Comparative evaluation of metformin & sitaformin in classic PCOS patients undergoing intracytoplasmic sperm injection: A randomized controlled pilot study

Polycystic ovarian syndrome (PCOS) is a disorder with long term effects on women’s overall health1. Women with PCOS often suffer from hormonal imbalance that causes changes in follicular growth leading to pregnancy or infertility problems2. Among women with PCOS, the quality of the oocyte is one of the most consequential factors determining the rate of success in intracytoplasmic sperm injection (ICSI). Women with PCOS are distinguished by an increased number of oocytes during the course of ovulation stimulation; nonetheless, these oocytes are often of low quality, leading to poor fertilization, cleavage and implantation3. In such patients, more than half of the oocytes retrieved from stimulated cycles exhibit at least one abnormal morphological characteristics4.

Previous studies emphasize the role of insulin resistance and glucose intolerance in the pathogenesis of PCOS5. Therefore, there is an increasing interest in using drugs such as metformin, which is part of insulin-sensitizing and anti-diabetic drugs, in the treatment of PCOS6.

Metformin, an anti-diabetic agent of the biguanide class is an adenosine monophosphate (AMP) - activated protein kinase (AMPK)-activator which is extensively used for the treatment of type 2 diabetes mellitus (T2DM). It has been reported that treatment of PCOS patients with metformin can ameliorate hyperandrogenaemia, can restore menstrual cycles, can affect the endometrial thickness and blood flow and eventually improves ovulation, implantation and pregnancy rates7. Therefore, metformin, is presently being considered as a first line of ovulation-inducing drug in infertile women with PCOS.

Recently, sitagliptin, an oral drug used in T2DM, has been considered in PCOS clinical trials8,9. Sitagliptin belongs to the family of dipeptidyl peptidase-4 (DPP-4) inhibitors10. DPP-4 plays a key role in glucose homeostasis and in the adjustment of insulin secretion11. Important substrates of DPP-4 include the glucose-dependent insulin tropic polypeptide and the glucagon-like peptide1 (GLP-1). Both these peptides are important components of the incretin pathway and are necessary for regulation of insulin secretion and uptake in the muscle cells and adipocytes and also for regulation of fatty acid metabolism8. A previous study showed that the concentrations and the serum activity of DPP4 are increased in women with PCOS10. Therefore, reducing its levels by inhibitors such as sitagliptin can improve the symptoms of PCOS.

Despite the proven efficacy of metformin in diabetic patients, combined therapy with a DPP4 inhibitor, such as sitagliptin is considered to control the blood levels of glucose, particularly as evidence of its safety and effectiveness has become available. Sitaformin is the first combined drug introduced for diabetic patients12. Since the effects of sitaformin treatment compared to the treatment with metformin alone on the oocyte and embryo quality during the course of ovulation induction in patients with PCOS have not been assessed yet, in the present study, the efficacy of sitaformin was evaluated and compared with that of metformin, on the oocyte and embryo quality during the course of ovulation induction in classic PCOS patients undergoing ICSI.

Material & Methods

Study design: This three-arm randomized placebo-controlled pilot study was conducted at the Infertility Clinic, Mahdiyeh Educational Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran after approval from the Institutes Ethics Committee. The trial was registered with www.clinicaltrials.gov (reg. no.-NCT04268563).

A total of 60 infertile PCOS patients (age: 25-35 yr), who were planned to undergo ICSI during January 2020 and May 2020, were included in this study. Written informed consent was obtained from all the participants. All the participants were asked to avoid any changes in their diet and usual physical activity and also not to take any new pharmacotherapy during the study period.

Inclusion criteria: Patients who fulfilled the classic PCOS diagnostic criteria13 were included in the study.

Exclusion criteria: Hypersensitivity to either metformin or sitaformin presence of infertility factors other than anovulation, pelvic organic pathologies, congenital adrenal hyperplasia, thyroid dysfunction, Cushing’s syndrome, hyperprolactinaemia, androgen-secreting neoplasia, T2DM, male infertility, consumption of medications affecting carbohydrate metabolism and hormonal analogues other than progesterone two months prior to enrolment in the study, severe hepatic, pancreatitis, kidney or gall bladder diseases and serum follicle-stimulating hormone (FSH) level >12 mIU/ml were considered as the exclusion criteria. Semen samples were assessed according to the World Health Organization (WHO) guidelines14 and individuals with abnormal semen parameters were excluded from the study.

Patient recruitment and randomization: Afterenrolment of the participants by a nurse, they were examined by a gynaecologist. Simple randomization method was followed in this study15.

A total of 60 patients were enrolled and divided into three groups (n=20 each): (i) metformin treated group receiving metformin (Glucophage, Merck, West Drayton, UK; 500 mg, twice daily); (ii) sitaformin-treated group treated with sitaformin (Janumet, Merck, West Drayton, UK. 50/500 mg, twice daily); and (iii) placebo administered twice daily. In all groups, drug treatment was administered two months prior to the start of the ovulation cycle and was continued until the day of the oocyte aspiration. The tablets of metformin, sitagliptin-metformin and placebo were placed in similar containers and given to participants by midwife. Both participants and physician were blinded to the treatment regimen. Before starting treatment, participants were asked to report any adverse effects experienced during the treatment, and they were evaluated for any presenting complaints at the end of the treatment period. Fifteen participants (5 per group) dropped out of the study. So, in all 45 participants remained in the study and were evaluated further (Figure).

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Figure

Flow chart of the participants.

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Ovulation induction: Participants in all groups post drug treatment were stimulated with recombinant follicle stimulating hormone (rFSH; 150 IU Gonal-F, Merck Serono Ltd., UK) from day three of the menstrual cycle till the day of hCG injection. The menstrual cycle was induced by progesterone (Medrofem^®, Iran Hormone, Iran) followed by injections of cetrorelix acetate 0.25 mg/day (Cetrotide, Merck Serono), a GnRH antagonist, when there were follicles >12-mm diameter on ultrasound scan and were carried on till the day of human chorionic gonadotropin (hCG) injection. Recombinant hCG (10,000 IU Ovitrelle, Merck Serono) was injected to the participants in all groups when the diameter of two or three follicles reached at least 18 mm on ultrasound scan. Oocyte retrieval was performed approximately 36-40 h after hCG administration using transvaginal ultrasound guidance. The drug treatment was stopped on the day of oocyte aspiration.

Assessment of baseline and clinical features: For each participant weight, height and body mass index (BMI) were recorded and fasting blood sample (8 ml) was collected once prior to the start of treatment and once on the day of oocyte aspiration. The blood samples were allowed to clot at room temperature for 15-30 min and serum was separated by centrifugation at 1500 g for 10 min at RT (EBA 20, Hettich, UK). The serum samples were stored at −70°C for future analysis.

Serum levels of total testosterone (TT), estradiol (E2) and fasting insulin were measured in all samples using enzyme immunoassay (Demeditec Diagnostics GmbH, Germany).

Oocyte retrieval, intracytoplasmic sperm injection (ICSI) and embryo culture was carried out according to the protocol followed in Mahdia Hospital, as described by Daneshjou et al16.

Statistical analysis: Data were analyzed using the SPSS software version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Kolmogorov-Smirnov test was employed to assess the normality of continuous variables, and data were reported as the mean±standard deviation (SD). One-way ANOVA, Tukey’s and Dunnett’s T3 tests for post hoc analysis and chi-squared test was used for statistical analysis where appropriate. P≤0.05 was considered as significantly different.

Results

Semen parameters: The semen analysis of the male partners of the study participants was normal and there were no significant differences in the characteristics of semen parameters between the three groups. Semen volume was 3.0±0.7, 3.2±1.0 and 3.2±0.8; sperm concentration was 12.6±3.0, 15.4±5.0, 14.5±4.1; total count was 40.6±7.2, 39.7±8.3, 37.1±4.4; sperm motility was 45.9±6.8, 43.6±6.0, 42.7±5.1 while, normal forms were 5.7±2.0, 5.8±2.3, 6.4±3.0 for placebo, metformin and sitaformin groups, respectively. .

Clinical and demographic characteristics: There were no significant differences in the age (29.53±2.97, 28.93±2.76, 30.26±3.15), duration of marriage (7.73±2.28, 7.46±2.35, 7.33±2.02) and duration of infertility (5.86±1.99, 5.93±2.73, 5.53±2.09) between the three groups (placebo, metformin and sitaformin, respectively). Furthermore, there were no significant differences in weight, height and BMI before and after the treatment between these groups (Table I).

Biochemical characteristics: No significant differences were identified in the serum levels of insulin, estradiol and TT among the three groups before treatment. Serum levels of insulin and TT reduced significantly after treatment in both metformin and sitaformin treated groups compared to the placebo (P<0.05). Further, there was no significant difference in serum levels of estradiol among the three groups after treatment (Table II).

Evaluation of the quality of the oocytes and embryos: There was a significant difference in the number of immature oocytes (MI + GV stage) and normal MII oocytes in the sitaformin and metformin group compared to the placebo group (P<0.05). In addition, there was a significant difference in the number of MI + GV stage, grade I embryos, fertilization and cleavage rates in the sitaformin group compared to the metformin and placebo groups (P<0.05).

There was no significant difference in the total number of the oocytes retrieved and the number of abnormal mature oocytes nor the transferred embryos (grade I + II) or the biochemical change and clinical pregnancy rate or in the rate of ovarian hyperstimulation syndrome or in the duration of stimulation, among any of the groups (P>0.05) (Table III).

Discussion

In the classic type of PCOS, hyperandrogenism, hyperinsulinaemia and insulin resistance are frequent manifestation1, which affect the quality of oocytes and embryos and are considered the cause of ovulatory dysfunction. Previous studies have shown that metformin treatment in women with PCOS can reduce hyperandrogenism and insulin resistance and is effective in improving ovarian function16,17. In the present study also, we showed that metformin caused a significant decrease in the serum levels of insulin and TT compared to the placebo group. Metformin also increased the number of MII oocytes retrieved and normal MII oocytes significantly when compared to the placebo. These findings are consistent with the findings from other studies16,18,19. In addition, the results of this study showed that metformin affected neither the number of oocytes retrieved or the rate of fertilization, cleavage and pregnancy nor the quality of embryos. While these findings are in agreement with the findings of previous studies2,20, some other studies have reported that metformin can increase the number of oocytes retrieved21 and the rate of fertilization19. The differences in the results achieved by various studies are probably due to the differences in treatment duration or the administered dose of metformin as well as genetic differences in the study population.

Studies have investigated the effect of sitagliptin on metabolic and hormonal factors in PCOS patients22-24. A study by Palma et al24 had shown that sitagliptin decreased insulin levels in PCOS patients, perhaps by influencing pancreatic beta-cell function. Sitagliptin selectively and competitively inhibits the enzyme DPP-4 and ultimately increases incretins such as GLP-125. By suppressing glucagon secretion from islet cells and facilitating glucose uptake by liver and muscle, incretin leads to a decrease in glucose production from hepatic sources26,27. In addition, previous studies have pointed to the effect of GLP-1 on the activation of anti-apoptotic pathways24,28. Therefore, sitagliptin may reduce apoptosis in ovarian granulosa cells by increasing GLP-1 and ultimately improve oocyte quality29. In a study by Wang et al27, in the theca cells of PCOS rat models, the expression of TGF-β1 was higher than in the control group, and it is possible that the high expression of TGF-β1 was related to the occurrence of hyperandrogenism. DPP-4 levels are excessively reduced by sitagliptin, which may reduce the levels of factors related to the TGF-β1/Smad2/3 signaling pathway and ultimately reduce androgen levels indirectly. Ferjan et al30 have shown that sitagliptin was able to preserve the androgen profile more effectively than the control. Sitagliptin can also decrease AGE (advanced glycation end) products and improve ART outcomes. AGEs can induce oxidative stress as well as activate inflammatory pathways in vascular endothelial cells by binding to RAGE (receptor for advanced glycation end products)31. Carani and Dipti32 have also shown that sitagliptin recuperates oxidative stress and inflammatory cytokine expression in the ovary of PCOS rats. Therefore, sitagliptin may improve the quality of oocytes and embryos by decreasing the inflammatory factors.

Since metformin and sitagliptin have different mechanisms of action; therefore, combined use of the two drugs in the form of sitaformin can be more effective in achieving much greater improvements not only in controlling glycaemic conditions in patients but also in increasing the mature oocytes and quality of embryos in PCOS patients. Sitaformin significantly decreases glycosylated haemoglobin (HbA_1c) and fasting plasma glucose, and it also improves proinsulin/insulin ratio with a lower incidence of abdominal pain and diarrhoea compared to metformin monotherapy in patients with T2DM33,34. Al Hussona et al25 have shown that metformin in combination with sitagliptin has a greater effect than metformin alone on oxidative stress and pro-inflammatory markers in patients with T2DM. Our results also indicated that treatment with sitaformin caused a significant decrease in the serum level of insulin and TT and also in the number of immature oocytes compared to the placebo. Sitaformin was further able to significantly increase the number of MII oocytes retrieved, normal MII oocytes, oocyte fertilization and cleavage rate and also the number of grade I embryos compared to the placebo. Moreover, sitaformin significantly increased good quality embryos and also significantly decreased the number of immature oocytes compared to the metformin-treated group. These results are indicative of the synergic effect of metformin and sitagliptin when combined together.