactivity disorder (ADHD) among 106 198 children to 47 540 nurses from the Nurses Health Study.

Study population

The proposed methods are motivated by a study of the effect of diethylstilbestrol exposure on thirdgeneration ADHD diagnosis in the Nurses Health Study II. The data consist of K=61 485 female nurses aged 25–42 in 1989 who returned a series of questionnaires in subsequent years and had no multiple sameyear births. In 2005 and 2013, nurses reported whether their children had been diagnosed with ADHD and analysis is restricted to concordant responses. The data are hierarchical in nature, with N = 106 198 children clustered within families identified by their mothers (nurses).

A key feature of the data is that cluster size (number of children) is potentially informative, as seen in Table 3: ADHD prevalence ranged from 5.62% in only-children to 3.22% in children from families of five or more children. Some of this relationship may be due to diethylstilbestrol exposure, whose rate was highest for nurses with no children (2.79%) and decreased to 1.18% for those with five or more children. Critically, 23% of nurses reported no live births and were thus excluded from previous analyses. To explore the impact of this decision on the conclusions of the analysis, we now consider the full population of nurses that met the eligibility criteria, this time including those without children.


The primary aim of the study was to quantify the effect of diethylstilbestrol on third-generation ADHD diagnosis, and we compared results of each analysis approach considered in the simulations. Logistic outcome models were adjusted for nurse’s exposure to diethylstilbestrol, smoking status, and year of birth. For the joint models, we modeled cluster size using a zero-inflated Poisson model (where the Poisson component adjusted for the same covariates and the zero inflation adjusted for exposure) in order to permit informative and non-informative emptiness. For the joint model that ignores empty clusters, we assumed a Poisson distribution, with a minimum size of one. We adopted a random intercepts model with exposure-dependent variance (as in the simulations), permitting correlation to depend on diethylstilbestrol exposure.


Estimates of marginal parameters can be found in Table 4. Diethylstilbestrol had a moderate adverse population-averaged (marginal) effect onA DHD risk, and estimates varied only somewhat across analyses: the independence estimating equations odds ratio estimate was 1.46 [95% confidence interval (CI) (1.19–1.78)] and was slightly larger than the cluster size weighted estimating equations estimate of 1.39 (1.13–1.71). Because these estimates are consistent for distinct parameters only under informative cluster size, these results (in light of the large sample size) suggest weak informativeness. As such, emptiness did not seem to have a large impact here, and the joint marginalized estimate fell between those of the estimating equations [1.41; 95% CI (1.14–1.73)].

Conditional parameter estimates can be found in Tables 5. The cluster-specific (conditional) estimates of the exposure-ADHD odds ratio were naturally much larger, but still varied little across analyses, ranging from 2.39 (1.38–4.12) under the outcome-only GLMM to 2.33 (1.33–4.06) under the complete joint model. The other covariate-outcome associations varied negligibly across conditional analyses.

Despite discrepant levels of correlation by exposure level (σ0 and σ1 are estimated to be 2.03 and 1.66 under the joint model), the variation in exposure effects across analyses is modest. This is because although there was a strong potential for informative cluster size (see Table 3), the actual level of informativeness was low (the estimate of the scaling parameter for the unexposed was −0.01).



Pharmacologic and Environmental Endocrine Disruptors in the Pathogenesis of Hypospadias

a Review – Current environmental health reports, 2018



Endocrine disrupting chemicals (EDCs) potentially have a role in causing hypospadias malformation through modifiable in-utero exposure. Considering the emerging literature on the role of potential endocrine disrupting substances on the occurrence of hypospadias and the potential to inform public health efforts to prevent the occurrence of these malformations, we have summarized the current literature, identified areas of consensus, and highlighted areas that warrant further investigation.


Pharmaceuticals, such as diethylstilbestrol, progestin fertility treatments, corticosteroids, and valproic acid, have all been associated with hypospadias risk. Data on exposure to dichlorodiphenyltrichloroethane and hexachlorobenzene pesticides, as well as non-persistent pollutants, particularly phthalates, is less consistent but still compelling. Improving exposure assessment, standardizing sample timing to relevant developmental windows, using clear case identification and classification schemes, and elucidating dose-response relationships with EDCs will help to provide clearer evidence. Promising directions for future research include identification of subgroups with genetic hypospadias risk factors, measurement of intermediate outcomes, and study of EDC mixtures that will more accurately represent the total fetal environment.

Exogenous Sex Steroids

Estrogens were the first chemicals to be studied in the context of maternal exposure and hypospadias. The synthetic nonsteroidal estrogen, diethylstilbestrol (DES), is a known carcinogen formerly administered to pregnant women to prevent miscarriage prior to evidence of adverse health effects and a lack of efficacy for that indication.

An early cohort study conducted in the Netherlands identified four cases of hypospadias among 205 sons of women exposed to DES in utero (~ 2% prevalence) versus 8 cases out of 8729 sons of mothers without DES exposure (0.09% prevalence). This study observed a strong association (prevalence odds ratio (pOR) 21.3; 95% CI 6.5–70.1) between maternal in utero DES exposure and hypospadiac son.

In a US cohort study, maternal DES exposure was related to a higher but not statistically significant increase in risk of offspring hypospadias with ten cases per 2552 live births from exposed mothers and three cases per 1336 live births from unexposed mothers (pOR 1.7; 95% CI 0.4–6.8).

A case-control study surveying 834 mothers with 251 hypospadiac children observed that women exposed to DES in utero were nearly five times more likely to have infants with hypospadias (OR 4.9; 95% CI 1.1–22.3).

A French, multigenerational cohort study also observed a relation between maternal DES exposure during pregnancy and increased prevalence of hypospadias for the next two generations, suggesting that the underlying biological mechanism may be epigenetic

Thus, studies on DES raise the possibility of an association with hypospadias. While no longer prescribed, DES is similar in chemical structure with other xenobiotic compounds and thus, the epidemiologic findings are still of relevance. It is important to note, however, that the studies of DES exposure all suggest a possible epigenetic effect on the development of hypospadias in later generations. While DES is historically important and chemically relevant to this discussion, its effect may derive from interaction with the maternal oocyte rather than the developing male penis itself.


  • Pharmacologic and Environmental Endocrine Disruptors in the Pathogenesis of Hypospadias: a Review, Current environmental health reports, NCBI PubMed PMID: 30578470, 2018 Dec.
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Pregnancy Drugs, Fetal Germline Epigenome, and Risks for Next-Generation Pathology

A Call to Action, Environmental and Molecular Mutagenesis,
Escher J, Robotti S, 19 March 2019.


Drugs taken during pregnancy can affect three generations at once:

  • the gestating woman (F0),
  • her exposed fetus (F1),
  • and the fetal germ cells that confer heritable information for the grandchildren (F2).

Unfortunately, despite growing evidence for connections between F0 drug exposures and F2 pathology, current approaches to risk assessment overlook this important dimension of risk.

In this commentary, we argue that the uniquemolecular vulnerabilities of the fetal germline, particularly with regard to global epigenomic reprogramming, combined withempirical evidence for F2 effects of F1 in utero drug and other exposures, should change the way we consider potential long-term consequences of pregnancy drugs and alter toxicology’s standard somatic paradigm.

Specifically, we

  1. suggest that pregnancy drugs common in the post-war decades should be investigated as potential contributorsto the “missing heritability” of many pathologies now surging in prevalence;
  2. call for inclusion of fetal germline risks in pregnancy drugsafety assessment;
  3. and highlight the need for intensified research to ascertain generational impacts of diethylstilbestrol (DES), a vanguard question of human germline toxicity.

Only by fully addressing this important dimension of transplacental exposure can we responsibly evaluate safety of drug exposures during pregnancy and convey the full scope of risks, while also retrospectively comprehending the generational legacy of recent history’s unprecedented glut of evolutionarily novel intrauterine exposures.

The imperative to intensify research on diethylstilbestrol F2s

“Finally, we ask that research to ascertain generational impacts of diethylstilbestrol (DES) should be intensified and broadened in scope. The DES disaster presents a paradigmatic question of human germline toxicity, and a unique opportunity to better understand generational impacts of this drug, and also the broader phenomenon of hormone disruption in humans. But while research on DES F2s has linked the exposure to a variety of pathologies, including increased risks of infertility, hypospadias, tumor growth, ovarian cancer, menstrual irregularities, and ADHD, by and large the research has been limited compared to the magnitude of exposure and the breadth of possible ensuing pathologies.

For example, the issue of neurodevelopmental outcomes and socio-sexual behavior strikes us as very important and mostly unexplored. Only one study has attempted to ascertain F2 neurodevelopmental impairments, and it indeed detected a link. Though a recent study found no evidence of same-sex orientation in female F2s borne of female DES exposed F1s, no other study has probed socio-sexual outcomes in F2s other either sex, whether through male or female F1s. We note that a recent animal study found these types of F2 effects when F0 dams were exposed to EDCs. In other words, research must think more broadly about F2 pathologies precipitated by DES exposure to also encompass the brain, cognitive ability,behavior, sexuality, and other crucial endpoints beyond the standard teratogenesis paradigm.

The urgency of more F2 research is critical not just for our affected families, but for the science of endocrine disruption generally and an entire population increasingly worried about generational effects of hormone-disrupting chemicals. DES is the bellwether of hormone disruption—given the combined existence of exposure records and possibilities for ascertainment of F2 outcomes, it uniquely shines a light on this crucial scientific question of germline and heritable effects of disrupted hormone signaling.

The primary research study looking at DES F2 outcomes in the United States is the National Cancer Institute (NCI) DES Combined Cohort Follow-Up Study formed in 1992. It consists of eight different cohort studies which totaled about 21,000 participants at inception and included DES-exposed F0 mothers, DES F1 daughters, and DES F1 sons, as well as unexposed mothers, daughters and sons. The oldest cohort started in 1971, the most recent in 1984, and two are British studies. Almost all the studies look at F2 outcomes via F1 exposed females, but not the F1 exposed males. In the initial decade, all participants of the largest of the eight cohorts, the Diethylstilbestrol Adenosis Project (DESAD), were physically examined yearly. Since that time the research inputs consist of questionnaires every five years.
While some subjects have been lost to the study due to location mobility and death, extensive attempts are made to seek all participants. Decreasing ability to do robust follow-up concerns us. We are just beginning to appreciate the effects on F2s, and indeed many effects on F1s as well, and while several other teams have published significant DES studies, many of those other efforts lack access to a database of people with confirmed medical histories of DES exposure, which presents a limitation.

A withering of efforts from what had been the primary source of research on the effects of DES represents a loss of an opportunity to learn critical lessons for understanding what may be at stake when early germ cells undergo programming in a foreign hormonal milieu.”



Grandmaternal DES and ADHD in Children

Dr Kioumourtzoglou‘s reply to comments in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

We appreciate the interest that Drs Ryan and Smith and Dr Costas have expressed in our article.

Drs Ryan and Smith raise the issue of exposure misclassification and the potential problem of overreporting of grandmaternal diethylstilbestrol (DES) exposure by mothers whose children have attention-deficit/hyperactivity disorder (ADHD).

First, we were not clear in the article, but the κ of 0.74 for nurse and grandmother reporting was specific for DES.

We also found that the DES-specific κ value did not vary by ADHD status.

Concern for biased recall does need to be considered, and our results should indeed be interpreted cautiously, while hopefully spurring more work in this area.

That said, some aspects of our study differ from the military study Drs Ryan and Smith reference.

When the nurses reported their mothers’ DES exposure (1993), there had been no human studies of any third-generation effects and possibly only 1 mouse study of tumors. Thus, the level of attention to potential anthrax effects on those directly exposed was not there for third-generation DES effects in 1993.

In addition, the nurses were not queried about ADHD in their children until, at earliest, 12 years after the question about their mothers’ DES exposure (2005). So the possibility of priming the nurse to think about a possible DES-ADHD association by asking about exposure and outcome at the same time was avoided.

Additionally, the specificity of elevated effects in the first trimester would argue against recall bias. In our models, we simultaneously included terms for unknown trimester DES exposure and exposure in other trimesters as a check for confounding, which also works as a check for recall bias in this case.

Indeed, one might expect that nurses falsely recalling grandmaternal DES exposure would be more likely to report that they did not know the trimester of exposure, yet that group did not exhibit increased odds of ADHD.

In addition to these points, in responding to this letter we conducted the same analysis restricted to the 18 792 F0 women (42 097 F2 children) who provided information themselves about DES use during pregnancy with F1, instead of F1 reporting. In these analyses, we found virtually the same result, although with wider 95% CI as expected with the smaller numbers: adjusted odds ratio, 1.31 (95% CI, 1.00-1.71).



Grandmaternal DES and ADHD in Children – Costas’ Question

Dr Costas editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“I read with interest the article “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits” by Kioumourtzoglou et al,  published in JAMA Pediatrics.

Using a valuable cohort of 47 450 women in the Nurses’ Health Study II, the authors found that exposure to the potent synthetic estrogen diethylstilbestrol during pregnancy was associated with increased risk of attention-deficit/hyperactivity disorder (ADHD) in the grandchildren.

They reasonably conclude that “diethylstilbestrol exposure is associated with multigenerational neurodevelopmental deficits.”

This result is a valuable contribution to the literature on etiology of neurodevelopmental disorders, adding further evidence for the role of endocrine-disrupting chemicals as risk factors for ADHD.

The etiology of ADHD is complex, involving a combination of genetic as well as environmental risk factors.

Heritability for ADHD has been estimated as 70% to 80%.

Ehe genetics basis of ADHD is partly owing to many variants of individual low effect scattered along the genome.” …



Grandmaternal DES and ADHD in Children – Ryan’s Question

Drs Ryan and Smith editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“We read with interest the publication by Kioumourtzoglou et al, identifying a link between grandmaternal diethylstilbesterol (DES) exposure and attention-deficit/hyperactivity disorder in children.

We certainly agree with the JAMA Pediatrics Editorial that this transgenerational association is novel and provocative.” …


  • Continue readingGrandmaternal Diethylstilbesterol and Attention-Deficit/Hyperactivity Disorder in Children, on JAMA Pediatrics doi:10.1001/jamapediatrics.2018.3737, December 2018.
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Effect of DES exposure on fetal testis development and function

Human Reproduction Update, human experimental data systematic review, 2019



Overall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.


The objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.


For identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. Abstracts were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.


A total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.


Direct experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility.


Development of the male reproductive system and its subsequent function is impacted by events that occur in utero. Perturbations in testicular development or function during fetal life may result in male reproductive disorders that present postnatally. This includes anatomical abnormalities identified at birth, such as cryptorchidism and hypospadias, or disorders presenting in adulthood, including testicular cancer or infertility. These associated disorders are collectively referred to as the testicular dysgenesis syndrome (TDS). The development of TDS has been shown in rats to be influenced by a reduction in androgen production or action during a key period of fetal life, known as the masculinization programming window (MPW). The increasing incidence of TDS disorders over recent decades, highlights the potential importance of environmental impacts in their etiology. Environmental factors that have been proposed to affect fetal testis development and predispose to TDS disorders include environmental chemicals (e.g. plasticizers and pesticides), pharmaceuticals (e.g. analgesics, metformin and diethylstilboestrol) and lifestyle factors (e.g. diet, alcohol and smoking).

Pharmaceuticals : Diethylstilboestrol

Diethylstilboestrol (DES) is a synthetic estrogen that was used clinically to prevent spontaneous miscarriage and pre-term labor from the 1940 s until the early 1970 s. DES was withdrawn from clinical use after the demonstration of a causal role in the development of vaginal carcinoma in girls born to exposed mothers. In addition to the effects on female offspring, an association with structural abnormalities of the male reproductive tract was also described including epididymal cysts, microphallus and testicular hypoplasia.

Animal studies

Animal studies involving in-vitro culture of rat and mouse fetal testis, have reported a reduction in testosterone production following exposure to DES, similar to the results of previous in-vitro studies involving fetal mice and in-vivo studies in rats.


For TDS disorders, which are linked to a reduction in androgen action during fetal life, there is conflicting evidence regarding their association with maternal DES exposure. Three studies have reviewed the literature relating to exogenous estrogen exposure and male reproductive disorders. Whilst early studies reported that hypospadias was significantly associated with DES exposure, it has subsequently been pointed out that this related to urethral abnormalities resulting from exposure to exogenous estrogens (including DES), which may have resulted from abnormalities in penile development rather than an effect on urethral formation as a result of reduced androgen exposure. The meta-analysis of all available evidence revealed a significant association between DES exposure and hypospadias; however, it was concluded that any effect of DES on hypospadias is likely to be small. For cryptorchidism, an increased risk in association with DES exposure is reported; however, this was dependent on the statistical model used and was indicative of heterogeneity. A subsequent cohort study has reported an association between in-utero exposure to DES and an increased risk of cryptorchidism, however, only for those in whom the initial exposure occurred prior to the 11th week of gestation with no significant association following exposure after 11 GWs. Studies have demonstrated no effect of prenatal DES exposure on sperm counts or fertility; however, this is in contrast to a previous study demonstrating an association between prenatal exposure to DES and semen parameters in adult men. Importantly, this study included analysis of men born to a large cohort of mothers who participated in an RCT involving DES exposure during pregnancy.

Experimental evidence from human studies

To date, only two studies have investigated the effect of DES exposure on the human fetal testis (Table VI). In-vitro organ culture of first trimester human fetal testis exposed to DES (10−5 to 10−6 M) for 3 days did not alter testosterone production. Interestingly, this study compared effects of DES exposure in rodent and human fetal testis demonstrating contrasting results between species using an identical experimental system.

In a separate study using the xenograft model, exposure to DES (100μg/kg, three times weekly) for 35 days resulted in no significant difference in testosterone production by second trimester (15–19 GW) testis tissue. Interestingly, host mouse seminal vesicles were significantly increased in weight, which was indicative of increased testosterone production from the xenografted tissue over the entire grafting period. The reason for this unexpected increase in testosterone is unclear.


Whilst rodent studies have indicated a profoundly negative effect of DES exposure on testosterone production by the fetal testis, experimental studies utilizing human fetal testis tissues have failed to identify similar effects, which may relate to the presence of ESR1 in rodent Leydig cells, and the absence of this estrogen receptor in human fetal testis. Epidemiological data suggests that any potential effect of DES exposure on male reproductive development is likely to be of small magnitude. Taken together the results suggest an important species difference in terms of DES effects on fetal testosterone production which may explain why this frequently results in the development of male reproductive disorders in rodents, whilst associations between DES and subsequent male reproductive disorders in humans are rather modest. Whilst DES is unlikely to be used in pregnant women in the future, the findings of this study offer some reassurance regarding the potential of low-level exposure to environmental estrogens to affect human male reproductive development, given their extremely low potency compared with DES and the high exposures that resulted from therapeutic use of DES.


  • Full study (free access) : Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data, Human Reproduction Update,, 14 March 2019.
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