A Comparative Study on Therapeutic Effect of Metformin and Sitagliptin/metformin in Infertile Women with Polycystic Ovary Syndrome (PCOS) undergoing intracytoplasmic sperm injection (ICSI)
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Abstract Objective: The aim of this study was to investigate the effects of sitagliptin/metformin (sitaformin), metformin and sitagliptin on PCOS patients undergoing ICSI. Design: A randomized, placebo-controlled pilot study Intervention (s): Participants were randomly assigned to receive metformin, sitagliptin, sitaformin or placebo Treatment was carried out 2 months before the start of the ovulation cycle and continued until the day of ovum pick up. Result(s): The serum levels of homeostatic model assessment for insulin resistance (HOMA-IR) and free androgen index (FAI) decreased significantly in the treated groups compared to the placebo. The serum and the follicular fluid (FF) levels of leptin also decreased significantly in the sitaformin group when compared to the metformin and sitagliptin groups. Moreover, the serum and FF levels of anti-mullerian hormone (AMH) and malondialdehyde (MDA) had a significant decrease in the sitaformin and sitagliptin group compared to the placebo. The mRNA expression and protein levels of GDF9 and BMP15 in the cumulus cells increased significantly in the sitaformin group compared to metformin and sitagliptin groups. The expression level of GDF9 and BMP15 mRNA were positively correlated with the fertilization rate and grade I embryos. Conclusion (s): Sitaformin improves hormonal factors and levels of GDF9 and BMP15 in PCOS compared to metformin and sitagliptin, which can increase the rate of fertilization and grade I embryos.
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Patients in all groups were pretreated for two months with either placebo, metformin, sitagliptin, or sitaformin from the 3rd day of the menstrual cycle. The menstrual cycle was induced by progesterone. All patients were stimulated using a protocol of ovarian downregulation with a GnRH antagonist. Oocyte retrieval was carried out transvaginally under ultrasound guidance 36–40 hours after hCG administration. The follicular fluid (FF), from the first aspiration with no visible blood contamination, was collected. Fasting blood samples were also collected from each participant once before starting the treatment and once on the day of oocyte aspiration. Following follicular puncture, cumulus oocyte complexes (COCs) were collected and cultured for at least three hours in a total global medium covered with mineral oil. Then, cumulus granulosa cell surrounding the oocytes were stripped individually. After harvesting the remaining cumulus cells of the oocytes with hyaluronidase enzyme 80 IUmL-1, the collected oocytes were examined and evaluated separately. Mature (MII) oocytes were identified by the first polar body under a stereomicroscope. Only those oocytes that had extruded the first polar body (MII oocyte) were used for ICSI. Four hours after oocyte retrieval, a single motile sperm with normal morphology was immobilized and used to inseminate the oocyte. Fertilization was assessed the next day by the presence of two pronuclei (2PN). Embryo quality was assessed on the 3rd day of insemination and graded. Total RNA was extracted from the cumulus cells with Trizol. Complementary DNA synthesis was performed using oligo dT primers and was reverse-transcribed using Superscript II. To determine the relative expression of target genes, quantitative real-time polymerase chain reaction (RT-qPCR) was carried out using SYBR-Green/ ROX qPCR master mix assay by gene-specific primers. GAPDH was used as the reference gene. Total proteins were extracted from cumulus cells using RIPA lysis and the extraction buffer Kit, according to the manufacturer’s instruction. Proteins Concentration were determined according to Bradford’s method using bovine serum albumin (BSA) as the reference standard. Total proteins were electrophoresed in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to polyvinylidene fluoride membranes probed with specific antibodies. Membranes were developed using enhanced chemiluminescence reagents and the intensity of immunoreactive polypeptides was analyzed subsequent to observing the bands developed on a photographic film. Protein bands on the photographic film were quantified by densitometry scanning after background subtraction.