Efficacy of vitamin D supplementation and metformin compared to metformin alone in infertile females with polycystic ovary syndrome: a randomized open label trial.

doi:10.21203/rs.3.pex-2199/v1

Method Article

Efficacy of vitamin D supplementation and metformin compared to metformin alone in infertile females with polycystic ovary syndrome: a randomized open label trial.

https://doi.org/10.21203/rs.3.pex-2199/v1

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Background: WHO has declared Infertility as the fifth-highest serious global disability with prevalence of approximately 22% in Pakistan; 80% due to polycystic ovarian syndrome (PCOS). PCOS is characterized by “the presence of any two out of three criteria, that is oligo and/or anovulation, excess androgen activity and/or polycystic ovarian morphology on ultrasound”. Vitamin D deficiency (VDD) [serum 25(OH) D <20 ng/ml] is vital for female health and becoming public health problem even in sun-drenched cities. With high prevalence (58%) of VDD in Pakistani PCOS. The hypothesis: “VD supplementation can improve fertility by correcting in IR, ovulatory dysfunction, endometrial thickness and hyperandrogenism in primary infertile PCOS patients of our population”.

We aim to evaluate impact of oral VD supplementation on fertility potential of VDD PCOS (VD <20 ng/ml) in a 2-years randomized clinical trial; Group-1 VDD control PCOS (n=100) and Group-2 VDD intervention group (n=100). Group-1will receive 1.5 g/day metformin therapy for 24 weeks. Group-2 will be given 50,000 I.U of VD once weekly for 8 weeks, followed by 50,000 I.U once every fortnight from 9- 24 weeks plus metformin therapy.

Impact will be assessed by comparing trans-vaginal ultrasound (TVS; Day 12) at the start with TVS at end point (24 weeks) to observe change in dominantfollicle size (≥14 mm) and endometrial thickness (≥8 mm).

Discussion: The proposed interventions will help to evaluate the impact of vitamin D supplementation on follicular maturation and endometrial thickness and PCOS parameters including lipid index and insulin resistance.

Trial registration: ClinicalTrials.gov, NCT03989778

Infertility as declared by WHO ¹ is the fifth-highest serious global disability with 22% prevalence in Pakistan ². There are many causes of infertility, however Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility among women of reproductive age with metabolic manifestations, such as obesity, Insulin resistance (IR), hyperandrogenism, risks of cardiovascular disease and type 2 Diabetes ^{3, 4}. Anovulatory infertility in PCOS is attributed to IR preventing growth of ovarian follicles to a size of ≥14 mm, and hyperandrogenism disturbing endometrial blood flow/thickness required for embryo implantation. Recently, vitamin D deficiency (VDD) has been implicated in PCOS as a risk factor for glucose intolerance, with low levels associated with IR and increased diabetes risk. However, discrepancy in the findings in studies evaluating effects of vitamin D (VD) supplementation on glucose homeostasis, hormonal status, lipids and biomarkers of inflammation and oxidative stress are noted. Moreover, it has been shown to play a role in egg quality and fertility.

PCOS prevalence has increased (52- 59%) due to a change in eating habits; increased intake of sugar, fast foods and soft drinks superimposed on a sedentary lifestyle and lack of exercise ⁴ causing anovulatory infertility in 80% of cases ^5-8. In our population at a cut off of 20ng/ml (50nmol/l), 58- 59% PCOS had VDD ^{7, 9}. Impaired VD status is an important risk factor for IR, leading to PCOS, and its associated metabolic and hormonal abnormalities ¹⁰ ¹¹. Together, PCOS and moderate VDD may exacerbate cardiovascular and metabolic characteristics in women with PCOS ¹⁰. In addition to global studies, a strong and independent association between VDD and risk of PCOS has been identified in Pakistani population ¹².

Evidence supports that VD supplementation can improve fertility of PCOS, but the cause effect relationship is not delineated. The hypothesis: “VD supplementation can improve fertility by correcting IR, ovulatory dysfunction, endometrial thickness and hyperandrogenism in primary infertile PCOS patients of our population” is supported by our research; improvement in metabolic and reproductive characteristics of PCOS-induced animal model with VD therapy, association of VD with maturity of oocytes and clinical pregnancy¹³.

Contextual Problem:

VDD is a prevalent in women with PCOS in Pakistan^{14, 15}. A “D-Health Trial” conducted nationally has confirmed role, safety and dose of VD (4000IU/day) supplementation in mothers¹⁶. Role of VD in fertility is supported only by observational studies ¹⁷. Gaps on “proof-of-concept”, “cause-effect relationship”, “optimal dose of dietary VD” ¹⁸ with lack of published guidelines for VD supplementation for fertility. Large, multi-center, placebo-controlled clinical trials in infertile females with PCOS are therefore essential to establish role and dose of VD supplementation to correct VD status before pregnancy and improve “Quality of Life” of these females ¹⁹.

Randomized control trials (RCT) on VD replacement prove that VD improved serum insulin, triglycerides, very low-density lipoproteins, and cholesterol levels in women with VDD and PCOS without effecting fasting blood glucose and other lipid profiles ²⁰. Another study reported that VD supplementation had no significant effect on metabolic and endocrine parameters in PCOS with exception of reduced plasma glucose ²¹. Regarding its impact on fertility, Aramesh et al, proved that VD increases levels of Anti Mullerian Hormone (AMH) without changing the antral follicle count/ovarian reserve ²². Lerchbaum et al,. reported showed improvement in LH/FSH ratio in PCOS females with 24 weeks VD treatment in RCT ²³. VD supplementation improved ovulation and helped in conception of PCOS during in-vitro fertilization (IVF) ^{22, 24-26}. VD administration (50,000 IU / week) decreased the hirsutism scores, androgen levels and improved fertility in overweight PCOS females ²⁷. The rationale for combination of Metformin with VD is supported from an Indian study where the combination enhanced follicular maturation in PCOS patients ²⁸. The likelihood of improvement in endometrial thickness is supported by Asadi et al,. ²⁹.

Study Rationale:

According to WHO more than 180 million couples in developing countries suffer from primary or secondary infertility with 22% prevalence in Pakistan. ^{1, 30}. High prevalence in developing countries is due to insufficient basic knowledge, lack of exploration of infertility causes, unproductive treatment plans, minimal focus on infertility care, and associated socio-cultural beliefs/stigmas. Infertility has resulted in an impact on health whereas social stigma and psychological trauma of childlessness has led to demotivation, isolation and abandonment of the infertile couples from the society. Increased infertility prevalence with exacerbation of psychological and economic burden of developing countries has motivated researchers to look for the relationship of infertility with various treatable/modifiable causative factors. One of these factors is VDD, affecting one billion people around the globe with an alarming VDD in Pakistan despite exposure to sunrays ^{31, 32}. Impaired VD status is an important risk factor leading to development of PCOS, anovulatory infertility and increased metabolic and hormonal abnormalities ^{10, 11}. Observational national studies on VDD association with risk of PCOS and work of our research group on animal model highlight the need of a RCT ^{12, 33}.

We aim to evaluate impact of oral VD supplementation on fertility potential in women with VDD and PCOS. The proposed interventions will help to evaluate the impact of vitamin D supplementation on follicular maturation and endometrial thickness and PCOS parameters including lipid index and insulin resistance.

Aim & Objectives:

Aim: To evaluate impact of oral VD supplementation on fertility potential of women with PCOS in a randomized clinical trial setting for two years.

Primary Objectives: To study the effect of oral supplementation of VD in infertile females with PCOS from the time of enrollment to its completion (24 weeks) on fertility potential, by comparing size of dominant follicles (≥ 14 mm) and endometrial thickness (≥ 8mm) by day-12 using Trans Vaginal Scan (TVS).

Secondary Objectives: To study the effects of oral supplementation of VD in infertile women with PCOS from the time of enrollment to its completion (24 weeks) on:

· IR, by comparing results of HOMA-IR (serum insulin, fasting blood glucose).

· Lipid Accumulation Product (LAP index) ^{34, 35}.

Study Outcome: Accomplishing these aims will inform us to report VDD as a predisposing factor for infertility and recommend VD for prevention and treatment of infertile women with PCOS.

Methods:

Study design: This will be a multidisciplinary individual randomized open label trial to be conducted by Departments of Biological & Biomedical Sciences, Pathology and Laboratory Medicine, Community Health Sciences, Gynecology & Obstetrics, Medicine (Endocrinology) and Radiology in Aga Khan University Hospital (AKUH) for a period of 2 years following approval from Clinical Trials Unit (CTU) and Ethical Review Committee of AKUH (ERC- 2021-2033-19498).

Inclusion criteria: Women presenting at the Gynecology & Endocrine Clinic, from all ethnic background, age range 20-36 years with primary infertility and diagnosed PCOS with presence of at least 2 of the following 3 elements: hyperandrogenism, chronic anovulation and polycystic ovaries (from reports of available routine TVS, as standard of care) with VDD (serum levels <20 ng/ml) will be included in the study after informed consent.

Exclusion criteria: Exclusion of subjects will be done by the treating physicians (Co-Principal Investigators (Co-PIs)) at the respective clinics after clinical, biochemical, and radiological evaluation. All women meeting any of the following criteria will be excluded from the study:

· Women with secondary infertility

· Hypercalcemia (plasma calcium concentrations >2.65 mmol/L)

· Tuberculosis or other granulomatous disorders, women on VD replacement, oral contraceptives, hormonal replacement therapy, glucocorticoids, calcium supplementation, insulin-sensitizing drugs (incretin mimetic drugs, thiazolidinedione, sulfonylurea), lipid-lowering drugs or other drugs affecting insulin sensitivity or serum androgens (e.g., niacin, corticosteroids, beta-blockers, calcium channel blockers, thiazide diuretics), anti-epileptics, anti-retroviral, cholestyramine, anti-fungal, statins, H2 receptor blockers, immunosuppressants, chemotherapeutic agents, antimicrobials (rifampicin, isoniazid, hydroxychloroquine) or any other drug modifying lipid metabolism in the previous three months prior to study

· Women with history of congenital adrenal hyperplasia, Cushing’s syndrome, androgen-secreting tumors, type 2 Diabetes Mellitus, renal, hepatic, or thyroid disorders, hyperparathyroidism, malabsorption syndromes, chronic kidney disease, hepatic failure, cystic fibrosis, vaginal bleeding of unknown etiology or /and suffering from COVID-19 (within 3 months).

Recruitment of subjects: It will be a purposive sampling and women diagnosed with PCOS based on Rotterdam criteria with confirmed VDD (VD <20 ng/ml) will be recruited. VD will be tested in Clinical laboratory of AKUH before recruitment.

Sample size calculation; Total of 97 individuals in each group (VD supplementation with metformin and Metformin only) will achieve 80% power when anticipated change in follicular size is (>14 mm) (11) and two-sided level of significance is 0.05. Sample size of 200; 100 participants in each group with a dropout rate of 3% will be considered.

Distribution of subjects in study years: A total of 200 subjects (3% drop out rate); 100 in each arm will be recruited; constituting control and intervention groups 50 (each) in years I and II respectively.

Randomization: Subjects will be brought to Clinical Trial Unit (CTU) of AKU which has full-fledged clinical space for consultation, trial, and laboratory support. Randomization will be done using the permuted block randomization technique generating blocks with equal representation of all arms in a 1:1 ratio (Table 1). The randomization list is confidential and will be kept within the premises of CTU under restricted access.

Table 1: Treatment Allocation during the study period (24 weeks) in all study years

Group-1 (Control)

Group-2 (Intervention)

Metformin therapy 1.5 grams/ day (24 weeks)

Vitamin D capsules 50,000 I.U once weekly (Initial 8 weeks)

Vitamin D capsules 50,000 I.U once every fortnight (9-24 weeks).

No VD therapy

Metformin therapy 1.5 grams / day (24 weeks)

o Study starts point: Randomization will be followed by detailed history, physical examination, biophysical and biochemical parameters estimation. Anthropometric parameters including the weight and height of each patient will be measured, and body mass index (BMI) will be calculated as weight (kg)/height (m)². Waist circumference (WC) will be measured as an indicator of abdominal obesity, at the midway level between the lateral lower rib margin and the superior anterior iliac crest, in a standing position. Drug for 12 weeks will be dispensed.

· Study mid-point (follow-up visit at 12 weeks): At 12 weeks follow up, compliance to supplementation and occurrence of any adverse effect will be assessed by research team allocated members. Moreover, drug for remaining 12 weeks will be dispensed and any other queries of participants will be responded. Special instructions to women in control group not to take any form of VD during the study period will be given (Fig 1).

· Study end-point (follow-up visits at 24 weeks): Trans Vaginal Scan [TVS; Ultrasound examination]: will be done on Day 12 of cycle after 24 weeks treatment (study end point) in both groups at Clinical imaging services of the Radiology Department of AKUH using a high frequency transvaginal probe (5-11 MHz) of Toshiba Xario 200 or Aplio 500 machine in a private room by senior sonographer/radiologist with more than 5 years of experience using standard operating procedures. Women will be asked to empty urinary bladder before scan. In the examination room, the individual will be requested to undress from waist down, lie down on the examination table in knees flexed position and covered with a sheet. The TVS probe will be covered with two condoms and lubricating gel before insertion into the vagina. Client will be informed that the probe insertion may be mildly uncomfortable temporarily but relieved after the procedure. The test will take approximately 20 minutes and the results will be available in 24 hours. Change in follicular size and endometrial thickness will be assessed by comparison of TVS done at 24th week with the base line TVS reports of these females (retrieved from the desk records).

· VD estimation of Group-2 (Intervention): Women will be inquired about clinical manifestations of hypervitaminosis D; anorexia, weight loss, weakness, fatigue, disorientation, vomiting, dehydration, polyuria and constipation. Serum calcium and albumin will be tested if required. All the participants will be informed about the outcome of the results. Vitamin D estimation will be done at study end point using human, 25-hydroxyvitamin D ELISA Kit (Cat. No E1919Hu, Bioassay Technology Laboratory) with the detection range of 7pmol/L-1500pmol/L with an intra and inter-assay coefficient of variation (CV) of <8% and <10% respectively.

Fig 1: Intervention Model of the clinical trial unit for the proposed project

Biochemical Testing:

For the biochemical parameters, blood samples (10 cc) will be collected by vein puncture in gel vacutainer for triglycerides and insulin, fluoride for glucose after 8 to 12 hours of fasting. Serum will be separated and stored at -40 for further analysis. Fasting insulin, glucose and Triglycerides will be tested by Enzyme Linked Immunoassay (ELISA) in Multi-disciplinary lab as per manufacturer’s protocol. ³⁴.

HOMA-IR will be calculated using fasting glucose levels [mmol/L] × fasting insulin levels μU/mL]/22.5

Lipid accumulation product (LAP) index will be calculated, (waist circumference [cm] − 58) × (triglyceride concentration [mmol/L]) in women³⁵. Waist circumference will be measured.

Table 2: Methods used to achieve respective Objectives

Objectives

Key Indicators

Methods Used: Comparison at start and end point (24 weeks) in both study groups:

To study the effect of oral supplementation of Vitamin D on:

Fertility potential

Improvement in follicular size and endometrial thickness

Trans Vaginal Scan (TVS) findings using a high frequency transvaginal probe (5-11 MHz) of Toshiba Xario 200 or Aplio 500 machine in radiology clinic

Insulin Resistance

Reduction in :

· Homeostatic Model Assessment of Insulin Resistance. (HOMA-IR Range: (0.5–1.4)

· Lipid Accumulation Product (LAP index)

· HOMA-IR calculated by; fasting glucose levels [mmol/L] × fasting insulin levels μU/mL]/22.5 Fasting Blood Glucose and Insulin will be conducted in Multi-disciplinary lab by ELISA kits(37).

· Lipid accumulation product (LAP) index, (waist circumference [cm] − 58) × (triglyceride concentration [mmol/L]) in women (38). Waist circumference will be measured and Triglyceride concentration will be conducted in Multi-disciplinary lab by ELISA kits.

· Ethical Considerations: Research will be carried out in compliance with ethical principles for medical research involving human subjects of Helsinki Declaration. They will have a counselling session at the time of enrollment in the study with the Research Associate in which they will be told about;

o purpose of the study trial

o Chances of inclusion in the study on the basis of randomization by computer

o Request to avoid ovulation therapy during the study period (24 weeks)

o Report to the investigators if a spontaneous conception occurs

o Report to the investigators for any adverse effects

o An informed consent will then be signed by the participants. It would be made explicit that non-participate will not result in any loss of benefit. All the information related to participants will be kept confidential. Their names and identity will not be recorded. Each individual will be given a unique research I.D. Sample will be collected in a manner which confers minimal discomfort to the participants.

· Data acquisition and Management: An electronic data management system (DMS) will be created, and all information will be stored at the Department of Biological & Biomedical Sciences (BBS), Aga Khan University. Each participant will be assigned a unique study ID. The true identity of any subject will neither be linked to an electronic file nor shared with anyone. Initially all information will be entered in Microsoft excel. Data will be properly coded, decoded and then analyzed for final interpretation. It will be stored and archived for a period of 5 years only. The access to the hard copies and identities will be restricted to the PI only.

· Plan of Data Analysis: The data analysis will be divided into two steps. In the first step descriptive findings by study arms will be reported as numbers and percentages for categorical variables, mean & standard deviation for symmetric data and median & inter-quartile range for asymmetric data. In the second step, Generalized Estimating Equation (GEE) for linear models at univariate and multivariable analyses will be used because the follicular size, endometrial thickness, improvement in IR and FAI (outcome variables) will be taken at two time points (baseline and 24 weeks). The relationship of variables with intervention at univariate level and will also be observed after adjusting for confounding and/or plausible interactions at multivariable level. Multicollinearity among independent variables will be assessed using Pearson correlation coefficient, Cramer’s V or Phi, Eta and Kendall’s Tau for variables eligible for multivariable analysis. This criterion will be set at a p-value <0.20. A p-value of less than 0.05 will be treated as significant for the multivariable analysis. It will be conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS version 21; IBM Corp Inc., Armonk, NY). Descriptive analysis of categorical data will be presented in terms of frequencies and percentages, whereas that for continuous variables, will be expressed as mean ± standard deviation. Mann–Whitney U test and Pearson’s χ2 test of independence will be used for comparing continuous and categorical variables wherever applicable. In all calculations, a p-value of <0.05 will be considered significant.

Discussion: This RCT will evaluate the hypothesis that interactive effect of vitamin D and insulin sensitizing agent Metformin, will substantially improve fertility potential and metabolic parameters in PCOS patients having VDD. A multipronged approach is required to manage PCOS related infertility with the aim to reduce associated hormonal changes, to improve endometrial dysfunction and to enhance follicular development. Such interventions help in achieving a favorable milieu for ovulation inducing drugs for better pregnancy and ovulation rates.

Studies have shown promising effect of metformin in improving endometrial dysfunction and ovulation rates in PCOS patients ³⁶. Similarly, observational studies have supported vitamin D inverse association with body mass index, waist circumference and IR^{37, 38}. However, Rashidi H et al.³⁹ found no such correlation between vitamin D, high cholesterol levels and IR. The emerging evidence is supporting vitamin D supplementation in improving anovulation and use of vitamin D supplementation with clomiphene is beneficial in improving pregnancy rates ⁴⁰. Recently, a systematic review of RCTs evaluated vitamin D use with placebo or with Metformin and reported beneficial effect of vitamin D on follicular development versus no use and improved menstrual regularity in patients with combined use of vitamin D and Metformin ⁴¹.

However, there is need of further evidence for managing PCOS to better understand the effect of these two modalities on follicular size, endometrial thickness, IR and biochemical parameters of metabolic syndrome including LAP index. Therefore, this randomized trial will provide evidence to solve this puzzle and to better understand the combined effect of Vitamin D and Metformin as this synergistic effect has its impact ranging from restoration of ovulation to increase endometrial receptivity leading to successful pregnancy outcome. The evidence generated from this RCT will help physicians to provide appropriate treatment to their PCOS patients having dire need of improving fertility.

Limitations

· A dropout rate of only 3% is considered due to budget constraints, which is likely to be higher than this assumption. A proper follow-up plan of the subjects will be closely monitored.

o Since it is not a double-blind placebo controlled randomized trial, it will be very difficult to stop someone else prescribing VD to women in the control arm. This will be dealt with frequent discussions and reminders to females with PCOS in control arm.

o Restriction of specific fertility treatment plans during the study period (6 months) may disappoint the infertile couples who will be counselled to have patience during the study with the hope that both drugs have been reported to improve fertility once are taken in exact dosage and for recommended time duration.

o No fertility treatment is given to these females, yet the chance of normal conception cannot be overlooked since Metformin’ and ‘Vitamin D’ both increase the fertility potential. Conception as a result will therefore increase the “Intention to treat analysis” in the respective arms and decrease ‘end-to end’ sample size.

o The beneficial effects of this safe and less expensive treatment on human reproduction will be determined. However, VD treatment alone may not be able to show drastic improvements in reproductive issues; A message to be communicated loud and clear to avoid over expectations of the subjects.

Outcome Measures

Public Health: After identification of pathophysiology and causal relationship of VDD with infertility in PCOS females, VD could be added for correction of IR, hyperandrogenism and hyperinsulinemia in spontaneous and assisted cycles to improve follicular maturity and endometrial thickness for successful conception, if indicated in the trial. VD supplementation will also prevent associated comorbidities; type 2 diabetes, MS and cardiovascular diseases in these females. Moreover, added to lifestyle modifications in unmarried females with clinical manifestations of PCOS for prevention of infertility in the years ahead.

Social Impact: This study will help to reduce the chances of subfertility, hence medical, social, psychological and economic burden of developing countries.

Educational Impact: Proven the results, awareness sessions and seminars to emphasize on correction of VDD will be organized.

Collaboration: The multi-disciplinary project will promote team building to accomplish key tasks and shared ideas on varied responsibilities within AKUH.

Capacity Building: The RA will have an exposure to galaxy of researchers; endocrinologist, gynecologist, radiologist, basic scientist and statistician. Knowledge sharing and working under their guidance will lead to personal and professional development.

Policy Development: In Pakistan, investments in the Health sector are viewed as an integral part of the government’s poverty alleviation endeavor. An effective nutritional strategy with nutrition interventions, supplements and nutritional awareness plans to control VDD in females of reproductive age at all levels of the community can therefore be proposed. A structured public health program could be launched to educate masses on awareness of all the nutritional, environmental and other life style risk factors for improved fertility outcome. Hence, at policy level health professionals should be educated about the modifiable risk factors associated with female infertility. They should target infertile couples, initiate interactive counseling sessions and education campaigns to enhance awareness on the correction of VD by diet, physical activity and supplementation if required.

Public Private Partnership for Primary Health Care: Based on coordination with the public sector, we will be able to have a holistic perspective of PCOS with VDD and plans to improve primary health care can be suggested with partnership to move forward for health and wellbeing of infertile females
Decelerations

Ethics approval and consent to participate

Ethics approval is given by the Aga Khan University Ethical Review Committee (2021-2033-19498). A data collector will be trained to obtain informed assent/consent from study participants. For participants younger than 18 informed assent along with parental/guardian consent will be obtained, which will indicate their willingness to participate in this study. Voluntary participation and the right to ask any questions and to decline participation at any time will be emphasized during the data collection.

Consent for publication

Not applicable.

Availability of data and materials

Materials described in this paper pertain to the study protocol only and no raw data are reported. The datasets will be collected and analyzed and can be made available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study is funded by the University Research Council of Aga Khan University (URC-AKU). The funding of this trial covers the cost of research staff, consumables, supplies, equipment, and transport to other clinics.

Authors' contributions

All authors have contributed substantially to this protocol. RR, SN, HNK and AHK conceptualized, designed, and drafted the manuscript of this study. RR is the principal investigator, constructed the conduct plan and analytical strategies, reviewed the manuscript several times, and provided feedback. HNK, AS, AA, MA, and SA led the overall methodological development from clinical, laboratory, and nutritionist perspectives and provided feedback on the proposal development. MA was involved in liaison with the CTU in the randomization process and assisted in assembling the study tools. All authors (RR, SN, HNK, AS, AA, MA, SA and AHK) have read this manuscript and approved the final version

of the paper.

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Table 1: Treatment Allocation during the study period (24 weeks) in all study years Table 2: Methods used to achieve respective Objectives
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Efficacy of vitamin D supplementation and metformin compared to metformin alone in infertile females with polycystic ovary syndrome: a randomized open label trial.

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Efficacy of vitamin D supplementation and metformin compared to metformin alone in infertile females with polycystic ovary syndrome: a randomized open label trial.

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