Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting.

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 µM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

The (decision) tree of fertility: an innovative decision-making algorithm in assisted reproduction technique.

PURPOSE: Several mathematical models have been developed to estimate individualized chances of assisted reproduction techniques (ART) success, although with limited clinical application. Our study aimed to develop a decisional algorithm able to predict pregnancy and live birth rates after controlled ovarian stimulation (COS) phase, helping the physician to decide whether to perform oocytes pick-up continuing the ongoing ART path. METHODS: A single-center retrospective analysis of real-world data was carried out including all fresh ART cycles performed in 1998-2020. Baseline characteristics, ART parameters and biochemical/clinical pregnancies and live birth rates were collected. A seven-steps systematic approach for model development, combining linear regression analyses and decision trees (DT), was applied for biochemical, clinical pregnancy, and live birth rates. RESULTS: Of fresh ART cycles, 12,275 were included. Linear regression analyses highlighted a relationship between number of ovarian follicles > 17 mm detected at ultrasound before pick-up (OF17), embryos number and fertilization rate, and biochemical and clinical pregnancy rates (p < 0.001), but not live birth rate. DT were created for biochemical pregnancy (statistical power-SP:80.8%), clinical pregnancy (SP:85.4%), and live birth (SP:87.2%). Thresholds for OF17 entered in all DT, while sperm motility entered the biochemical pregnancy's model, and female age entered the clinical pregnancy and live birth DT. In case of OF17 < 3, the chance of conceiving was < 6% for all DT. CONCLUSION: A systematic approach allows to identify OF17, female age, and sperm motility as pre-retrieval predictors of ART outcome, possibly reducing the socio-economic burden of ART failure, allowing the clinician to perform or not the oocytes pick-up.

The effect of the 2004 Italian legislation on perinatal outcomes following assisted reproduction technology.

OBJECTIVE: To assess the perinatal outcomes of the first three years under the 2004 Italian reproductive legislation obligating transfer of all embryos resulting from insemination of < or =3 oocytes. STUDY DESIGN: We compared the perinatal results of clinical assisted reproductive technology (ART) pregnancies during the three years following the new Italian legislation with the previous three years. RESULTS: There were 583 and 571 clinical pregnancies during the respective periods. Before the law, the overall embryonic and fetal loss rates were significantly higher resulting in a significantly lower rate of live born infants and significantly fewer clinical pregnancies with at least one live born infant. Quadruplet and quintuplet pregnancies were entirely eliminated following the 2004 law but the neonatal mortality rate was not different between the two study periods. CONCLUSION: The 2004 Italian infertility legislation led to improved quantitative and qualitative outcomes of ART.

The effect of legislation on outcomes of assisted reproduction technology: lessons from the 2004 Italian law.

OBJECTIVE: To evaluate the effect of the 2004 Italian regulations (insemination of

Collagen content and growth factor immunoexpression in uterine lower segment of type IA osteogenesis imperfecta: Relationship with recurrent uterine rupture in pregnancy.

OBJECTIVE: The purpose of this study was to evaluate collagen content and platelet-derived growth factor, vascular endothelial growth factor, and connective tissue growth factor expression in the myometrium of the uterine lower segment from a patient with type IA osteogenesis imperfecta with recurrent uterine rupture and to evaluate the existence of a relationship between the rare recurrent uterine rupture and the tissue disorders of type IA osteogenesis imperfecta. STUDY DESIGN: Collagen content and platelet-derived growth factor, vascular endothelial growth factor, and connective tissue growth factor expression in the uterine lower segment were assessed in the patient with type IA osteogenesis imperfecta and in eight otherwise healthy ("control") patients. RESULTS: Type IA osteogenesis imperfecta contained less total collagen amount, with no difference in type III collagen expression and showed increased levels of platelet-derived growth factor and vascular endothelial growth factor in myometrial smooth muscle cells. No difference was observed in connective tissue growth factor expression. CONCLUSION: These findings confirm the diminished collagen amount in myometrium from osteogenesis imperfecta and show the presence of additional pathogenetic mechanisms. A relationship is hypothesized between the underlying myometrial biochemical modifications and the recurrent uterine rupture.

Immunohistochemical detection of insulin-like growth factor type I receptor and uterine volume changes in gonadotropin-releasing hormone analog-treated uterine leiomyomas.

OBJECTIVE: This study was undertaken to assess the relationship between insulin-like growth factor (IGF) type I receptor (IGF-I-R) expression in uterine leiomyomas after gonadotropin-releasing hormone (GnRH) analog administration and modifications in uterine size. STUDY DESIGN: Forty-six women with menorrhagia for uterine leiomyomatosis were treated monthly with leuprolide acetate depot 3.75 mg before undergoing surgery. The uterine volume before and after therapy was assessed by transabdominal ultrasonography. Immunohistochemical detection of IGF-I-R was performed on leiomyoma tissue samples. The relationship between IGF-I-R levels and uterine volume changes was analyzed. RESULTS: Uterine volume decreased after therapy. Patients with a lower immunohistochemical expression of IGF-I-R showed a larger decrease in uterine size. CONCLUSION: The shrinkage in uterine volume induced by GnRH analogs seems to be related to the observed reduction in IGF-I-R levels. So, the IGF-I/IGF-I-R system might be involved in leiomyoma growth, and the pharmacologic action of GnRH analogs on uterine leiomyomas might also be related to the effects on IGF-I-R expression.

Relationship between platelet-derived growth factor expression in leiomyomas and uterine volume changes after gonadotropin-releasing hormone agonist treatment.

The unopposed estrogen effect is the main cause of leiomyoma growth and is at the basis of the clinical use of gonadotropin-releasing hormone (GnRH) agonists. Platelet-derived growth factor (PDGF) has been indicated as the main growth factor involved, in vitro in the proliferation response of leiomyoma smooth muscle cells to estrogen stimulation. The aim of this article is to evaluate the mitogenic action of PDGF in vivo by studying the relationship between PDGF expression in leiomyomas and post-GnRH analogue treatment changes in uterine volume. Thirty-nine patients suffering from uterine leiomyomas were treated with leuprorelin acetate depot 3.75 mg for three cycles; 31 untreated patients were enrolled as control group. Uterine volume was determined twice by ultrasonography in each patient, the first time at admission and the second time after treatment in the study group and after 3 months in the control group. The change in the uterine volume was then evaluated. Patients underwent surgery, and PDGF immunohistochemical detection was performed on the obtained fibroid samples. Uterine volume decreased significantly after treatment, whereas just a poor modification was found in the controls. The decrease in the uterine volume was found to be statistically related to PDGF expression. Thus PDGF levels decreased in treated patients as compared with controls. The decreased PDGF production in leiomyomas after GnRH analogue treatment and the relationship between decreased PDGF expression and greater shrink age in uterine volume suggest that PDGF might have a mitogenic action on leiomyomas in vivo.