The Effects of Vitamin D on Angiogenic Factors in Women With Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) affects 5 to 10% of women of reproductive age. It is characterized by a cluster of hyperandrogenism, hyperinsulinemia, menstrual dysfunction, hirsutism and infertility. Although the pathogenesis of PCOS is unknown, accumulating evidence suggests that the dysregulation of some angiogenic factors, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), may be implicated. TGF-βs and VEGF exert a diverse range of biological functions regulating cell proliferation, angiogenesis, fibroblast activation and tissue fibrosis. PCOS ovaries show all the hallmarks of TGF-β and VEGF upregulation, including increased collagen deposition in ovarian stroma and theca, supported by increased vascularity. Consistent with this, The investigators recently showed that TGF-β1 is increased in serum of PCOS women while its circulating receptor soluble endoglin (sENG) is decreased, resulting in greater TGF-β1 bioavailability. Furthermore, it has been shown that women with PCOS have increased VEGF levels in the serum and/or follicular fluid. PCOS patients also have decreased vitamin D levels, and vitamin D treatment has been previously shown to improve various clinical parameters in PCOS women, including glucose intolerance, hypertension and androgen levels. Interestingly, vitamin D has been shown to decrease TGF-β1 and VEGF levels in several diseases, including myelofibrosis and various human cancer cells. Therefore, the investigators hypothesize that vitamin D treatment of PCOS women will result in a decrease of serum TGF-β1 levels and/or VEGF levels concomitant with improvement in clinical disease parameters. In addition, the investigators hypothesize that improvement in clinical disease parameters will correlate with changes in serum VEGF levels and TGF-β1 bioavailability. Our aim in the present study is to investigate the effects of vitamin D treatment on serum VEGF and TGF-β1/sENG levels in PCOS women, and assess whether changes in these angiogenic factors following vitamin D treatment correlate with clinical disease in these women. For this end, PCOS patients who are vitamin D-deficient will be treated with vitamin D and their serum levels of VEGF, TGF-β1 and its sENG receptor will be measured before and after treatment. In addition, clinical disease parameters will be recorded before and 4 months after treatment, including serum glucose and insulin levels, serum androgen levels, and blood pressure.

The proposed study aims to identify a putative link between vitamin D, VEGF, and TGF-β1 in the context of PCOS, and provide a novel molecular explanation for the beneficial clinical effects of vitamin D on PCOS patients.

This study is a randomized, single blind, placebo-controlled trial to evaluate the effect of vitamin D on vitamin D-deficient women with PCOS. 93 reproductive-aged women diagnosed with PCOS presenting to Maimonides Medical Center for annual check-up between October 2013 and March 2015 were screened for vitamin D deficiency (defined as 25 hydroxy-vitaminD \[25OH-D\] levels \<20 ng/mL). All participants signed the informed consent and the study was approved by the international review board (IRB) of Maimonides Medical Center. PCOS was diagnosed according to the Rotterdam Consensus (ESHRE/ASRM criteria), i.e. the presence of two of three criteria: oligo- or anovulation, signs of clinical hyperandrogenism, and/or biochemical signs of hyperandrogenism and polycystic ovaries on ultrasonography after exclusion of specific identifiable disorders (thyroid disorder, hyperprolactinemia, congenital adrenal hyperplasia, androgen-secreting tumors, and Cushing's syndrome). The investigators included women aged between 18 and 38 years who were not: 1) pregnant, postpartum, breastfeeding, or 2) taking any vitamin D supplements, metformin or any hormonal therapy.

Interventions and blood collection:

68 women diagnosed with PCOS and vitamin D deficiency were enrolled. Participants were allocated to each group according to a computer-generated list using ratio 2/1 (Vitamin D/placebo). Women allocated to vitamin D group received one capsule 50.000 IU of vitamin D3 once weekly for eight weeks. The vitamin D supplementation regimen was extracted from the Endocrine Society guidelines. Women in the placebo group received once capsule of placebo once weekly for eight weeks. The placebo was prepared at Maimonides Medical Center's pharmacy. To ensure compliance, The investigators called each participant once weekly and reminded her to take her pill. Fasting blood samples were collected by venipuncture before starting and within two weeks after completing the treatment (vitamin D or placebo). Blood samples were allowed to clot for 30 minutes at room temperature before centrifugation at 1,200 rpm for 10 minutes. Serum was stored at -80°C in aliquots until assayed.

The assays of all measured hormones, 25OH-D, VEGF, TGF-β1, sENG, and AMH:

Serum 25OH-D levels were measured before and after completing the treatment. The levels were determined by the ADVIA Centaur vitamin D assay (Siemens Healthcare Diagnostics). Dehydroepiandrosterone sulfate (DHEAS), testosterone, sex hormone-binding globulin (SHBG), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured using IMMULITE 2000 XPi immunoassay system (Siemens Healthcare USA). Insulin and prolactin concentrations were quantified by DXL 800 immunoassay analyzer according to manufacturer's protocols (Beckman Coulter). Insulin resistance was calculated according to the homeostatic model assessment (HOMA) (29) by using the following formula: Insulin resistance (HOMA IR) = \[fasting insulin (µU/mL) x fasting glucose (mmol/L)\]/22.5. 17OH-progesterone level was determined by ELISA assay (Eagle BioSciences). AMH concentration was measured using the ultrasensitive AMH/MIS CLIA kit (AnshLabs). TGF-β1 concentration was measured using Human TGF-beta1 Quantikine ELISA kit according to manufacturer's protocols (R\&D Systems). sENG levels were quantified by Human Endoglin/CD105 Quantikine ELISA kit (R\&D Systems). VEGF concentration was quantified using Human VEGF Quantikine ELISA kit according to manufacturer's protocols (R\&D Systems). The inter-assay and intra-assay coefficients of variation for all assays were less than 10%.

Clinical parameters:

All the clinical parameters were evaluated before and four months after the completion of treatment. These parameters included blood pressure (BP), Ferriman-Gallwey score (FGS) (hirsutism score), acne status, and interval between periods.

Statistical analysis:

Data were tested for normality. All values were expressed as mean ± standard error of the mean (SEM). A paired student's t-test was used to compare pre- and post-treatment serum levels and clinical parameters. Correlation between changes in angiogenic factors and changes in clinical disease parameters was analyzed using Pearson's test and linear regression. X2-test was used to evaluate the changes in acne after treatment. SigmaStat (SPSS Science, Chicago, IL) was used for statistical analysis. P\<0.05 was considered to be statistically significant.

Eligibility Criteria:
Inclusion Criteria:

* Women with PCOS who have vitamin D deficiency (serum 25-hydroxyvitamin D\<20 ng/mL)

Exclusion Criteria:

* Pregnant, postpartum, breast feeding
* Taking Metformin, vitamin D, or any hormonal therapy