The Effects of Vitamin D on Angiogenic Factors in Women With Polycystic Ovary Syndrome
Condition: Polycystic Ovary Syndrome; Vitamin D Deficiency
MeSH terms: Polycystic Ovary Syndrome; Vitamin D Deficiency; Syndrome
Study type: Interventional
Phase: Phase 4

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Vitamin D3; Placebo
Intervention type(s): Drug; Other
Intervention description(s): Women allocated to vitamin D arm received one capsule 50.000 IU of vitamin D3 once weekly for eight weeks.; Women in the placebo arm received once capsule of placebo once weekly for eight weeks
Intervention MeSH terms: Vitamin D; Cholecalciferol
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Single (Participant)
Primary purpose: Treatment