BackgroundSeveral studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat.ObjectivesUsing the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate—mono-2-ethylhexyl phthalate (MEHP)—an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function.MethodsHuman fetal testes were recovered during the first trimester (7–12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH).ResultsWhatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10−4 M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10−4 M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3–positive germ cells, without modification of their proliferation.ConclusionsThis is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.

The bispecific antibody Emicizumab is increasingly used for hemophilia A-treatment. However, its specificity for human factors IX and X (FIX, FX) has limited its in vivo functional analysis to primate models of acquired hemophilia. Here we describe a novel mouse model allowing to examine Emicizumab function. Briefly, FVIII-deficient mice receive Emicizumab intravenously 24h before performing a tail clip-bleeding model. A second infusion with human FIX and FX is administered 5 min before bleeding. This approach generates consistent levels of Emicizumab (0.7-19 mg/dL for 0.5-10 mg/kg dose) and of both FIX and FX (85 and 101 U/dL respectively, after dosing 100 U/kg). Plasmas from these mice display FVIII-like activity in a diluted aPTT and in thrombin generation assays, similar to human samples containing Emicizumab. Emicizumab doses of 1.5 mg/kg and higher significantly reduced blood loss in a tail clip-bleeding model using FVIII-deficient mice. However, reduction was incomplete compared to mice treated with human FVIII concentrate, and no difference in efficacy between doses was observed. From this model, we deducted a FVIII-like activity of Emicizumab that corresponds to a dose of 4.5 U FVIII/kg (i.e. 9.0 U/dL). Interestingly, combined with a low FVIII dose (5 U/kg), Emicizumab provided additive activity to allow a complete bleeding arrest. This model could be useful for further in vivo analysis of Emicizumab.

BackgroundCadmium (Cd) is a common environmental pollutant and a major constituent of tobacco smoke. Adverse effects of this heavy metal on reproductive function have been identified in adults; however, no studies have examined its effects on human reproductive organs during development.ObjectivesUsing our previously developed organ culture system, we investigated the effects of cadmium chloride on human gonads at the beginning of fetal life, a critical stage in the development of reproductive function.MethodsHuman fetal gonads were recovered during the first trimester (7–11 weeks postconception) and cultured with or without Cd. We used different concentrations of Cd and compared results with those obtained with mouse fetal gonads at similar stages.ResultsCd, at concentrations as low as 1 μM, significantly decreased the germ cell density in human fetal ovaries. This correlated with an increase in germ cell apoptosis, but there was no effect on proliferation. Similarly, in the human fetal testis, Cd (1 μM) reduced germ cell number without affecting testosterone secretion. In mouse fetal gonads, Cd increased only female germ cell apoptosis.ConclusionsThis is the first experimental demonstration that Cd, at low concentrations, alters the survival of male and female germ cells in humans. Considering data demonstrating extensive human exposure, we believe that current environmental levels of Cd could be deleterious to early gametogenesis.

BackgroundPhthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro.Methodology/Principal FindingsTestes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10−4M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro.Conclusions/SignificanceWe evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.

Previously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P<0.0001), compatible with a slower clearance of von Willebrand factor in SR-AI-deficient mice. Interestingly, mutants associated with increased clearance (von Willebrand factor/p.R1205H and von Willebrand factor/p.S2179F) had significantly increased binding to purified SR-AI and SR-AI expressed on macrophages. Accordingly, propeptide/antigen ratios for these mutants were reduced in SR-AI-deficient mice. In conclusion, we have identified SR-AI as a novel macrophage-specific receptor for von Willebrand factor. Enhanced binding of von Willebrand factor mutants to SR-AI may contribute to the increased clearance of these mutants.

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The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10⁻⁵ M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation and cultured in the presence or not of 10⁻⁵ M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with ¹⁴C-MEHP. A 10⁻⁵ M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10⁻⁵ M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells.