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Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Human male reproductive disorders (cryptorchidism, hypospadias, testicular cancer and low sperm counts) are common and some may be increasing in incidence worldwide. These associated disorders can arise from subnormal testosterone production during fetal life. This has resulted in a focus on in-utero environmental influences that may result in reproductive effects on the offspring in later life. Over recent years, there has been a dramatic increase in the scientific literature describing associations between in-utero environmental exposures (eg, industrial chemicals and pharmaceuticals) and subsequent reproductive outcomes in male offspring. This includes studies investigating a potential role for in-utero analgesic exposure(s) on the fetal testis; however, providing definitive evidence of such effects presents numerous challenges. In this review, we describe an approach to assessing the potential clinical relevance of in-utero (and postnatal) environmental exposures on subsequent male reproductive function using exposure to the analgesic paracetamol as an example.

Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks. With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.

Analgesic exposure during pregnancy may affect aspects of fetal gonadal development that are targeted by endocrine disruptors. We investigated whether therapeutically relevant doses of acetaminophen and ibuprofen affect germ cell (GC) development in human fetal testes/ovaries using and xenograft approaches. First-trimester human fetal testes/ovaries were cultured and exposed to acetaminophen or ibuprofen (7 d). Second-trimester human fetal testes were xenografted into mice and exposed to acetaminophen (1 or 7 d), or ibuprofen (7 d). To determine mechanism of action, a human GC tumor–derived cell line (NTera2) exhibiting fetal GC characteristics was used in addition to and rat models. Gonocyte (TFAP2C ) number was reduced relative to controls in first-trimester human fetal testes exposed in vitro to acetaminophen (-28%) or ibuprofen (-22%) and also in ovaries exposed to acetaminophen (-43%) or ibuprofen (-49%). Acetaminophen exposure reduced gonocyte number by 17% and 30% in xenografted second-trimester human fetal testes after treatment of host mice for 1 or 7 d, respectively. NTera2 cell number was reduced following exposure to either analgesic or prostaglandin E (PGE ) receptor antagonists, whereas PGE agonists prevented acetaminophen-induced reduction in NTera2 cell number. Expression of GC pluripotency genes, and genes that regulate DNA/histone methylation, also differed from controls following analgesic and PGE receptor antagonist exposures. Gene expression changes were observed in rat fetal testis/ovary cultures and after in vivo acetaminophen exposure of pregnant rats. For example, expression of the epigenetic regulator , was increased following exposure to acetaminophen in human NTera2 cells, rat fetal testis/ovary cultures, and in fetal testes and ovaries after in vivo exposure of pregnant rats, indicating translatability across experimental models and species. Our results demonstrate evidence of PGE -mediated effects of acetaminophen and ibuprofen on GC/NTera2 cells, which raises concerns about analgesic use during human pregnancy that warrant further investigation. https://doi.org/10.1289/EHP2307.

Abstract Context The endocrine function of human fetal adrenals (HFAs) is activated already during first trimester, but adrenal steroidogenesis during fetal life is not well characterized. Objective This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from first and second trimesters. Design and Setting Male and female HFA from gestational weeks (GWs) 8 to 19 were examined, including a total of 101 samples from 83 fetuses. Main Outcome Measure(s) Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS. Results Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2, and SULT2A1 were significantly higher in second trimester compared to first trimester (P < 0.05), whereas expression levels of 3β-HSD2 and ARK1C3 were unaltered between GWs 8 and 19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout the investigated period, with most enzymes expressed primarily in the fetal zone, except 3β-HSD1/2, which was expressed mainly in the definitive zone. Abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1071 to 2723 ng/g tissue), and testosterone levels were the lowest (2 to 14 ng/g tissue). Conclusions The expression profiles of HFA steroidogenic enzymes are distinct from first to second trimester, with no major differences between male and female samples. Intra-adrenal steroid hormone concentrations confirm that cortisol is produced throughout first and second trimesters, suggesting continued regulation of the hypothalamus-pituitary-adrenal axis during this entire period. Investigation of steroidogenic enzyme expression and steroid hormone secretion demonstrates that human fetal adrenal steroidogenesis is actively regulated throughout the first and second trimesters.

There is increasing evidence to suggest that fetal events can predetermine reproductive health and general wellbeing in adulthood, a process termed 'fetal programming'. This refers to the association between altered fetal growth/development and health disorders in adulthood e.g. the metabolic syndrome, which is linked to low male testosterone levels. Studies from both Europe and the USA have shown that adult male testosterone levels have been declining, independent of age. As low testosterone levels in aging men are associated with increased morbidity and mortality, this highlights the importance of investigating how testosterone levels are determined or potentially ‘programmed’ during fetal development. Evidence from human and rodent studies have shown that reduced fetal androgen exposure results in lower adult testosterone levels, although the mechanism(s) is unknown, to date. One way to explain how a fetal insult (e.g. androgen deficiency) could affect (testosterone producing) adult Leydig cells in adulthood, is if their progenitor cells were present during fetal life and were thus affected by such an insult. This hypothesis has been unexplored to date, due to the lack of a unifying/defining marker for adult Leydig progenitor cells. An earlier study promoted the hypothesis for the studies in this thesis, namely that chicken ovalbumin upstream promoter transcription factor-II (COUP-TFII) might constitute such a marker, as inducible knockout of COUP-TFII in pre-pubertal male mice results in failure of adult Leydig cells to develop. Therefore, the hypothesis which was explored in this thesis was that 'fetal programming' of COUP-TFII+ adult Leydig progenitor cells prior to their differentiation into adult Leydig cells, would explain how fetal events could predetermine adult testosterone levels. To investigate whether adult Leydig cells (ALC) develop from COUP-TFII+ interstitial cells, firstly an adult Leydig cell ablation/regeneration model was used, which involved a single injection of ethane dimethane sulphonate (EDS). This identified that in rats, ALC derive from COUP-TFII+ interstitial cells which do not express any other phenotypical adult Leydig or interstitial cell markers prior to differentiation. Secondly, COUP-TFII+ adult Leydig progenitor cells are abundant in the fetal testis and conserved across species, including man. Thirdly, fetal interstitial cells which differentiated into ALC, as evident from an ALC lineage tracer model, also expressed COUP-TFII. Overall, these findings suggest that the COUP-TFII+ interstitial cells which differentiate into ALC are 'adult Leydig progenitor cells'. The findings from this thesis also show that the identified adult Leydig progenitor cells express the androgen receptor (AR) in fetal life. Furthermore, experimental reduction of androgen action in fetal life in transgenic mice (AR knockout) or chemical manipulations to reduce fetal testosterone levels (di(n-butyl) phthalate; DBP exposure) resulted in a similar reduction (~40%) in progenitor cell numbers from birth through to adulthood. A parallel reduction of adult Leydig cell numbers across postnatal development was found in mice, but not rats, but as a result of altered fetal androgen action, both models showed evidence for compensated adult Leydig cell failure. This is defined as normal/low testosterone and elevated luteinising hormone (LH) levels. Cell-selective knockout of AR in peritubular myoid (PTM) cells (PTM-ARKO) or Sertoli cells (SC-ARKO) did not affect the numerical development of adult Leydig progenitor cells. To manipulate testicular testosterone action in postnatal life, rats were exposed to a potent AR antagonist, flutamide, which reduced the number of adult Leydig progenitor cells but did not affect ALC number/function. However, the combination of fetal DBP+postnatal flutamide exposure reduced adult Leydig progenitor cells and resulted in compensated ALC failure. Overall, these studies highlight the importance of fetal androgens for the normal development of adult Leydig progenitor cells and for the subsequent development of normally functioning adult Leydig cells. As fetal deficits in androgen exposure resulted in adult Leydig cell dysfunction, this thesis also investigated three separate models to determine whether increased fetal androgen exposure could increase/enhance adult Leydig progenitor cell development, resulting in a 'gain of adult Leydig cell function'. In the first model to increase fetal androgen exposure, pregnant dams injected with testosterone propionate (TP; 20mg/kg/day e14-21.5) were discarded, due to confounding factors including fetal growth restriction and aromatisation of TP. The second model utilised dihydrotestosterone (DHT; 10mg/kg/day), administered to pregnant dams, but there were no effects found in adulthood to male offspring. It was concluded that the administered dose was not sufficient to increase intratesticular testosterone levels in the fetus. The third model utilised an inducible nitric oxide synthase knockout (iNOS-/-) mouse model, for which previous evidence showed increased testis weight, Leydig and Sertoli cell number (~50%), and normal testosterone but low LH levels in adulthood. Stereological quantification showed an increase in the number of adult Leydig progenitor cells in postnatal, but not fetal life, which resulted in the conclusion that the observed changes were a consequence of postnatal effects. Finally, a potential mechanism to explain how DBP-induced androgen deficiency in fetal life, could result in adult Leydig cell dysfunction in adulthood was investigated. Analysis of testicular genes in adulthood, involved in the steroidogenic pathway, showed a reduction in 3b-hsd and StAR. The reduced StAR expression was associated with increased repressive histone methylation (H3K27me3) in its proximal promoter region, as demonstrated by a chromatin immunoprecipitation (ChIP) assay, qPCR, and densitometrical analysis. Accordingly, adult Leydig cells were shown to express increased H3K27me3 by immunohistochemistry, a change also evident in adult Leydig progenitor cells in the fetal testis. This would provide a potential mechanism to explain how fetal events can 'programme' adult Leydig cell testosterone production, namely via an epigenetic change to adult Leydig progenitor cells. In summary, the results in this thesis show how fetal events, including androgen action on progenitor cells, can potentially programme adult Leydig cell function and thus determine testosterone levels. As testosterone is crucial to man, the findings reported in this thesis may have important implications for the general health and longevity of man.

Context: The endocrine function of human fetal adrenals (HFA) is activated already during first trimester, but changes in adrenal steroidogenesis during fetal life are not well characterized. Objective: This study aimed to investigate HFA steroidogenesis by analyzing adrenal glands from 1st and 2nd trimester. Design and Setting: Male and female HFA samples from gestational week (GW) 8-19 were examined, including a total of 101 samples from 83 fetuses. Main Outcome Measure(s): Expression level of steroidogenic genes and protein expression/localization were determined by quantitative PCR and immunohistochemistry, respectively, and intra-adrenal steroid levels were quantified by LC-MS/MS. Results: Transcriptional levels of StAR, CYP11A1, CYP17A1, CYP21A2, CYP11B1/2 and SULT2A1 was significantly higher in 2nd trimester compared with 1st trimester (P<0.05), while expression levels of 3β-HSD2 and ARK1C3 were unaltered between GW 8-19. All investigated steroidogenic proteins were expressed in a distinct pattern throughout GW 8-19 with most enzymes expressed primarily in the fetal zone, except 3β-HSD1/2 which was mainly expressed in the definitive zone. The abundant steroidogenic enzyme expression was reflected in overall high intra-adrenal tissue concentrations of mineralocorticoids, glucocorticoids, and androgens; cortisol was the most abundant (1,071-2,723 ng/g tissue, in average) and testosterone levels the lowest (2-14 ng/g tissue, in average). Conclusions: The expression profiles of HFA steroidogenic enzymes are distinct from 1st to 2nd trimester, with no major differences between male and female samples. The intra-adrenal steroid hormone concentrations confirms that cortisol is produced throughout 1st and 2nd trimester, suggesting continued regulation of the HPA axis during this entire period.

Objectives: There is no consensus on the optimal treatment of young in-season athletes with anterior shoulder instability and limited data are available to guide return to play and treatment. The purpose of this study was to examine the likelihood of return to sport following an in-season shoulder instability event based on the type of instability (subluxation vs. dislocation). Additionally, injury factors and patient reported outcome scores administered at the time of injury were evaluated to assess the predictability of eventual successful return to sport and time to return sport during the competitive season following injury. Methods: Over two academic years, 45 contact intercollegiate athletes were prospectively enrolled in a multicenter observational study to assess return to play following in-season anterior glenohumeral instability. The primary outcomes of interest were the ability to return to sport and time lost from sport following an acute anterior shoulder instability event. Baseline data collection included sport played, previous instability events, direction of instability, and type of instability (subluxation or dislocation). Patient reported outcome scores specific to the shoulder were obtained at the time of injury and included the Western Ontario Shoulder Instability Index (WOSI), Single Assessment Numeric Evaluation (SANE), Simple Shoulder Test (SST), and American Shoulder and Elbow Score (ASES). All observed patients underwent a standardized accelerated rehabilitation program without shoulder immobilization, following the initial shoulder instability event. Subjects were followed during the course of their competitive season to determine return to play success and recurrent instability. Results: Thirty-three of 45 (73%) athletes returned to sport for either all or part of the season after a median 5 days lost from competition (IQR=13)(Fig 1). Of the 33 athletes returning to in-season sport following an instability event, 63% (22/33) completed the season. Athletes with a subluxation were 5.3 times more likely (OR=5.32, 95%CI: 1.00, 28.07, p=0.049) to return to sport following an initial in-season shoulder instability event when compared to those with dislocations. Logistic regression analysis suggests that the WOSI (OR=1.05; 95% CI 1.00, 1.09; p=0.037) and SST (OR=1.03, 95% CI 1.00, 1.05; p=0.044) administered after the initial instability event are predictive of ability to return to play . For every 1 point higher the WOSI scaled score at the time of injury, the athlete was 5% more likely to return to play during the same season. Time loss from sport following a shoulder instability event was inversely correlated with the WOSI (p=0.039), SST (p=0.007), and ASES (p=0.02) scores at the time of initial injury. The SST demonstrated the strongest correlation with time lost from sport, and for every 10 points higher the SST scale score was at the time of injury an athlete returned to sport 1.2 (95%CI: 0.4, 1.9) days sooner (p=0.004). Based on the logistic regression analysis, time lost from sport is predicted using the SST score after the initial instability event (Table 1). Conclusion: In the largest prospective study evaluating shoulder instability in mid-season contact athletes, we demonstrate that 73% of athletes return to play after one week. While the majority of athletes who return to sport complete the season, recurrent instability events are common regardless of whether the initial injury was a subluxation or dislocation.

Objectives: Debate continues regarding the optimal treatment of intercollegiate contact athletes with in-season anterior shoulder instability. Comparative evaluation of successful return to play (RTP) without recurrence in subsequent seasons after the index instability event has not been prospectively evaluated in this patient population. The purpose of this study was to examine return to sport and recurrent instability in the season that follows the index anterior in-season instability event. Methods: Over two academic years, 45 contact intercollegiate athletes treated nonoperatively or with arthroscopic stabilization were prospectively followed in a multicenter observational study to evaluate RTP and recurrent instability in the intercollegiate season following an initial in-season anterior glenohumeral instability event. The primary outcome of interest was successful RTP, defined as the ability to return to sport in the subsequent season without recurrent instability. Baseline data collection included sport played, previous instability events, direction of instability, type of instability (subluxation or dislocation), and treatment method (nonoperative or arthroscopic stabilization). The decision to pursue operative or nonoperative treatment was made at the discretion of the patient and surgeon. All nonoperatively treated athletes underwent a standardized accelerated rehabilitation program without shoulder immobilization. Surgical stabilization was performed arthroscopically in all cases and successful RTP was evaluated during the next competitive season after complete rehabilitation. All subjects were actively monitored during the course of their competitive season to determine return to play success and recurrent instability events. Results: A total of 45 intercollegiate contact athletes were treated for in-season anterior shoulder instability. Thirty-nine athletes had remaining NCAA eligibility and were followed through the subsequent competitive season. Of these, 10 elected to be treated nonoperatively while 29 elected for surgical repair. Of the group selecting nonoperative treatment, only 4 (40%) successfully RTP without recurrence during the subsequent season. Of the 29 athletes treated surgically, 26 (90%) were able to successfully RTP without recurrence the following season. Two athletes were cut from the team and one athlete sustained a recurrent instability event requiring revision stabilization. Athletes who underwent surgical reconstruction prior to the following season were 5.8 (95%CI: 1.77, 18.97, p=0.004) times more likely to complete the subsequent season without recurrent instability. Of the 29 athletes electing surgical stabilization, there was no difference (RR=0.95, 95%CI: 0.10, 9.24, p=1.00) in RTP between the 9 stabilized following a single instability event (90% RTP) and the 20 stabilized following multiple in-season recurrent instability events (89% RTP). Conclusion: Collegiate collision athletes with in-season shoulder instability are significantly more likely to successfully return to sport without subsequent instability events the following season if they undergo surgical repair in the off season.