Effect of Metformin and Its Combination with Probiotic on Menstrual Irregularity and TSH Levels in Patients with Polycystic Ovarian Syndrome: A Randomized Controlled Trial

Background: Polycystic ovarian syndrome (PCOS), the far most common endocrine disorder among sexually active women. The disease is typically characterized by irregular menstrual cycles and appears to be influenced by associated thyroid dysfunction. Aim: The goal of this research was to assess and compare the effects of Metformin and its combination with Probiotic on monthly cycle irregularity and TSH levels in PCOS women. Original Research Article Zafar et al.; JPRI, 33(33A): 29-38, 2021; Article no.JPRI.70009 30 Methodology: This was a single-center study conducted at Karachi's Gynecological Outpatient department from January 2019 to September 2019. A total of 52 PCOS patients aged 18 to 40 years were included in this trial, which followed Rotterdam criteria. After providing written and consent form, individuals were randomized into one of the two groups and received Metformin 500 mg TD (n = 26) or Metformin Combination (n = 26). Results: After three months of treatment, both groups improved in terms of menstrual cycle irregularity and TSH levels, but the combination treatment improved the most. Conclusion: Probiotics may be considered in conjunction with Metformin for improving TSH levels to achieve better results.


INTRODUCTION
Polycystic ovarian syndrome (PCOS) is a commonly diagnosed reproductive disorder and one of the leading causes of ovulatory infertility. PCOS is well-defined by the Rotterdam criteria as a combination of oligo / amenorrhea, clinical or biochemical signs of hyperandrogenism, and polycystic ovary on ultrasonography [1]. Being a heterogeneous disorder, it affects many body functions, leading to a variety of health complications such as infertility, menstrual dysfunction, androgenic symptoms, metabolic syndrome, and autoimmune disease.
Thyroid autoimmunity is associated with an increased risk of infertility, spontaneous miscarriage, and metabolic dysfunctions, all of which are common in PCOS [2,3]. In women with PCOS Subclinical hypothyroidism (SCH) and thyroid autoimmunity are more common than in women of the general population [4,5]. Recent studies have explored the relationship between thyroid autoimmunity and metabolic parameters in PCOS, particularly dyslipidemia and insulin resistance [6,7].
Thyroid hormones can act as insulin agonists in muscle and antagonists in the liver, so thyroid deficiency may result in a decline in glucose production and utilization [8,9]. Moreover, there are studies that have identified insulin resistance(IR),as a primary factor in the pathogenesis of PCOS, which might also play a key role in developing hypothyroidism [10,11].
The most frequently recommended drug for the treatment of insulin resistance is Metformin. It is considered a safer drug, with no clinically relevant pharmacologic interaction that has been described yet [12]. In the recent years, there has been a growing body of evidence supporting Metformin's beneficial effects in PCOS [13]. Multiple studies resulted in consensus statements and recommendations for Metformin use in PCOS patients [14,15].
Spite of the fact, that Metformin was instituted in clinical practice approximately five decades ago for the management of diabetes mellitus [16] however, its effect on clinically significant menstrual cycle irregularity was first reported in 1994 due to improvements in insulin resistance [17]. This was only lately, that this drug was observed to modify the thyroid hormone profile resulting in a substantial reduction of serum TSH levels [18,19].
There are number of adverse effects associated with Metformin which are commonly GI in nature: diarrhea, nausea, and/or abdominal discomfort. These effects are usually mild, transitory, and dose-related, but then they can arise in up to 50% of patients taking the drug [20,21]. And, even at low dosages, about 5% of individuals can't endure the medication [20].
Because of the increasing likelihood of side effects, the use of this drug has been limited. As a result of the challenges, dietary approaches are becoming more popular for managing different diseases [22].
Considering that, Probiotics emerge as effective nutritional supplements because of their possible future benefits to human health or preventative medicine [22]. Probiotics are characterized as live microorganisms that, when ingested in adequate amounts, present a health beneficial effect on the host [23]. They are not delegated drugs under current worldwide law. Yet, they are still found to reduce pathogenic bacterial colonization in the intestinal tract, improve physiological gut barrier function, and impact the production of proinflammatory and anti-inflammatory cytokines [23-25].
Moreover, there seems to be currently little scientific proof of Probiotics interacting with concurrent drug ingestion, attributed to their impact on bacterial enzymatic activity. Given the popularity of probiotic-containing products, it is critical to identify any potential interactions with pharmacological therapies [26,27].
In view of above circumstances, prescriptions for insulin-sensitizing drugs, most notably Metformin, are not common among hypothyroid patients however, combination of Metformin and Probiotic have also not been used previously. Therefore, the purpose of this study is to directly investigate the effect of Metformin, alone or in combination with Probiotic, on the management of menstrual irregularities and TSH levels during PCOS treatment.

Trial Design and Participants
It's an open label, randomly allocated, parallelarm, and non-inferiority research in which 52 new participants with established PCOS, aged 18 to 40 years, were registered. This singlecenter cross-sectional study was conducted on women attending a gynecological clinic at a hospital in Karachi.
The participants were listed via convenient sampling technique between January 2019 to September 2019. It was the non-probability sampling technique so we cannot rule out the chances of biasness The sample size was calculated by Sealed Envelope calculator version 201: (Significance level (alpha) 1%, 99% confidence interval Power (1-beta) 90, Percentage success in control group: 12%.

Consent
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

Inclusion Criteria
Newly diagnosed PCOS patients were enrolled according to the Rotterdam criteria. According to the criteria, two of the following three characteristics are required to detect PCOS cases: • Oligo-ovulation or anovulation: (Oligomenorrhea i.e. more than 45 days or less than 8 cycles per year and Amenorrhea i.e. more than 3 months in women with previous periodic menses) for a period of 6 months. • Hyperandrogenism: clinical (including signs such as hirsutism) or biochemical (including a raised free androgen index or free testosterone) hyperandrogenism. • Polycystic ovaries on ultrasonography: >12 follicles in one or both ovaries, 2-9 mm in diameter and/or increased ovarian volume >10 m 3 ).
Menstrual cycle and serum TSH levels were evaluated before and 3 months after starting Metformin and its combination with Probiotic treatment. Blood samples for assaying TSH were drawn between 08:00 and 09:00 a.m., after an overnight fast.

Statistical Analysis
SPSS 20 was used to analyze the data. The numeric factor was represented as mean standard deviation, while the categorical variable was represented as frequency and percentage. Normal distribution was verified by using the Shapiro -Wilk test. The paired-t test was used to compare the pre andpost result of all numerical parameter. The Independent Samples t Test was used to test the differences between the means of two groups. A p-value of 0.01 was deemed statistically significant.

RESULTS
At first, 60 patients were able to enroll in this trial; however, 8 participants were excluded, including those who refused to sign the consent form (n= 5) and those who did not return for follow-up (n= 3). As previously stated, all eligible participants were randomly assigned to one of two interventional groups: Metformin and Combination (Met/Pro) ( Fig. 1).

Menstrual Cycles:
The 26 patients in both groups had irregular menstrual cycles at the start of the study. The current study found that symptoms improved in 13 women in the Metformin group (50%) and 20 women in the Met/Pro group (77%) after 12 weeks of treatment (Fig. 2).
The mean age of the patient in the Metformin group and Combination group is 27.2 ± 4.6 and 25.1 ± 5.3 respectively. Both the groups showed no significant change in the age.
At the start there was no significant difference between the two treatment groups. The TSH levels were decreased significantly after 12 weeks of intervention in both the treatment groups. The Combination group showed more prominent results than Metformin alone however, no significant change exist between the two groups after treatment.

DISCUSSION
PCOS is thought to be a multifactorial dysfunction with a heterogeneous etiology. It has also been related to hypothyroidism in 6.3% of women with PCOS Thyroid-stimulating hormone (TSH) is a pituitary hormone that stimulates the thyroid gland to produce thyroxine (T4), followed by triiodothyronine (T3), which stimulates the metabolic processes of nearly all other tissues of the body [33]. It is indeed a glycoprotein hormone that is produced by thyrotrope cells within the anterior pituitary gland that helps regulate the thyroid's endocrine function [34].
Regardless of the fact that the etiology of hypothyroidism and PCOS is exclusively different, both of these conditions share many characteristics [19]. Thyroid levels that are too high or too low can cause ovulatory dysfunction and menstrual irregularities [36].
The purpose of this study was to see how adding a Probiotic to Metformin and Metformin alone affected PCOS women with menstrual irregularities and TSH levels.
Metformin is used for not just the prevention and treatment of hyperglycemia-related disorders, but it is also used for the management of other metabolic, endocrine, and non-endocrine diseases [37]. Current medical research is revealing new aspects in the pharmacology of Metformin. As, it is known that hypothyroidism has been linked to polycystic ovarian syndrome (PCOS

CONCLUSION
In patients with polycystic ovarian syndrome, the use of Probiotics in addition to Metformin had a more favorable effect on cycle regularity and TSH levels reduction. More research is needed to determine the mode of action of these supplements in relation to Thyroid modulation.

LIMITATIONS
It was an only centered study and because we used a non-probability survey method, there is indeed a possibility of selection bias.

FUTURE RECOMMENDATIONS
More far multi-center and long-term clinical studies are needed to validate the findings and different amounts of Metformin and Probiotics must be evaluated for unrivalled results.

CONSENT
The authors obtained written and informed consent from the patients and kept it.