Leopoldina Scotti

Administration of a gonadotropin-releasing hormone agonist affects corpus luteum vascular stability and development and induces luteal apoptosis in a rat model of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotropins followed by the administration of human chorionic gonadotropin (hCG) to trigger the final steps of oocyte maturation. Gonadotropin-releasing hormone (GnRH) analogs are thought to be effective in preventing this complication and a clinical trial has found a lower incidence of OHSS in patients treated with these molecules. Our aim was to analyze the in vivo effect of a GnRH-I agonist on corpus luteum development and regression, ANGPT-1, ANGPT-2 and Tie-2 protein expression and luteal blood vessel stabilization, the expression of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 side-chain cleavage enzyme (P450scc) and cell proliferation, in ovaries from an OHSS rat model. To this end immature female Sprague-Dawley rats were hyperstimulated and treated with a GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) administration until the day of hCG injection for 5 consecutive days. Blood and tissue samples were collected 48h after hCG injection. Vascular endothelial growth factor VEGF levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxylin and eosin (H&E) stained slides. Luteal blood vessel stability, cell proliferation and apoptosis were assessed by immunohistochemistry for SMCA, PCNA, and TUNEL, respectively. P450scc, StAR, FLK-1, ANGPT-1, ANGPT-2, Tie-2 and PCNA protein levels were evaluated by Western blot from dissected corpora lutea (CL). The treatment with the GnRH-I agonist significantly decreased serum progesterone and estradiol levels as well as P450scc and StAR protein expression in the untreated OHSS group. In addition, the agonist significantly decreased the number of CL in the OHSS group, as compared with the untreated OHSS group. In the OHSS group, the area of periendothelial cells in the CL was larger than that of the control group. However, the treatment with the GnRH-I agonist significantly reduced the area of periendothelial cells in the CL in the OHSS group. The luteal levels of ANGPT-1 and its receptor Tie-2 significantly increased in the OHSS group when compared with the control group. Conversely, the administration of the GnRH-I agonist significantly decreased the levels of these factors in the CL from the OHSS group, as compared with the untreated OHSS group. In addition, the treatment with the GnRH-I agonist reduced the diameter of CL and decreased CL cell proliferation as compared with that observed in the untreated OHSS group. Finally, the GnRH-I agonist increased apoptosis in the CL from the OHSS group. In conclusion, these results show that GnRH-I agonist exerts diverse actions on the CL from a rat OHSS model. The decrease in P450scc, StAR, ANGPT-1 and Tie-2 expression, blood vessel stability and luteal proliferation leads to CL regression in the ovaries from OHSS rats. Moreover, our results suggest that the downregulation of ANGPT-1 and its receptor is a possible mechanism whereby GnRH-I agonists could prevent early OHSS.

Local VEGF inhibition prevents ovarian alterations associated with ovarian hyperstimulation syndrome.

The relationship between human chorionic gonadotropin and ovarian hyperstimulation syndrome (OHSS) is partially mediated by vascular endothelial growth factor A (VEGF). The aim of this study was to investigate the effects of VEGF inhibition on the development of corpora lutea (CL) and cystic structures, steroidogenesis, apoptosis, cell proliferation, endothelial cell area, VEGF receptors (KDR and Flt-1), claudin-5 and occludin levels in ovaries from an OHSS rat model. The VEGF inhibitor used (VEGF receptor-1 (FLT-1)/Fc chimera, TRAP) decreased the concentrations of progesterone and estradiol as well as the percentage of CL and cystic structures in OHSS rats, and increased apoptosis in CL. Endothelial cell area in CL and KDR expression and its phosphorylation were increased, whereas claudin-5 and occludin levels were decreased in the OHSS compared to the control TRAP reversed these parameters. Our findings indicate that VEGF inhibition prevents the early onset of OHSS and decreases its severity in rats.

Platelet-derived growth factor BB and DD and angiopoietin1 are altered in follicular fluid from polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, and is characterized by abnormalities in ovarian angiogenesis, among other features. Consistent with this association, follicular fluid (FF) concentration and ovarian expression of vascular endothelial growth factor (VEGF) are increased in PCOS patients. In this study, we examined the protein levels of platelet‐derived growth factor (PDGF) BB and DD (PDGFBB and PDGFDD), angiopoietin 1 and 2 (ANGPT1 and ANGPT2), and their soluble receptor sTIE2 in FF from PCOS and control patients undergoing assisted reproductive techniques. We also analyzed the effect of FF from PCOS and control patients on tight and adherens junction protein expression in an endothelial cell line. PDGFBB and PDGFDD were significantly lower whereas ANGPT1 concentration was significantly higher in FF from PCOS patients than from control patients. No changes were found in the concentration of ANGPT2 or sTIE2. Expression of claudin‐5 was significantly increased in endothelial cells incubated for 24 hr in the presence of FF from PCOS versus from control patients, while vascular‐endothelial cadherin, β‐catenin, and zonula occludens 1 expression were unchanged. The changes observed in the levels of PDGF isoforms and ANGPT1 may prevent VEGF‐induced vascular permeability in the PCOS ovary by regulating endothelial‐cell‐junction protein levels. Restoring the levels of angiogenic factors may provide new insights into PCOS treatment and the prevention of ovarian hyperstimulation syndrome in affected women. Mol. Reprod. Dev. 81: 748–756, 2014.

Involvement of the ANGPTs/Tie-2 system in ovarian hyperstimulation syndrome (OHSS)

Ovarian hyperstimulation syndrome (OHSS) is a disorder associated with ovarian stimulation. OHSS features are ovarian enlargement with fluid shifting to the third space. Disturbances in the vasculature are considered the main changes that lead to OHSS. Our aim was to analyze the levels of angiopoietins 1 and 2 (ANGPT1 and 2) and their soluble and membrane receptors (s/mTie-2) in follicular fluid (FF) and in granulosa-lutein cells culture (GLCs) from women at risk of developing OHSS. We also evaluated the effect of ANGPT1 on endothelial cell migration. In ovaries from an OHSS rat model, we analyzed the protein concentration of ANGPTs, their mTie-2 receptor, and platelet-derived growth factor PDGF-B, -D and PDGFR-β. ANGPT1 levels were increased in both FF and GLCs from women at risk of OHSS. Incubation of these FF with an ANGPT1 neutralizing antibody decreased endothelial cell migration. In the ovaries of OHSS rat model, mTie-2 protein levels increased and PDGF-B and -D decreased. In summary, these results suggest that ANGPT1 could be another mediator in the development of OHSS.

Inhibition of platelet-derived growth factor (PDGF) receptor affects follicular development and ovarian proliferation, apoptosis and angiogenesis in prepubertal eCG-treated rats.

The platelet-derived growth factor (PDGF) system is crucial for blood vessel stability. In the present study, we evaluated whether PDGFs play a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. We examined the effect of intrabursal administration of a selective platelet-derived growth factor receptor (PDGFR) inhibitor (AG1295) on follicular development, proliferation, apoptosis and blood vessel formation and stability in ovaries from rats treated with equine chorionic gonadotropin (eCG). The percentages of preantral follicles (PAFs) and early antral follicles (EAFs) were lower in AG1295-treated ovaries than in control ovaries (p < 0.01-0.05). The percentage of atretic follicles (AtrFs) increased in AG1295-treated ovaries compared to control (p < 0.05). The ovarian weight and estradiol concentrations were lower in AG1295-treated ovaries than in the control group (p < 0.01 and p < 0.05, respectively), whereas progesterone concentrations did not change. AG1295 decreased the proliferation index in EAFs (p < 0.05) and increased the percentage of nuclei positive for cleaved caspase-3 and apoptotic DNA fragmentation (p < 0.01-0.05). AG1295 increased the expression of Bax (p < 0.05) without changes in the expression of Bcl-2 protein. AG1295-treated ovaries increased the cleavage of caspase-8 (p < 0.05) and decreased AKT and BAD phosphorylation compared with control ovaries (p < 0.05). AG1295 caused a decrease not only in the endothelial cell area but also in the area of pericytes and vascular smooth muscle cells (VSMCs) in the ovary (p < 0.05). Our findings suggest that the local inhibition of PDGFs causes an increase in ovarian apoptosis through an imbalance in the ratio of antiapoptotic to proapoptotic proteins, thus leading a larger number of follicles to atresia. PDGFs could exert their mechanism of action through an autocrine/paracrine effect on granulosa and theca cells mediated by PDGFRs. In conclusion, these data clearly indicate that the PDGF system is necessary for follicular development induced by gonadotropins.

Inhibition of angiopoietin-1 (ANGPT1) affects vascular integrity in ovarian hyperstimulation syndrome (OHSS)

Ovarian hyperstimulation syndrome (OHSS) is a complication of ovarian stimulation with gonadotrophins following human chorionic gonadotrophin (hCG) administration. The relationship between hCG and OHSS is partly mediated via the production of angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietins (ANGPTs). Here, we investigated the effect of ANGPT1 inhibition on ovarian angiogenesis in follicular fluid (FF) from women at risk of OHSS, using the chorioallantoic membrane (CAM) of quail embryos as an experimental model. We also analysed cytoskeletal changes and endothelial junction protein expression induced by this FF in the presence or absence of an ANGPT1-neutralising antibody in endothelial cell cultures. The presence of this antibody restored the number of vascular branch points and integrin αvβ3 levels in the CAMs to control values. ANGPT1 inhibition in FF from OHSS patients also restored the levels of claudin-5, vascular endothelial cadherin and phosphorylated β-catenin and partially reversed actin redistribution in endothelial cells. Our findings suggest that ANGPT1 increases pathophysiological angiogenesis in patients at risk of OHSS by acting on tight and adherens junction proteins. Elucidating the mechanisms by which ANGPT1 regulates vascular development and cell-cell junctions in OHSS will contribute to identifying new therapeutic targets for the treatment of human diseases with aberrant vascular leakage.

Local administration of platelet-derived growth factor B (PDGFB) improves follicular development and ovarian angiogenesis in a rat model of Polycystic Ovary Syndrome

Alterations in ovarian angiogenesis are common features in Polycystic Ovary Syndrome (PCOS) patients; the most studied of these alterations is the increase in vascular endothelial growth factor (VEGF) production by ovarian cells. Platelet-derived growth factor B (PDGFB) and D (PDGFD) are decreased in follicular fluid of PCOS patients and in the ovaries of a rat model of PCOS. In the present study, we aimed to analyze the effects of local administration of PDGFB on ovarian angiogenesis, follicular development and ovulation in a DHEA-induced PCOS rat model. Ovarian PDGFB administration to PCOS rats partially restored follicular development, decreased the percentage of cysts, increased the percentage of corpora lutea, and decreased the production of anti-Müllerian hormone. In addition, PDGFB administration improved ovarian angiogenesis by reversing the increase in periendothelial cell area and restoring VEGF levels. Our results shed light into the mechanisms that lead to altered ovarian function in PCOS and provide new data for potential therapeutic strategies.

Allopregnanolone alters follicular and luteal dynamics during the estrous cycle

BackgroundAllopregnanolone is a neurosteroid synthesized in the central nervous system independently of steroidogenic glands; it influences sexual behavior and anxiety. The aim of this work is to evaluate the indirect effect of a single pharmacological dose of allopregnanolone on important processes related to normal ovarian function, such as folliculogenesis, angiogenesis and luteolysis, and to study the corresponding changes in endocrine profile and enzymatic activity over 4 days of the rat estrous cycle. We test the hypothesis that allopregnanolone may trigger hypothalamus - hypophysis - ovarian axis dysregulation and cause ovarian failure which affects the next estrous cycle stages.MethodsAllopregnanolone was injected during the proestrous morning and then, the animals were sacrificed at each stage of the estrous cycle. Ovarian sections were processed to determine the number and diameter of different ovarian structures. Cleaved caspase 3, proliferating cell nuclear antigen, α-actin and Von Willebrand factor expressions were evaluated by immunohistochemistry. Luteinizing hormone, prolactin, estrogen and progesterone serum levels were measured by radioimmunoassay. The enzymatic activities of 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid oxidoreductase and 20α-hydroxysteroid dehydrogenase were determined by spectrophotometric assays. Two-way ANOVA followed by Bonferroni was performed to determine statistical differences between control and treated groups along the four stages of the cycle.ResultsThe results indicate that allopregnanolone allopregnanolone decreased the number of developing follicles, while atretic follicles and cysts increased with no effects on normal cyclicity. Some cysts in treated ovaries showed morphological characteristics similar to luteinized unruptured follicles. The apoptosis/proliferation balance increased in follicles from treated rats. The endocrine profile was altered at different stages of the estrous cycle of treated rats. The angiogenic markers expression increased in treated ovaries. As regards corpora lutea, the apoptosis/proliferation balance and 20α-hydroxysteroid dehydrogenase enzymatic activity decreased significantly. Progesterone levels and 3β-hydroxysteroid dehydrogenase enzymatic activity increased in treated rats. These data suggest that allopregnanolone interferes with steroidogenesis and folliculogenesis at different stages of the cycle.ConclusionAllopregnanolone interferes with corpora lutea regression, which might indicate that this neurosteroid exerts a protective role over the luteal cells and prevents them from luteolysis. Allopregnanolone plays an important role in the ovarian pathophysiology.

Lysophosphatidic acid induces the crosstalk between the endovascular human trophoblast and endothelial cells in vitro: BELTRAME et al.

Spiral artery remodeling at the maternal–fetal interface is crucial for successful pregnancy and requires the interaction between the first trimester trophoblast and the endothelial cells of the maternal vessels. However, the precise mechanism of this dialog has yet to be determined. The current study investigated whether lysophosphatidic acid (LPA) modulates trophoblast–endothelial crosstalk in vitro. HTR‐8/SVneo trophoblast cell line (H8) was seeded on top of Geltrex, incubated with LPA or LPA + NS‐398 (selective cyclooxygenase‐2 inhibitor), LPA + 1400W (selective inducible nitric oxide synthase inhibitor) or LPA + IL‐6 neutralizing antibody and assayed for tube formation to model the acquisition of trophoblast endovascular phenotype. The supernatants were collected and used as conditioned media (CM). To test trophoblast–endothelial crosstalk, the endothelial cell line EA.hy926 was incubated with trophoblast CM. The CM from LPA‐induced tubulogenesis stimulated endothelial cells migration and did not modify the apoptosis. Soluble factors derived from cyclooxygenase‐2 and IL‐6 pathways were involved in H8–EA.hy926 interaction under the LPA effect. Moreover, LPA increased the levels of IL‐6 mRNA by cyclooxygenase‐2 pathway in H8 cells. Collectively, LPA promotes trophoblast–endothelial crosstalk in vitro and induces the release of trophoblast soluble factors that stimulate endothelial cells migration without changes in apoptosis. The evidence presented here provides new insights about an active role of LPA as a lipid mediator regulating vascular remodeling at the maternal–fetal interface.