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.

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.

Inclusion Criteria:

• Clinical diagnosis of PCOS
• Approval for IVF/ICSI and total embryo freezing
• Consent to endometrial sampling

Exclusion Criteria:

• Women with hyperandrogenism or ovulatory dysfunction due to non-PCOS etiologies.
• Patients taking vitamin D, insulin sensitizers, lipid-lowering or other hormonal drugs in the last three months were not included.
• Those with a history of mechanical endometrial injury, polypectomy, hysteroscopic myomectomy, endometrioma resection, dilatation/curettage, ovarian drilling and salpingectomy were also excluded.
• Participants with premalignant or malignant endometrial pathology, those with a history of tubal factor infertility, congenital uterine anomaly or any chronic systemic diseases were not included.
• Azoospermia patients were excluded from the study because we preferred ejaculate sperm for ICSI in patients in the control group.

These patients are female patients who are infertile and planned for IVF/ICSI. The condition of being in the reproductive age of the patients was sought.