Endometrial Vitamin D Receptor in PCOS
Condition: Vitamin D Receptor Defect
Study type: Interventional

Summary: 
      Vitamin D receptor (VDR) is widely expressed not only in tissues responsible for calcium
      hemostasis, but also in reproductive organs, including the endometrium. The VDR, which is low
      in the proliferative phase, starts to increase in the early secretory phase and decreases
      again in the mid-secretory phase. Enzymes responsible for vitamin d (VD) production and
      metabolism are expressed locally in the endometrium. Binding of active VD to VDR in nuclear
      or plasma membrane increases receptivity gene expression and immunomodulators by activating
      genomic and nongenomic pathways. VDR expression defect has been reported in the decidual
      cells of recurrent miscarriages. There are no studies investigating the endometrial VDR
      expression pattern in polycystic ovary syndrome (PCOS) and its phenotypes. The VDR expression
      pattern in the endometrium of PCOS patients who underwent invitro fertilization
      /intracytoplasmic sperm injection (IVF/ICSI) and total embryo freezing was analyzed at both
      mRNA and immunohistochemical. The study group consisted of 44 PCOS patients who were referred
      to IVF/ICSI because they did not respond to first-line treatment with letrozole or clomiphene
      citrate (CC). Twenty patients who were scheduled for IVF/ICSI due to male factor infertility
      and who were matched with the study group in terms of age and body mass index (BMI) were
      included as the control group. Following egg collection, endometrial tissue was collected by
      pipelle cannula while the patient was under anesthesia. All good quality blastocysts of both
      groups were vitrified. The mRNA expression of vitamin D receptor transcript 2 (VDR-X2) and
      vitamin D receptor transcript 4 (VDR-X4) were determined by quantitative polymerase chain
      reaction (qPCR). The quantitative cycle equation method was used to calculate the differences
      between VDR-mRNA expressions. Endometrial VDR and progesterone receptor (PR) immunoreactivity
      were determined by immunohistochemistry.
    

Detailed description: 
      Study question: How does the expression of endometrial vitamin D receptor vary according to
      phenotypes in women with polycystic ovary syndrome (PCOS)?

      What is known already: VDR is widely expressed not only in tissues responsible for calcium
      hemostasis, but also in reproductive organs, including the endometrium. The VDR, which is low
      in the proliferative phase, starts to increase in the early secretory phase and decreases
      again in the mid-secretory phase. Enzymes responsible for VD production and metabolism are
      expressed locally in the endometrium. Binding of active VD to VDR in nuclear or plasma
      membrane increases receptivity gene expression and immunmodulators by activating genomic and
      nongenomic pathways. VDR expression defect has been reported in the decidual cells of
      recurrent miscarriage cases. There are no studies investigating the endometrial VDR
      expression pattern in PCOS and its phenotypes.

      Study design, size, duration: The study group consisted of 44 PCOS patients who were referred
      to IVF/ICSI because they did not respond to first-line treatment with letrozole or clomiphene
      citrate (CC). Twenty patients who were scheduled for IVF/ICSI due to male factor infertility
      and who were matched with the study group in terms of age and BMI were included as the
      control group. Those who met at least two of the criteria for hyperandrogenemia (HA),
      ovulatory dysfunction (OD) and polycystic ovarian morphology (PCOM) determined by European
      Society of Human Reproduction and Embryology/ American Society for Reproductive Medicine were
      considered PCOS. Following egg collection, endometrial tissue was collected by pipelle
      cannula while the patient was under anesthesia. All good quality blastocysts of both groups
      were vitrified.

      Participants/materials, setting, methods: The 44 participants in the PCOS group were divided
      into four phenotypic groups as follows according to the National Institutes of Health (NIH)
      consensus panel. Phenotype A: HA+OD+PCOM (classical/complete, n=17); phenotype B: HA+OD
      (classical/non-PCOM, n=10); phenotype C: HA+PCOM (ovulatory, n=9); and phenotype D: OD+PCOM
      (non-hyperandrogenic, n=8). The mRNA expression of vitamin D receptor transcript 2 (VDR-X2)
      and vitamin D receptor transcript 4 (VDR-X4) were determined by qPCR. The quantitative cycle
      equation method was used to calculate the differences between VDR-mRNA expressions.
      Endometrial VDR and progesterone receptor (PR) immunoreactivity were determined by
      immunohistochemistry. The histological score formula was used for the quantitative evaluation
      of VDR and PR immunoreactivity.

      Limitations, reasons for caution: The lack of an equal number of participants for each
      phenotype is our first limitation. Due to the collection of endometrial samples in the
      IVF/ICSI cycle, the possible effect of ovarian stimulation drugs and the associated increased
      estrogen levels on the endometrium has not been excluded. Changes in endometrial VDR-mRNA
      expression could not be confirmed by protein measurement.
    

Intervention name(s): endometrial sampling with pipelle
Intervention type(s): Procedure
Intervention description(s): Following oocyte retrieval while the patient was under anesthesia, endometrial tissue sampling was performed with the help of pipelle cannula. A portion of the endometrial tissue was put into RNA stabilization buffer (RNAlater) to be used in qPCR and stored at -20°C until the analysis. The remaining endometrial tissue was first fixed with 10% formalin, followed by paraffin blocking and prepared for immunohistochemical analysis.
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Intervention model description: While the study group consisted of PCOS patients, the control group was selected from non-PCOS patients with male factor infertility.
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: PCOS; Vitamin D; Endometrium; VD receptor; Hyperandrogenemia; Insulin resistance