N-Acetylcysteine Compared to Metformin, Improves The Expression Profile of Growth Differentiation Factor-9 and Receptor Tyrosine Kinase c-Kit in The Oocytes of Patients with Polycystic Ovarian Syndrome

Abstract

Background: Paracrine disruption of growth factors in women with polycystic ovarian syndrome (PCOS) results in production of low quality oocyte, especially following ovulation induction. The aim of this study was to investigate the effects of metformin (MET), N-acetylcysteine (NAC) and their combination on the hormonal levels and expression profile of GDF-9, BMP-15 and c-kit, as hallmarks of oocyte quality, in PCOS patients.

Materials and methods: This prospective randomized, double-blind, placebo controlled trial aims to study the effects of MET, NAC and their combination (MET+NAC) on expression of GDF-9, BMP-15 and c-kit mRNA in oocytes [10 at the germinal vesicle (GV) stage, 10 at the MI stage, and 10 at the MII stage from per group] derived following ovulation induction in PCOS. Treatment was carried out for six weeks, starting on the third day of previous cycle until oocyte aspiration. The expression of GDF9, BMP15 and c-kit were determined by quantitative real time polymerase chain reaction (RT-qPCR) and western blot analysis. Data were analyzed with one-way ANOVA.

Results: The follicular fluid (FF) level of c-kit protein significantly decreased in the NAC group compared to the other groups. Significant correlations were observed between the FF soluble c-kit protein with FF volume, androstenedione and estradiol. The GDF-9 expression in unfertilized mature oocytes were significantly higher in the NAC group compared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups. The c-kit expression in unfertilized mature oocytes were significantly lower in the NAC group compared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups (Registration number: IRCT201204159476N1).

Conclusion: We concluded that NAC can improve the quality of oocytes in PCOS.

Keywords: Gene Expression; Metformin; N-acetylcysteine; Oocyte; Polycystic Ovarian Syndrome.

Fig.1

Flowchart of participants in this study. MET; Metformin, NAC; N-acetylcysteine, and PLA; Placebo.

Fig.2

Correlation between parameters of follicular fluid (FF) in all the population. A. Soluble c-Kit with volume of FF, B. Estradiol (E2), and C. Androstenedione. R₂; Determination of coefficient.

Fig.3

Distribution of oocytes retrieved, quality of oocytes and embryos in polycystic ovary syndrome patients undergoing treatment of N-acetylcysteine (NAC), metformin (MET), NAC+MET and Placebo (control). Data are the mean ± SEM. Statistical analyses were performed by ANOVA followed by Tukey’s test for multiple comparisons. Means without a common letter are significantly different (P<0.05).

Fig.4

Effects of NAC and MET on BMP-15, GDF-9 and c-kit mRNA and protein expression in immature oocytes (IMO, GV oocytes) of PCOS patients. Results of reverse transcriptase real-time polymerase chain reaction (PCR) for mRNAs of A. BMP-15, B. GDF-9, C. c-kit, in GV oocytes, D. Immunoblots of BMP-15, GDF-9 and c-kit from oocyte cell lysates. Densities of E. BMP-15, F. GDF-9 and G. c-kit protein bands in the experimental groups are shown. Means without a common letter are significantly different (P<0.05). NAC; N-acetylcysteine, MET; Metformin, GV; Germinal vesicle, and PCOS; Polycystic ovarian syndrome.

Fig.5

Effects of NAC and MET on BMP-15, GDF-9 and c-kit mRNA and protein expression in unfertilized mature oocytes (UMO, MII oocytes) of PCOS patients. Results of reverse transcriptase real-time polymerase chain reaction (PCR) for mRNAs of A. BMP-15, B. GDF-9, C. c-kit in MII oocytes, D. Immunoblots of BMP-15, GDF-9 and c-kit from oocyte cell lysates. Densities of E. BMP-15, F. GDF-9, and G. c-kit protein bands in the experimental groups are shown. Means without a common letter are significantly different (P<0.05). NAC; N-acetylcysteine, MET; Metformin, and PCOS; Polycystic ovarian syndrome.