In brief:Formation and function of the corpus luteum strongly rely on active angiogenesis. This study demonstrates the role of the hypoxia-inducible factor (HIF) in luteinization with no effect on fertility. Abstract:HIFs are transcription factors responsible for sensing low oxygen levels and, in response, inducing the transcription of numerous genes. One of the main processes stimulated by HIFs is the formation of new vessels to increase oxygen supply to the tissue. Formation of the corpus luteum strongly depends on the vasculature, and active angiogenesis occurs during luteinization. In this study, we aimed to analyze the role of HIF in the early formation of corpus luteum and its function, and in female fertility. To this aim, we superovulated mice using equine chorionic gonadotropin and human chorionic gonadotropin (hCG) and administered the HIF inhibitor acriflavine (ACF) to the mice 3 h before hCG. We found a decrease in ovarian HIF1A and VEGFA and in the vascular area in the animals treated with ACF. Moreover, we observed an increase in aberrant structures in the ovaries and in luteal cell apoptosis. Serum progesterone levels were decreased together with ovarian STAR expression. However, the animals treated with ACF during the early formation of the corpus luteum were completely fertile and no alterations were observed when the treated females were mated with fertile males. These results collectively suggest that HIF regulates gonadotropin-induced corpus luteum formation by acting on luteal blood vessel formation, luteal cell survival and progesterone synthesis. However, adequate HIF activity may not be essential to achieve and maintain pregnancy. These findings are significant to better understand the complex mechanisms of corpus luteum formation and identify potential abnormalities to allow better knowledge of ovarian physiology and pathologies in which this factor could be involved.

Endocrine dysfunctions account for 25% of infertility problems. Hormonal and molecular events involved in the process of folliculogenesis are crucial for acquiring oocyte competence, a key factor of fetal viability. In the present publication, we review a practical pathophysiological and therapeutic approach of the endocrine causes of female infertility. Frequently, these endocrine features have a genetic base, but identification is not accessible to most patients in the clinical practice. Older patients develop ovarian aging and endocrine disturbances, and these are key concepts within reproductive health. Hormonal profile assessment is a key aspect to diagnosing infertility. This publication will develop a guide for the management of endocrine disturbances. Most of the therapeutic strategies are grounded on pathophysiological mechanisms. A custom approach is recommended for each individual case. Discussion of risks and benefits with patients is always appropriate while keeping age, ovarian reserve, sperm quality, and potential endometrial receptivity in mind. Ovarian hyperstimulation syndrome is the most serious complication of ovulation induction, and its prevention must be the most important feature of the management. Worldwide debate in accessibility to treatments, cultural, financial, ethical, and regulatory aspects influences patient management and decision-making to this day.

Context The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5′UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. Aims To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. Methods We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. Key results We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. Conclusions The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. Implications Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

CONTEXT:The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17.AIMS:To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat.METHODS:We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages.KEY RESULTS:We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17.CONCLUSIONS:The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15.IMPLICATIONS:Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

Although advances in the prediction and management of ovarian hyperstimulation syndrome (OHSS) have been introduced, complete prevention is not yet possible. Previously, we and other authors have shown that vascular endothelial growth factor, angiopoietins (ANGPTs) and sphingosine-1-phosphate are involved in OHSS etiology. In addition, we have demonstrated that ovarian protein levels of platelet-derived growth factor (PDGF) ligands-B and-D decrease in an OHSS rat model, whilst PDGFR-beta and ANGPT2 remain unchanged. In the present work, we investigated the role of PDGF-B in OHSS by evaluating ligand protein levels in follicular fluid (FF) from women at risk of developing OHSS and by using an immature rat model of OHSS. We demonstrated that PDGF-B and PDGF-D are lower in FF from women at risk of developing OHSS compared to control patients (P<0.05). In the OHSS rat model, PDGF-B (0.5 mu g/ovary) administration decreased ovarian weight (P<0.05), reduced serum progesterone (P<0.05) and lowered the percentage of cysts (P<0.05), compared to untreated OHSS rats, but had no effect on the proportion of follicles or corpora lutea (CL). PDGF-B treatment also restored the expression of steroidogenic acute regulatory protein (P<0.05) and P450 cholesterol side-chain cleavage enzyme (P<0.01) to control levels. In addition, PDGF-B increased the peri-endothelial cell area in CL and cystic structures, and reduced vascular permeability compared to untreated OHSS ovaries. Lastly, PDGF-B increased the levels of junction proteins claudin-5 (P<0.05), occludin (P<0.05) and beta-catenin (P<0.05), while boosting the extracellular deposition of collagen IV surrounding the ovarian vasculature (PP<0.01), compared to OHSS alone. In conclusion, our findings indicate that PDGF-B could be another crucial mediator in the onset and development of OHSS, which may lead to the development of novel prediction markers and therapeutic strategies.

The aim of this study was to investigate whether the Notch pathway is modulated in response to the downregulation of the Wnt/Β-catenin system in corpora lutea (CLs) from superovulated rats. To this end, we analyzed the effect of in vitro CL Wnt/Β-catenin inhibition on the expression of Notch members and on luteal function. Mechanically isolated rat CLs were cultured with ICG-001, a Wnt/B-catenin inhibitor. In this system, Wnt/B-catenin inhibition reduced progesterone production and decreased StAR protein levels. Besides, Wnt/B-catenin inhibition stimulated the Notch system, evidenced by an increase in Hes1 expression, and promoted the expression of selected Notch family members. At long incubation times, StAR levels and progesterone concentration reached the control values, effects probably mediated by the Notch pathway. These results provide the first evidence of a compensatory mechanism between Wnt/B-catenin signaling and the Notch system, which contributes to the homeostasis of luteal cells.

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that β‐catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of β‐catenin by means of two molecules, XAV939 and ICG‐001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG‐001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of β‐catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/β‐catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG‐001 and DAPT. ICG‐001 increased the Notch target genes Hes‐1 and Hey‐1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and β‐catenin signaling co‐operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.

STUDY QUESTIONIs ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy?SUMMARY ANSWERLocal administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology.WHAT IS KNOWN ALREADYCancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy.STUDY DESIGN, SIZE, DURATIONThe 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration.PARTICIPANTS/MATERIALS, SETTING, METHODSAnimals were euthanized by cervical dislocation or cardiac puncture 2 weeks after surgery for collection of blood orovary and uterus samples, which were cleaned of adhering tissue in culture medium and used for subsequent assays. Ovaries were used for Western blotting or immunohistochemical and/or histological analyses or steroid extraction, as required (n = 5-8 per group). A set of mice (n = 3/group) was destined for oocyte recovery and IVF. Finally, another set (n = 5-6/group) was separated to study fertility parameters.MAIN RESULTS AND THE ROLE OF CHANCEThe number of primordial (P < 0.01), primary (P < 0.05) and preantral follicles (P < 0.05) were decreased in Cy-treated mice compared to control animals, while atretic follicles were increased (P < 0.001). In Cy + C1P mice, the ovaries recovered control numbers of these follicular structures, in both C1P doses studied. Cy affected AMH expression, while it was at least partially recovered when C1P is administered as well. Cy caused an increase in serum FSH concentration (P < 0.01), which was prevented by C1P coadministration (P < 0.01). E2 levels in Cy-treated ovaries decreased significantly compared to control ovaries (P < 0.01), whilst C1P restored E2 levels to those of control ovaries (P < 0.01). Cy increased the expression of BAX (P < 0.01) and decreased the expression of BCLX-L compared to control ovaries (P < 0.01). The ovarian BCLX-L:BAX ratio was also lower in Cy-treated mice (P < 0.05). In the Cy + C1P group, the expression levels of BAX, BCLX-L and BCLX-L:BAX ratio were no different than those in control ovaries. In addition, acid sphingomyelinase (A-SMase) expression was higher in Cy-treated ovaries, whilst remaining similar to the control in the Cy + C1P group. Cy increased the apoptotic index (TUNEL-positive follicles/total follicles) in preantral and early antral stages, compared to control ovaries (P < 0.001 and P < 0.01, respectively). C1P protected follicles from this increase. No primordial or primary follicular cells stained for either cleaved caspase-3 or TUNEL when exposed to Cy, therefore, we have found no evidence for follicular reserve depletion in response to Cy being due to apoptosis. Cy caused evident vascular injury, especially in large cortical stromal vessels, and some neovascularization. In the Cy + C1P group, the disruptions in vascular wall continuity were less evident and the number of healthy stromal blood vessels seemed to be restored. In Cy-treated ovaries α-SMA-positive cells showed a less uniform distribution around blood vessels. C1P coadministration partially prevented this Cy-induced effect, with a higher presence of α-SMA-positive cells surrounding vessels. By H&E staining, Cy-treated mice showed endometrial alterations compared to controls, affecting both epithelial and stromal compartments. However, C1P allowed that the stromal tissue to maintain its loose quality and its glandular branches. Cy-treated animals had significantly lower pregnancy rates and smaller litter sizes compared with control mice (P = 0.013 and P < 0.05, respectively), whereas cotreatment with C1P preserved normal fertility. Furthermore, a higher (P < 0.05) proportion of abnormal oocytes was recovered from Cy-treated mice compared to the control, which was prevented by C1P administration.LARGE SCALE DATAN/A.LIMITATIONS REASONS FOR CAUTIONThe results of this study were generated from an in-vivo animal experimental model, already used by several authors. Further studies on C1P functions in female reproduction in pathological conditions such as chemotherapy-induced ovarian failure and on the safety of use of this sphingolipid are required.WIDER IMPLICATIONS OF THE FINDINGSThe present findings showed that C1P administration prior to Cy might be a promising fertility preservation strategy in female patients who undergo chemotherapy.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by grants from ANPCyT (PICT 2015-1117), CONICET (PIP 380), Cancer National Institute (INC) and Roemmers Foundation, Argentina. The authors declare no conflicts of interest.

Ovarian cancer is the fifth leading cause of cancer-related deaths in women. In the past 20 years, the canonical types of drugs used to treat ovarian cancer have not been replaced and the survival rates have not changed. These facts show the clear need to find new therapeutic strategies for this illness. Thus, the aim of the present study was to investigate the effect of a gamma-secretase inhibitor (DAPT) in combination with the Platelet-derived growth factor B (PDGFB) on an ovarian cancer xenograft model. To achieve this goal, we analyzed the effect of the administration of DAPT alone and the co-administration of DAPT and recombinant PDGFB on parameters associated with tumor growth and angiogenesis in an orthotopic experimental model of ovarian cancer. We observed that the dose of DAPT used was ineffective to reduce ovarian tumor growth, but showed anticancer activity when co-administered with recombinant PDGFB. The administration of PDGFB alone normalized tumor vasculature by increasing periendothelial coverage and vascular functionality. Interestingly, this effect exerted by PDGFB was also observed in the presence of DAPT. Our findings suggest that PDGFB is able to improve tumor vascularity and allows the anticancer action of DAPT in the tumor. We propose that this therapeutic strategy could be a new tool for ovarian cancer treatment and deserves further studies.

STUDY QUESTION Can the bioactive lipid sphingosine-1 phosphate (S1P) act as an endothelial barrier-enhancing molecule and, in turn, restore the vascular integrity and homoeostasis in a rat model of ovarian hyperstimulation syndrome (OHSS). STUDY ANSWER In vivo administration of S1P may prevent the early onset of OHSS and decrease its severity. WHAT IS KNOWN ALREADY Although advances in the prediction and treatment of OHSS have been made, complete prevention has not been possible yet. S1P in follicular fluid from women at risk of developing OHSS are lower in comparison from women who are not at such risk and administration of S1P in an OHSS rat model decreases ovarian capillary permeability. STUDY DESIGN, SIZE, DURATION We used an animal model that develops OHSS in immature Sprague-Dawley rats. The rats were randomly divided into three groups: the control group, which was injected with 10 IU of pregnant mare's serum gonadotropin (PMSG), and 10 IU of hCG 48 h later; the OHSS group, which was injected with excessive doses of PMSG (50 IU/day) for four consecutive days, followed by hCG; and the OHSS + S1P group, which was injected with the same doses of PMSG and hCG as the OHSS group and then treated with 5 μl S1P (1 mM) under the bursa of both ovaries, whereas the other groups of animals received the S1P vehicle. PARTICIPANTS /MATERIALS, SETTING, METHODS Rats were killed by decapitation 48 h after the hCG injection for ovary, endometrium and blood collection. The ovaries were weighed and then used for subsequent assays, while the serum was used for hormone assays. One of the ovaries from each rat (n = 6) was used for Western immunoblot and the other for immunohistochemical analysis. Statistical comparisons between groups were carried out. MAIN RESULTS AND THE ROLE OF CHANCE S1P administration reduced the ovarian weight (P < 0.05), and decreased the concentration of serum progesterone in the OHSS group compared to the OHSS group without treatment (P < 0.001). The percentage of antral follicles in the OHSS group was lower than that in the control group. S1P increased the percentage of antral follicles (P < 0.05) and decreased the percentage of corpora lutea (P < 0.01) and cystic structures in the OHSS group (P < 0.05). S1P had no effect on the expression levels of the enzymes 3β-hydroxysteroid dehydrogenase (3βHSD) or cholesterol side-chain cleavage enzyme (P450scc), but reduced the levels of steroidogenic acute regulatory protein (StAR) in OHSS rat ovaries (P < 0.05). S1P decreased the endothelial (P < 0.05) and periendothelial (P < 0.01) cell area in OHSS rat ovaries. S1P restored the levels of N-cadherin and VE-cadherin proteins to control values. Furthermore, S1P enhanced the levels of claudin-5, occludin (P < 0.05) and sphingosine-1-phosphate receptor 1 (S1PR1) in OHSS (P < 0.01). In addition, no histological differences were found in endometrium between OHSS and S1P-treated OHSS animals. LIMITATIONS REASONS FOR CAUTION The results of this study were generated from an in vivo OHSS experimental model, which has been used by several authors and our group due to the similarity between the rat and human angiogenic systems. Further studies in patients will be needed to evaluate the effects of S1P in the pathogenesis of OHSS. WIDER IMPLICATIONS OF THE FINDINGS These findings concern the pathophysiological importance of S1P in OHSS. More studies on the regulation of endothelial cell barrier function by S1P in reproductive pathological processes and its therapeutic application are required. LARGE SCALE DATA N/A. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported by grants from ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundations, Argentina. The authors declare no conflicts of interest.

Tankyrases are physiological regulators of Axin, a protein involved in several cellular processes, including Wnt signaling. Here, we investigated the effect of a specific Tankyrase inhibitor (XAV939) in follicular-luteal dynamics, and its possible relationship with ovarian vascular development. Studies were designed to analyze the effect of intrabursa administration of XAV939 in gonadotropin-treated prepubertal rats. In particular, we examined follicle and corpus luteum development, steroidogenesis, angiogenic markers, and apoptotic parameters. We found that in vivo inhibition of Wnt signaling impaired corpus luteum development, with a decrease in the number of corpora lutea balanced by a high number of cysts; decreased circulating progesterone levels, likely due to a decrease in Steroidogenic acute regulatory protein content in the corpus luteum; and increased pro-apoptotic parameters. In addition, Extracellular signal-regulated kinase phosphorylation, Vascular endothelium growth factor 120 content, and endothelial cell area were diminished in corpora lutea of inhibitor-treated ovaries. Thus, Wnt/β-catenin signaling appears to participate in the regulation of corpus luteum development and luteal cell function.

Running title: Wnt/β-catenin in corpus luteum development and function. Accialini Paula, Irusta Griselda, Bechis Andres, Bas Diana, Parborell Fernanda, Abramovich Dalhia, Tesone Marta 2 Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina. 2 Departamento de Quıímica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina. Abbreviations: FSH, Follicle-stimulating hormone; LH, Luteinizing hormone; cytochrome P450scc, cytochrome P450 side-chain cleavage (also known as CYP11A1); STAR, Steroidogenic acute regulatory protein; VEGF, Vascular endothelial growth factor. Supported by Universidad de Buenos Aires (UBA 20020090300061), CONICET (PIP

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.

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.

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. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Polycystic ovary syndrome (PCOS) is a frequent pathology that affects more than 5% of women of reproductive age. Among other heterogeneous symptoms, PCOS is characterized by abnormalities in angiogenesis. Metformin has been introduced in the treatment of PCOS to manage insulin resistance and hyperglycemia. Besides its metabolic effects, metformin has been shown to improve ovulation, pregnancy and live birth rates in PCOS patients. In the present study, we used a dehydroepiandrosterone-induced PCOS rat model to analyze the effect of metformin administration on ovarian angiogenesis. We found that metformin was able to restore the increased levels of vascular endothelial growth factor, angiopoietin (ANGPT)1, and ANGPT1/ANGPT2 ratio and the decreased levels of platelet-derived growth factor B and platelet-derived growth factor D observed in the dehydroepiandrosterone-treated rats. These effects could take place, at least in part, through a decrease in the levels of serum insulin. We also found an improvement in follicular development, with a lower percentage of small follicles and cysts and a higher percentage of antral follicles and corpora lutea after metformin administration. The improvement in ovarian angiogenesis is likely to restore the accumulation of small follicles observed in PCOS rats and to reduce cyst formation, thus improving follicular development and the percentage of corpora lutea. These results open new insights into the study of metformin action not only in glucose metabolism but also in ovarian dysfunction in PCOS women.

SUMMARYPolycystic 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. © 2014 Wiley Periodicals, Inc.