Human Fetal Testis Research Articles

Dynamics of HSD17B3 expression in human fetal testis: implications for the role of Sertoli cells in fetal testosterone biosynthesis.

Introduction: Androgens play a pivotal role in shaping male sexual characteristics, with testosterone being an essential hormone in orchestrating various developmental processes. Testosterone biosynthesis involves a series of enzymatic reactions, among which the 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) holds significance. While its role in adult Leydig cells is well established, its localization and importance during the fetal period remain less known, especially in humans. This study aims to delineate the dynamics of HSD17B3 expression in human fetal testes to clarify the contribution of specific cell types to testosterone biosynthesis. Methods: Using immunofluorescence staining, we investigated the expression pattern of HSD17B3 in human fetal and adult testicular tissues. Results and discussion: The findings of this study revealed a distinct temporal and cellular expression pattern of HSD17B3 protein in the fetal period. We detected its expression exclusively in Sertoli cells, the highest during the second trimester. This unique localization suggests the inclusion of fetal Sertoli cells in testosterone production during the critical masculinization-programming window. Furthermore, we demonstrated a shift in HSD17B3 expression from Sertoli cells to Leydig cells in adulthood, corroborating findings from rodent studies. This study sheds light on the intricate, still underexplored regulation of steroidogenesis during fetal development, whose disturbance might lead to testicular dysgenesis. Further research is warranted to elucidate the regulatory pathways governing the expression of HSD17B3 and its transition between Sertoli and Leydig cells, potentially paving the way for novel therapeutic interventions in disorders of sexual development.

Endocrine disruption and male reproductive disorders: unanswered questions.

Maternal exposure to endocrine-disrupting chemicals (EDCs) in human pregnancy is widely considered as an important cause of adverse changes in male reproductive health due to impaired foetal androgen production/action. However, the epidemiological evidence supporting this view is equivocal, except for certain phthalates, notably diethyl hexyl phthalate (DEHP). Maternal phthalate exposure levels associated with adverse reproductive changes in epidemiological studies are several thousand-fold lower than those needed to suppress foetal androgen production in rats, and direct studies using human foetal testis tissue show no effect of high phthalate exposure on androgen production. This conundrum is unexplained and raises fundamental questions. Human DEHP exposure is predominantly via food with highest exposure associated with consumption of a Western style (unhealthy) diet. This diet is also associated with increased exposure to the most common EDCs, whether persistent (chlorinated or fluorinated chemicals) or non-persistent (phthalates, bisphenols) compounds, which are found at highest levels in fatty and processed foods. Consequently, epidemiological studies associating EDC exposure and male reproductive health disorders are confounded by potential dietary effects, and vice versa. A Western diet/lifestyle in young adulthood is also associated with low sperm counts. Disentangling EDC and dietary effects in epidemiological studies is challenging. In pregnancy, a Western diet, EDC exposure, and maternal living in proximity to industrial sites are all associated with impaired foetal growth/development due to placental dysfunction, which predisposes to congenital male reproductive disorders (cryptorchidism, hypospadias). While the latter are considered to reflect impaired foetal androgen production, effects resulting from foetal growth impairment (FGI) are likely indirect. As FGI has numerous life-long health consequences, and is affected by maternal lifestyle, research into the origins of male reproductive disorders should take more account of this. Additionally, potential effects on foetal growth/foetal testis from the increasing use of medications in pregnancy deserves more research attention.

Isolation and In Vitro Culture of Germ Cells and Sertoli Cells from Human Fetal Testis.

In the human fetal testis, fetal germ cells (FGCs) are progressively surrounded by supporting Sertoli cells inside seminiferous cords. During the second trimester, the FGCs develop asynchronously and can be observed in several stages of development. However, the mechanism that regulates the transition between the different developmental stages as well as the formation of spermatogonia is currently not well understood. For this, it is necessary to develop suitable isolation protocols and a platform for in vitro culture of FGCs of different stages. Here, we report a method to isolate distinct populations of FGCs and Sertoli cells from second trimester human testis using a panel of conjugated antibodies for THY1, PDPN, ALPL, KIT, and SUSD2 for fluorescence-activated cell sorting (FACS) followed by in vitro culture up to 7days. This platform provides the base for cellular and molecular characterization of the different testicular cell populations to investigate the transition between FGCs and spermatogonia and shed some light on crucial processes of early human gametogenesis unknown until now.

Evaluation of Ex Vivo Adrenocorticotropic Hormone Responsiveness of Human Fetal Testis.

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero.

Antiepileptic drugs are endocrine disruptors for the human fetal testis ex vivo.

Valproic acid (VPA) has long been the most widely used antiepileptic drug (AED) for the treatment of epilepsy, bipolar psychiatric disorders, and migraine. However, long-term VPA treatment has several adverse effects on the male reproductive system notably on endocrine functions and/or spermatic parameters. In utero exposure of the fetus to VPA is well known to be associated with a higher risk of several congenital malformations including those of male reproductive organs. Subsequent generations of AEDs, such as carbamazepine (CARB) and lamotrigine (LAM), are considered safer and are currently recommended for women of child-bearing age with epilepsy. Because anomalies of the male genital tract mostly result from endocrine imbalance during fetal life, we hypothesized that AEDs could directly impair testis differentiation. We thus aimed at identifying and characterizing the effects of VPA, CARB, and LAM on the differentiation and function of the different testicular cell types, and at understanding the mechanisms underlying these effects. By using ex vivo culture of first-trimester human fetal testes, we show that VPA induces multiple endocrine disruptive effects, compared with the milder ones caused by CARB and LAM. AED also subtly altered the germ cell lineage in distinct manners. Transcriptomic analysis of VPA-induced alterations highlighted a very broad range of effects on the fetal testis. Overall, our results show that AEDs can behave as endocrine disruptors for the human fetal testis ex vivo. This is consistent with, and likely underlies, the VPA-induced male genital tract masculinization abnormalities observed in patients.

Expression of the endocannabinoid system and response to cannabinoid components by the human fetal testis

BackgroundCannabis consumption by pregnant women continues to increase worldwide, raising concerns about adverse effects on fetal growth and deleterious impacts on the newborn, in connection with evidence of placental transfer of cannabis compound. Cannabis action is mediated by the endocannabinoid system (ECS), which expression is well established in the brain but unknown in the developing testis. The fetal testis, whose endocrine function orchestrates the masculinization of many distant organs, is particularly sensitive to disruption by xenobiotics. In this context, we aimed to determine whether cannabis exposure has the potential to directly impact the human fetal testis.MethodsWe determined the expression of components of the ECS in the human fetal testis from 6 to 17 developmental weeks and assessed the direct effects of phytocannabinoids Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) on the testis morphology and cell functions ex vivo.ResultsWe demonstrate the presence in the human fetal testis of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and to a lower level anandamide (AEA), as well as a range of enzymes and receptors for the ECS. Ex vivo exposure of first trimester testes to CBD, THC, or CBD/THC [ratio 1:1] at 10−7 to 10−5 M altered testosterone secretion by Leydig cells, AMH secretion by Sertoli cells, and impacted testicular cell proliferation and viability as early as 72 h post-exposure. Transcriptomic analysis on 72 h-exposed fetal testis explants revealed 187 differentially expressed genes (DEGs), including genes involved in steroid synthesis and toxic substance response. Depending on the molecules and testis age, highly deleterious effects of phytocannabinoid exposure were observed on testis tissue after 14 days, including Sertoli and germ cell death.ConclusionsOur study is the first to evidence the presence of the ECS in the human fetal testis and to highlight the potential adverse effect of cannabis consumption by pregnant women onto the development of the male gonad.

P-739 High-throughput Single-cell Survey Reveal a Pathological Niche in Developing Down Syndrome Testis

Abstract Study question What are the characteristics of developing testes in DS, and how do these alterations affect gamete development and androgen production? Summary answer Molecular alterations start accumulating in every cell-type of DS fetal testis. Different cell components, compromised functions, and deviated cellular communications make up a pathological niche. What is known already DS is the most common genetic chromosomal disorder. The flat or slightly decreased live birth and the significantly prolonged lifespan contribute to a growing DS population. Their needs for reproductive care come into view. Reproductive health is crucial for spermatogenesis, male characteristics, and even brain behaviors. DS males experience normal puberty, but their sex hormones and associated gonadotropins have altered since the infant, implying testis niche cell dysfunction. Male infertility may result from germ cell and/or niche cell malfunction. A single-cell resolution survey could help thoroughly uncover the heterogeneity of DS by cell type. Study design, size, duration We applied high-throughput single-cell RNA sequencing to capture the transcriptomes of DS testis (n = 1). In parallel, testis collected from the fetus reported no genetic disorders were set as control (n = 1). Molecular and histological verifications were conducted with independent samples (3 versus 3). Samples enrolled were around 22 weeks of gestation. Participants/materials, setting, methods With ethics approval and informed consent obtainment, DS and control samples were included. DS fetus was confirmed with amniocentesis, while no genetic abnormality was reported during the prenatal examination of the diploid fetus. Single-cell RNA sequencing was conducted with the Micro-well platform, pseudotime trajectory analysis, cellular interaction analysis, and weighted gene co-expression network analysis were performed. Transmission electron microscopy, immunohistochemistry, Westerns, and qPCR were used to verify the main differences. Main results and the role of chance A total of 15349 cell transcriptomes, DS and control pooled, were compared and clustered. Major cell types were unabridged in both karyotypes. DS testis contains more Sertoli cells with an atypical expression profile, and more immune cells. DS cells exhibit a much higher level of ribosomal protein (RP genes and a significantlylower level of marker genes for each cell type, such as INSL3 for DS Leydig cell and PECAM1 for DS endothelial cell, etc. Corresponding to cell proportion difference, gene co-expression network and intercellular communication analysis also suggested an immune activation in DS. Furthermore, pseudotime analysis suggested a relative retardation and malfunction of DS cells. The main differences in gene expression were further verified. Cell proportion alteration was demonstrated with immunohistochemistry. Overwhelming RPs were shown by transmission electron microscopy. Western and qPCR were used to test the expression difference of RPs, cell-type specific functional genes, etc. Limitations, reasons for caution The sample size was limited, although verification experiments were applied with independent samples might increase the credibility. The sample size needs to be extended further. More precise molecular mechanisms of these alterations and clinical dysfunction remains to be elucidate. Wider implications of the findings This study described human fetal DS testis at single-cell level. It reveals a pathological niche of altered cell components, compromised cell function, and activated immune response. It provides further targets for complement and/or intervention to rescue the reproductive function and even improve brain behavior in DS patients, soon after birth. Trial registration number Not applicable

Reconstitution of Human Prospermatogonial Development from Human-Induced Pluripotent Stem Cells.

There is a scarcity of information regarding the molecular mechanisms underlying human germ cell development due to limitations in obtaining the relevant materials. Reconstitution of human germ cell development from pluripotent stem cells in vitro would provide critical insight into the etiology of various reproductive conditions and disorders, including infertility.Recently, we reported the in vitro reconstitution of human prospermatogonial development from human-induced pluripotent stem cells through human primordial germ cell (PGC)-like cells (hPGCLCs) using long-term cultured xenogeneic reconstituted testes. Here, we describe a method to generate M-prospermatogonia-like cells (MLCs) and T1-prospermatogonia-like cells (T1LCs), which closely resemble M- and T1-prospermatogonia present in second-trimester human fetal testes in vivo.

Expression of Intermediate Filaments in the Developing Testis and Testicular Germ Cell Cancer.

Cytokeratin and desmin expression have been associated with Sertoli cell maturity and the development of testicular germ cell cancer (TGCC). Thus, the present study aimed to characterize the expression of these intermediate filaments in normal testis development and TGCC. Cytokeratin and desmin were determined by immunohistochemistry and immunofluorescence in human fetal, and adult testis and tissue from patients with pre-invasive germ cell neoplasia in-situ (GCNIS) or invasive TGCC. Desmin was expressed in Sertoli cells of the human fetal testis, and the proportion of desmin expressing Sertoli cells was significantly reduced in the second trimester, compared with the first trimester (31.14% vs. 6.74%, p = 0.0016). Additionally, Desmin was expressed in the majority of Sertoli cells in the adult testis and TGCC samples. Cytokeratin was detected in Sertoli cells of human fetal testis but was not expressed in Sertoli cells of human adult testis. In patients with TGCC, cytokeratin was not expressed in Sertoli cells in tubules with active spermatogenesis but was detected in Sertoli cells in tubules containing GCNIS cells in patients with both pre-invasive and invasive TGCC. In conclusion, desmin was not associated with Sertoli cell maturation or progression to TGCC. However, cytokeratin appeared to be an indicator of impaired Sertoli cell maturation.

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo

BackgroundReduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats.MethodsThe effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry.ResultsKetoconazole reduced androgen production in testes from gestational weeks (GW) 7–21, which were subsequently divided into four age groups: GW 7–10, 10–12, 12–16 and 16–21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7–10 and 10–12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7–10 age group. Flutamide treatment in specimens aged GW 7–12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed.ConclusionsThis study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7–14 period—thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.

Organotypic Culture of Testicular Tissue from Infant Boys with Cryptorchidism.

Organotypic culture of human fetal testis has achieved fertilization-competent spermatids followed by blastocysts development. This study focuses on whether the organotypic culture of testicular tissue from infant boys with cryptorchidism could support the development of spermatogonia and somatic cells. Frozen-thawed tissues were cultured in two different media, with or without retinoic acid (RA), for 60 days and evaluated by tissue morphology and immunostaining using germ and somatic cell markers. During the 60-day culture, spermatocytes stained by boule-like RNA-binding protein (BOLL) were induced in biopsies cultured with RA. Increased AR expression (p < 0.001) and decreased AMH expression (p < 0.001) in Sertoli cells indicated advancement of Sertoli cell maturity. An increased number of SOX9-positive Sertoli cells (p < 0.05) was observed, while the percentage of tubules with spermatogonia was reduced (p < 0.001). More tubules with alpha-smooth muscle actin (ACTA, peritubular myoid cells (PTMCs) marker) were observed in an RA-absent medium (p = 0.02). CYP17A1/STAR-positive Leydig cells demonstrated sustained steroidogenic function. Our culture conditions support the initiation of spermatocytes and enhanced maturation of Sertoli cells and PTMCs within infant testicular tissues. This study may be a basis for future studies focusing on maintaining and increasing the number of spermatogonia and identifying different factors and hormones, further advancing in vitro spermatogenesis.

Cisplatin Effects on the Human Fetal Testis - Establishing the Sensitive Period for (Pre)Spermatogonial Loss and Relevance for Fertility Preservation in Pre-Pubertal Boys.

BackgroundExposure to chemotherapy during childhood can impair future fertility. Studies using in vitro culture have shown exposure to platinum-based alkylating-like chemotherapy reduces the germ cell number in the human fetal testicular tissues. We aimed to determine whether effects of exposure to cisplatin on the germ cell sub-populations are dependent on the gestational age of the fetus and what impact this might have on the utility of using human fetal testis cultures to model chemotherapy exposure in childhood testis.MethodsWe utilised an in vitro culture system to culture pieces of human fetal testicular tissues (total n=23 fetuses) from three different gestational age groups (14-16 (early), 17-19 (mid) and 20-22 (late) gestational weeks; GW) of the second trimester. Tissues were exposed to cisplatin or vehicle control for 24 hours, analysing the tissues 72 and 240 hours post-exposure. Number of germ cells and their sub-populations, including gonocytes and (pre)spermatogonia, were quantified.ResultsTotal germ cell number and number of both germ cell sub-populations were unchanged at 72 hours post-exposure to cisplatin in the testicular tissues from fetuses of the early (14-16 GW) and late (20-22 GW) second trimester. In the testicular tissues from fetuses of mid (17-19 GW) second trimester, total germ cell and gonocyte number were significantly reduced, whilst (pre)spermatogonial number was unchanged. At 240 hours post-exposure, the total number of germ cells and that of both sub-populations was significantly reduced in the testicular tissues from fetuses of mid- and late-second trimester, whilst germ cells in early-second trimester tissues were unchanged at this time-point.Conclusions In vitro culture of human fetal testicular tissues can be a useful model system to investigate the effects of chemotherapy-exposure on germ cell sub-populations during pre-puberty. Interpretation of the results of such studies in terms of relevance to later (infant and pre-pubertal) developmental stages should take into account the changes in germ cell composition and periods of germ cell sensitivity in the human fetal testis.

Activin A and Sertoli Cells: Key to Fetal Testis Steroidogenesis.

The long-standing knowledge that Sertoli cells determine fetal testosterone production levels is not widespread, despite being first reported over a decade ago in studies of mice. Hence any ongoing use of testosterone as a marker of Leydig cell function in fetal testes is inappropriate. By interrogating new scRNAseq data from human fetal testes, we demonstrate this situation is also likely to be true in humans. This has implications for understanding how disruptions to either or both Leydig and Sertoli cells during the in utero masculinization programming window may contribute to the increasing incidence of hypospadias, cryptorchidism, testicular germ cell tumours and adult infertility. We recently discovered that activin A levels directly govern androgen production in mouse Sertoli cells, because the enzymes that drive the conversion of the precursor androgen androstenedione to generate testosterone are produced exclusively in Sertoli cells in response to activin A. This minireview addresses the implications of this growing understanding of how in utero exposures affect fetal masculinization for future research on reproductive health, including during programming windows that may ultimately be relevant for organ development in males and females.

Development of the human fetal testis: Morphology and expression of cellular differentiation markers

A comprehensive immunohistochemical ontogeny of the developing human fetal testis has remained incomplete in the literature to date. We collected human fetal testes from 8 to 21 weeks of fetal age, as well as postnatal human testes at minipuberty, pre-pubertal, and pubertal stages. Immunohistochemistry was performed with a comprehensive panel of antigens targeting gonadocytes, Sertoli cells, fetal Leydig cells, peritubular myoid cells, and other hormonal and developmental targets.Testicular cords, precursor structures to seminiferous tubules, developed from 8 to 14 weeks of fetal age, separating the testis into the interstitial and intracordal compartments. Fetal gonadocytes were localized within the testicular cords and evaluated for Testis-Specific Protein Y, Octamer-binding transcription factor 4, Sal-like protein 4, and placental alkaline phosphatase expression. Fetal Sertoli cells were also localized in the testicular cords and evaluated for SRY-box Transcription Factor 9, inhibin, and anti-Mullerian hormone expression. Fetal Leydig cells were present in the interstitium and stained for cytochrome p450c17 and calretinin, while interstitial peritubular myoid cells were examined using smooth muscle α-actin staining. Androgen receptor expression was localized close to the testicular medulla at 8 weeks and then around the testicular cords in the interstitium as they matured in structure.Postnatal staining showed that Testis-Specific Protein Y remained positive of male gonadocytes throughout adulthood. Anti-Mullerian hormone, SRY-box Transcription Factor 9, and Steroidogenic factor 1 are expressed by the postnatal Sertoli cells at all ages examined. Leydig cell markers cytochrome p450c17 and calretinin are expressed during mini-puberty and puberty, but not expressed during the pre-pubertal period. Smooth muscle α-actin and androgen receptor were not expressed during mini-puberty or pre-puberty, but again expressed during the pubertal period.The ontogenic map of the human fetal and postnatal testicular structure and expression patterns described here will serve as a reference for future investigations into normal and abnormal testicular development.

β-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells.

Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. We investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780. In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780. Here we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.

Expression Patterns of Analgesic Metabolising Machinery in 1st and 2nd Trimester Human Fetal Liver and Gonads

Use of over-the-counter analgesics during pregnancy is widespread globally. Most analgesic compounds can freely diffuse through the placental feto-maternal interface and reach the developing fetus. Current literature suggests an endocrine disruptor (ED) potential of in utero exposure to these compounds. The liver is the primary site of contact with EDs in the fetus. Exposure of the fetal gonads can also alter reproductive function with potential intergenerational effects. We aimed to characterise the metabolic capability of these fetal organs. RNA sequencing was performed in 80 second trimester human fetal livers and 48 fetal gonads (balanced for fetal age and fetal sex). Samples were collected from elective terminations of normal pregnancies (liver 11-19 weeks, FeGo study: REC 04/S0802/21, and gonads 6-17 weeks, as previously described1. RNA was extracted and Illumina NextSeq was used to produce 76 bp single end (liver) or paired end 2x50 bp (gonads) sequencing reads. Reads were quality controlled, aligned to the human reference genome and quantified at gene regions. Statistical analyses involved an ANOVA model of two-way interactions between fetal sex and fetal age. All organs expressed phase I and II metabolising enzymes and drug transporters involved in the pharmacokinetic and pharmacodynamic pathways of over-the-counter analgesics. The human fetal liver expressed ABCC2, ABCC3, ABCC4 and ABCG2 receptors at similar levels between males and females. Expression of cytochrome p450 enzymes CYP2A6, CYP2C8, CYP2C9, CYP2E1, CYP3A4 involved in metabolism of the analgesics paracetamol and ibuprofen, all increased with gestational age in the liver. Expression of GSTM1, GSTP1, GSTT1, SULT1A1, SULT1A3, SULT1A4, SULT1E1, SULT2A1, UGT2B4, UGT2B7 and UGT2B15 metabolising enzymes also increased during gestation, while fetal hepatic GSTP1 expression showed a significant 2-way interaction between both sex and age. Fetal gonads expressed ABCC4 and ABCG2 transporters, with transcript levels demonstrating significant sex-specific and gestational age differences. Fewer analgesic metabolising enzymes were expressed in the gonads than the fetal liver, including CYP2E1, GSTP1 and SULT1A1, all significantly altered by gestation and fetal sex. Our results reveal expression of major analgesics metabolic and transport components within the human fetal liver, ovaries and testes between gestation weeks 7-19. Significant sex alterations in transcript levels also suggest sexually dimorphic metabolic activity of these organs during fetal life. In conclusion, analgesics can be transported into fetal liver and gonad cells and metabolised into bioactive forms, posing toxicity risks for the developing fetus.1. Lecluze E, Rolland AD, Filis P, et al. Dynamics of the transcriptional landscape during human fetal testis and ovary development. Hum Reprod. 2020;35(5):1099-1119.

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer

BackgroundClinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues.MethodsWe developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24–240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified.ResultsCisplatin exposure resulted in a significant reduction in the total number of germ cells (− 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (− 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (− 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure.ConclusionsThis is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

Dynamics of the transcriptional landscape during human fetal testis and ovary development

Dynamics of the transcriptional landscape during human fetal testis and ovary development

Expression of the Insulin-like Growth Factor System in First- and Second-Trimester Human Embryonic and Fetal Gonads.

Insulin-like growth factor (IGF) signaling is crucial for sex differentiation and development of Leydig and Sertoli cells in fetal mice testes. No such information is available for human embryonic and fetal testes and ovaries. To investigate presence and activity of the IGF signaling system during human embryonic and fetal ovarian and testicular development. Human embryonic and fetal gonads were obtained following legal terminations of pregnancies. Gene expression was assessed by microarray and qPCR transcript analyses. Proteins of the IGF system components were detected with immunohistochemistry and immunofluorescence analyses. Specimens were included from 2010 to 2017. University Hospital. Ovaries and testes from a total of 124 human embryos and fetuses aged 5 to 17 postconception weeks were obtained from healthy women aged 16 to 47 years resident in Denmark or Scotland. Gene expression analysis using microarray was performed in 46 specimens and qPCR analysis in 56 specimens, both sexes included. Protein analysis included 22 specimens (11 ovaries, 11 testes). IGF system members were detected in embryonic and fetal testes and ovaries, both at gene transcript and protein level. A higher expression of IGF regulators was detected in testes than ovaries, with a preferred localization to Leydig cells. These data indicate that the IGF system is active during very early gestation, when it may have a regulatory role in Leydig cells.

Molecular genetic analysis of AKR1C2-4 and HSD17B6 genes in subjects 46,XY with hypospadias

The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into 5α-dihydrotestosterone (DHT). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→DHT) mediated by SRD5A2 and 2) a "backdoor" pathway in which DHT is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate. We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias. The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies. Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes. Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically 5α-DHT. We selected four candidate genes involved in the "backdoor" pathway for the formation of 5α-DHT. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" 5α-DHT pathway during human sexual development, specifically, the differentiation of male external genitalia. These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization.