Efficacy of Vitamin D Supplementation in Females With Polycystic Ovary Syndrome: a Randomized Open Label Delayed-start Design
Phase: PHASE1
Conditions: Anovulatory Infertility
Sponsor: Aga Khan University

Cover Letter 
Title: 
Efficacy of Vitamin D Supplementation in Females with Polycystic Ovary 
Syndrome: A Randomized Open-Label Delayed-Start Design 
 
Clinical Trial Unit, 
I am writing to submit my study, titled "Efficacy of Vitamin D Supplementation in Females with 
Polycystic Ovary Syndrome: A Randomized Open-Label Delayed-Start Design," for consideration in 
the Clinical Trial Unit. I am excited to present this research project for potential inclusion in your 
esteemed program. 
Polycystic Ovary Syndrome (PCOS) is a prevalent and complex endocrine disorder that affects many 
women worldwide. The condition is characterized by a range of symptoms, including irregular 
menstrual cycles, hyperandrogenism, and polycystic ovarian morphology. There is a growing body of 
evidence suggesting a link between vitamin D deficiency and PCOS, as vitamin D plays a crucial role in 
various aspects of reproductive health and endocrine regulation. 
Our study aims to investigate the efficacy of vitamin D supplementation as a potential therapeutic 
intervention for females with PCOS. The randomized open-label delayed-start design allows for a 
comprehensive examination of the long-term effects of vitamin D supplementation on PCOS 
symptoms and associated comorbidities. By comparing the outcomes between immediate and 
delayed-start groups, we hope to gain insights into the optimal timing of vitamin D intervention and 
its impact on the progression of PCOS. 
The significance of this study lies not only in its potential to improve the quality of life for individuals 
with PCOS but also in its contribution to the broader field of reproductive medicine. If our findings 
support the efficacy of vitamin D supplementation, it could lead to the development of cost-effective 
and accessible treatments for a condition that affects millions of women worldwide. 
I believe that the Clinical Trial Unit is the ideal platform for conducting this research due to its 
commitment to advancing clinical science and improving patient care. Our study adheres to rigorous 
ethical and methodological standards, and we are confident that it aligns with the objectives and 
values of your organization. 
I kindly request that you consider our study for inclusion in the Clinical Trial Unit and appreciate the 
opportunity to discuss this research further. The date of ERC approval is 7th December, 2021 
Thank you for your time and consideration.  
Best Regards, 

 
 
 
 
 
 
 
 
 
 
Title: 
Efficacy of Vitamin D Supplementation in Females with Polycystic Ovary 
Syndrome: A Randomized Open-Label Delayed-Start Design 
Methodology 
Study Design: This will be a randomized open label, delayed-start design trial conducted at 
Aga Khan University at  Departments of: Biological & Biomedical Sciences, Pathology and 
Laboratory Medicine, Medicine (Endocrinology) and Family Medicine for a period of 2 years 
following approval from Clinical Trials Unit and Ethical Review Committee of Aga Khan 
University 
Sample Size calculation; The sample size was calculated by PASS 11 software. In an 
equivalence test of means using two one-sided tests on data from two-periods, the minimum 
sample size that we will require is 142, 71 in each group with an inflation of 20% for loss to 
follow-up to achieve 80% power at a 5% significance level when the true difference between 
the means is 0.4, the standard deviation of the paired differences is 2.60. 

Eligibility Criteria: Patients recently diagnosed with PCOS with presence of at least 2 of these 3 
elements: clinical or biochemical signs of hyperandrogenism, chronic anovulation and polycystic 
ovaries (1) (from reports of available routine TVS), with VDD serum levels VD <20 ng/ml(10), age range 
18- 45 years, from all ethnic background will be included. We will exclude patients who will be 
pregnant, with Hypercalcemia (plasma calcium concentrations> 2.65 mmol/L), Tuberculosis or 
other granulomatous disorders, Chronic liver disease or alanine transaminase (ALT) level 3 
times higher than the normal limit, chronic, Kidney disease or serum creatinine >2.0 mg/dL, on 
drrug Therapies i.e    participants who had received VD injection in the last 3 months prior to recruitment 
in the study, 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 blockers, immunosuppressant, chemotherapeutic 
agents, antimicrobials (Rifampicin, Isoniazid, Hydroxychloroquine) or any other drug modifying lipid 
metabolism in the previous 3 months prior to study , patients suffering from 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: All diagnosed PCOS subjects as per the Rotterdam criteria visiting 
Endocrine, Family Medicine and/or Gynae Obs clinic qualifying the inclusion criteria with VD 
levels <20 ng/ml (7, 9, 10) and age range 18 to 45 years will be invited to participate in the 
study after acquiring written informed consent.  
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. The randomization list will be generated using the 
AKUH developed randomization software (http://www.randomizer). The randomization list is 
confidential and will be kept within the premises of CTU under restricted access. Subjects will 
be randomized into Group A (Intervention) and B (Control) and will receive the treatment 
protocol 
Intervention Group  (Table 2) for the initial 12 weeks will receive: VD 
supplementation (600,000 IU I/M) once during the study period  and elemental calcium 
1000 mg/day . However from 12-24 weeks they will receive standard PCOS treatment,  
lucophage XR 750 mg once at dinner for 15 days then twice daily, capsule Progeffik 100 
mg once at night every 3 weeks, then 1 week off and calcium 1000 mg/day. 
Control Group  for the initial-- 12 weeks the control group will receive the standard 
treatment for PCOS , Glucophage XR 750 mg once at dinner for 15 days then twice daily 
and Capsule Progeffik 100 mg once at night every 3 weeks, then 1 week off   
However, from 12- 24 weeks they will receive  VD supplementation (600,000 IUI/M) 
once during the study period , calcium 1000 mg/day and standard PCOS  treatment will 
be continued  
 

 
 
 
 
 
 
 
 
 
 
Data Collection: Randomization and drug dispensing will be followed by detailed 
history, physical examination, biophysical and biochemical parameters estimation by 
research team. The data will be collected on the following variables ;  Body Mass index. 
2, Waist circumference (WC)Waist -Hip Ratio, Biochemical estimation ( results of 
following routine tests are available (Human Sex Hormone-Binding Globulin (SHBG), 
Total Testosterone (TT), Human Insulin and Glucose) to be included in the profile. For 

estimation of Total Antioxidant Capacity (TAC), serum calcium and phosphorous levels, 
phlebotomist will collect blood samples (3 cc) by vein puncture).  
Calculations; 
i. 
HOMA-IR : HOMA- IR index, a commonly used marker of IR, will be calculated 
using the formula: HOMA-IR = fasting glucose levels [mmol/L] × fasting insulin 
levels [μU/mL]/22.5 (34). 
ii. 
Free Androgen Index will be derived from TT and SHBG, normally measured 
in nanomoles per liter. FAI =100 (TT /SHBG) 
Outcome : Follow- up visits at 12 weeks (Study Mid Pont): At 12 weeks follow up, 
compliance to supplementation and occurrence of any adverse effect will be assessed by 
research team allocated members in the respective clinics. Moreover, drug for 12 weeks (as per 
group allocation) will be dispensed and any other queries of participants will be responded. 
Fasting blood sample (10 ml) will be collected for estimation of SHBG, TT, Human Insulin, 
Glucose, serum calcium and phosphorous and TAC in both groups and VD and serum albumin 
estimation in Group A. At this point of time, treatment protocol of both groups will be 
changed (Table 2). 
1. Follow- up visits at 24 weeks (study End Point):  After completion of 24 weeks study 
exposure, subjects will be informed about completion of study. Blood samples in fasting 
state will be collected for repeat analysis in Multi Disciplinary Lab (MDL) for all the 
biochemical parameters (FAI, HOMA IR and TAC) and VD and serum albumin 
estimation in Group B. Figure 6: Complete cycle of each patient in control and 
intervention group for a period of 24 weeks 

 
Biohazardous Samples: According to the WHO the serum samples have low viral concentration. 
However, the centrifugation is a risk for aerosol generation and COVID exposure. To minimize the risk, 
we will ; Train the research staff about the Basics of Biosafety, Biosecurity & Bio risk Management 
approach for collection & processing of samples. All samples will be treated as potentially infectious, 
use of proper PPE will be mandatory while handling samples. After centrifugation, staff will wait for 10 
minutes before opening the centrifuge. In case of Breakage the already defined laboratory protocols 
(available in safety manual) will be followed.  For liquid waste disposal, will use 5% Hypochlorite. 
Potential Risks: The potential risks likely to occur in this study are; Loss of patients during 
movement in respective clinics (Lost to follow up) Noncompliance of subjects, Biosafety 
Issues, At the time of collection of samples, unexpected delays in the processing of samples; 
during bench work in MDL lab and Drug Overdose  
Risk management Plan for Potential Risks: 
i. 
Movement of Subjects: We have hired 2 Research Assistants (RA) who will be in 
connection with the PI and Co-PIs (Gynecologists and Endocrinologists) to guide 
the included patients from clinics to CTU, blood collection point and follow up 
sessions at 12 and 24 weeks.  
ii. 
Compliance: PI students and RA will be in communication with the subjects on 
emails, phone calls and WhatsApp group, will send them reminders to take their 
medicine, come for follow up visits and inform if any side effects occur 

iii. 
Biosafety Issues: Collection of Samples: For the entire research procedure the risks 
of having blood drawn include temporary pain from the needle stick, bruising, and 
rarely, infection. Any gross reaction will be reported and informed to the concerned 
investigator. Unexpected load: Due to pandemic situation, there is an unexpected 
load on the centrifuge machine in MDL. We have therefore requested our mentor 
Dr Aysha Habib; Head of Chemical Pathology and Laboratory Medicine to allow 
centrifuge of our samples in the clinical lab of AKUH (if required). Bench work in 
MDL lab as per AKU COVID-19 Safety Rules: The research team will follow all 
the standard revised operating procedures (SOPs) required for biosafety of lab staff 
during the pandemic in their BSL2 facility. They include; judicious cleaning of labs, 
minimal 6 feet distance, cleaning of bench with 70% Isopropyl alcohol or 80% 
ethanol and follow all the other SOPs that are already implemented in the research 
areas from before the Covid-19 pandemic. 
iv. 
Drug Safety: The vitamin doses that are planned for use in this study are based upon 
scientific evidence and recommendations from international societies and are 
unlikely to cause hypervitaminosis D. However, if Hypervitaminosis D occur in a 
rare situation, it can lead to hypercalcemia which can manifest as polyuria, 
polydipsia, nausea, vomiting, or constipation, muscle weakness, bone pain, weight 
loss or poor appetite, confusion or disorientation. We will warn all our subjects to 
inform us in case if any of these symptoms appear and RA will be in touch with 
them. We will stop the treatment and report to the respective consultant. We will 
also assess VD status along with calcium and albumin levels after they have received 
VD and calcium supplementation. All study related adverse effects will be managed 
and reported to the ERC, Clinical affairs and pharmacy as per institutional policy. 

Investigator and study coordinator will follow up any adverse events and serious 
adverse events.  
Plan of analysis: Data will be analyzed using STATA version 15. The main analysis for each outcome 
will be performed at the subject level using the intention-to-treat (ITT) principle, meaning that all 
participants with a recorded outcome will be included in the analysis and will be analyzed according to 
the treatment group to which they were randomized. Results will be presented as mean and standard 
error/ median (IQR) for the outcomes. Pretreatment, 12 weeks  and 24 weeks post treatment levels of 
the outcomes will be assessed by repeated measure ANOVA/ Friedman test as appropriate. Categorical 
variables will be reported as frequency and percentages and will be assessed by chi-square/ fisher exact 
test. Unadjusted and adjusted beta coefficient with 95% CI will be reported by using Generalized 
estimating equation (GEE) to determine the association of various independent  factors  with the 
outcomes. We will adjust for the independent variables and determine the association of factors causing 
a greater decline in outcomes by multivariable GEE. Plausible interactions and confounders will also 
be assessed.  A p value of < 0.05 will be considered as significant throughout the study 
Ethical Considerations:  In this research, we will adhere to ethical principles outlined in the Helsinki 
Declaration for medical research involving human subjects. We will seek approval from the Aga Khan 
Ethical Review Committee. The participants will be informed about the study's purpose and the 
possibility of being part of the intervention group based on computer randomization. We will obtain 
verbal and written consent from eligible participant. To ensure confidentiality, the entire process from 
participant enrollment to testing and outcome assessment will be handled discreetly. Consent forms 
and completed questionnaires will be assigned unique codes and securely stored in locked cabinets 
accessible only to the Principal Investigator. Participant identifiers will be removed, and only codes will 
be used in subsequent steps. Serum samples will be collected and coded without mentioning names. 
These samples will be processed at MDL-AKU and disposed of following AKU clinical lab's institutional 
policies for biological material disposal, which involves sealing them in red bags and incineration. Hard 
copies  questionnaires will be stored and archived for a minimum of 7 years in line with AKU policy. 
Afterward, information will be deleted from computers, and the forms and questionnaires will be 
securely shredded. Results and data will be appropriately coded, decoded, and documented. 
 
Strength & Limitations: The delayed-start design has some methodological issues in comparison with 
standard parallel-group study design of RCT. Nonetheless the effects of VD develop over time and we 
do not want to deprive VD deficient PCOS from VD supplementation in both groups, therefore we have 
selected this study design; all subjects will be given same dose of VD either before or after standard 
PCOS care. To the best of our knowledge this is the first clinical trial which will confirm not only the 

impact of VD but also predict the time of its administration. Novelty of the study is that in addition to 
the routine parameters of IR and hyperandrogenism, this study will also estimate and document the 
improvement in OS as a result of the replacement therapy.