Effects of Metformin and Combination of Metformin and Pioglitazone on Plasma Interleukin-6 and Interleukin-8 Levels in Polycystic Ovarian Syndrome
Phase: EARLY_PHASE1
Conditions: Polycystic Ovarian Syndrome
Sponsor: Khyber Medical University Peshawar

RESEARCH PROTOCOL 
 
EFFECTS OF METFORMIN AND COMBINATION OF METFORMIN AND PIOGLITAZONE 
ON plasma IL-6 AND IL-8 LEVELS IN POLYCYSTIC OVARIAN SYNDROME 
 
 
 
 
Clinical trial registration number: NCT03117517 
 
Date: 03/15/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Protocol Number: KMU/CTU/2016/02 
 
Sponsor:  
Office of Research, Innovation and Commercialization 
Khyber Medical University, Peshawar, Pakistan 
Hayatabad, Phase 5. 
Collaborations: 
This study is a collaborative project between Khyber Medical University Peshawar; 
Mardan Medical Complex, Mardan. 
Coordinating Centre:  
Clinical Trial Unit, Khyber Medical University, Peshawar  
Pakistan 
Email: CTU@kmu.edu.pk 
Study Chair 
Dr Mohsin Shah 
Collaborative Group Chairs 
Dr Farhat Rehman 
CTU KMU Staff: 
Clinical Lead: Dr Asif Ali 
Statistician: Dr Omar Malik 
Project Manager: Dr Mohsin Shah, Dr Dure Shehwar Ali 
Emergency contact:  
Name: Dr Farhat Rehman; Phone: 0301 8192880 
  
 
 
 
 
 
 

 
TITLE: 
EFFECTS OF METFORMIN AND COMBINATION OF METFORMIN AND PIOGLITAZONE 
ON plasma IL-6 AND IL-8 LEVELS IN POLYCYSTIC OVARIAN SYNDROME 
 
Contents 
1. Introduction: .................................................................................................................................................... 4 
2. Aims and objectives ..................................................................................................................................... 5 
3. Hypothesis: ....................................................................................................................................................... 5 
4. Operational Definitions: ............................................................................................................................ 5 
5. Materials and Methods: .............................................................................................................................. 6 
6. Subject Population: ...................................................................................................................................... 7 
7. Data Collection Procedure: ................................................................................................................... 10 
8. Assessment Plan: ........................................................................................................................................ 12 
9. Outcomes and Endpoints: ...................................................................................................................... 14 
10. Safety Reporting: ..................................................................................................................................... 14 
11. Treatment Information: ....................................................................................................................... 15 
12. Data Analysis Procedure: .................................................................................................................... 15 
13. Study committees: ................................................................................................................................... 16 
14. Administrative aspects: ....................................................................................................................... 16 
15. References:.................................................................................................................................................. 17 
 
 
 

1. Introduction: 
Polycystic ovarian syndrome (PCOS) is a common endocrinopathy affecting women of 
reproductive age, with a frequency of about 4 to 7% (11). The syndrome is characterized 
by chronic oligo-anovulation, polycystic ovaries and hyperandrogenism (1).  The patient 
has signs and symptoms of infertility, increased risk of insulin resistance, irregular 
menstruation (oligo-amenorrhea), hyperinsulinemia, type II diabetes mellitus, insulin 
resistance, increase hair growth (1).  
Metformin, basically a biguanide, is an anti-hyperglycemic agent that improves glucose 
tolerance, increases the action of insulin at cellular levels without affecting insulin 
secretion (2). Metformin has been used in the management of insulin resistance, 
hyperinsulinemia and lipid abnormalities (2). A positive effect of metformin is proved by 
several studies on both metabolic and reproductive aspects in PCOS women (9). Metformin 
reduces obesity and decreases serum c-reactive protein levels in PCOS women (10). 
Pioglitazone is a thiazolidinedione (TZD) with hypoglycemic (anti-hyperglycemic, 
antidiabetic) action in the management of diabetes. It is also useful for reducing the 
cardiovascular risks associated with polycystic ovarian syndrome, having both anti-
inflammatory and anti-arteriosclerotic properties (3). Pioglitazone was suggested to 
reduce the incidence of diabetes mellitus by more than 50% with administration of 
pioglitazone in PCOS (3). Interestingly, the protection from developing diabetes mellitus 
these patients remained even when pioglitazone was stopped (3).  
 IL-6, a major pro-inflammatory cytokine, shows an essential part in endocrine system, 
particularly related to ovarian growth and the course of fertilization and implantation (1). 
IL-6 plays an important role in facilitating low grade chronic inflammation in patients with 
PCOS (1). IL-6 has been shown to be closely related to insulin resistance and 
cardiovascular abnormalities (4). Obesity, a major risk factor for type II diabetes, was 
reported to be associated with elevated IL-6 levels (4). IL-8 is a chemokine formed via 
macrophages and other cell types like endothelial cells, airway smooth muscle cells and 
epithelial cells (6). The levels of interleukin-6 and interleukin-8 were decreased in patients 
of PCOS after they reduced their insulin resistance and body weight (4). The manifestation 
of increased markers such as c-reactive protein, interleukin-6, interleukin-8 levels and 

raised leukocyte count is indication of low grade inflammation in women with PCOS (8). 
Elevated levels of interleukin-6 and interleukin-8 were related with an increased risk of 
atherosclerosis and future myocardial infarction (4).  
We designed this study to elucidate the role of metformin and combination of metformin 
and pioglitazone in reducing the levels of interleukin-6 and interleukin-8 in patients with 
PCOS. This will help us in deciding a better treatment regimen for patients with PCOS. 
Interleukin-6 and interleukin-8 may emerge as predictive biomarkers of treatment 
response.  
 
2. Aims and objectives 
OBJECTIVES: 
The objectives of this study are 
1. Primary 
To investigate the changes in the levels of interleukin-6 and interleukin-8 after 3 
months treatment with metformin alone and combination of metformin and 
pioglitazone in patients with PCOS. 
2. Secondary 
To evaluate insulin resistance in all the groups at baseline and after 3 months of 
treatment. 
 
3. Hypothesis: 
Combination of metformin and pioglitazone may decrease the levels of IL-6 and IL-8 in 
women with PCOS better than metformin. 
4. Operational Definitions: 
POLYCYSTIC OVARIAN SYNDROME:  Polycystic ovarian syndrome, or PCOS, is a condition 
characterized by chronic oligo-anovulation, polycytic ovaries and hyperandrogenism. 
METFORMIN: An oral hypoglycemic drug that decreases glucose production by the liver 

and increases peripheral glucose uptake, used to treat type 2 diabetes. 
PIOGLITAZONE: is a thiazolidinedione (TZD) with hypoglycemic (antihyperglycemic, 
antidiabetic) action to treat diabetes. 
INTERLEUKIN-6 is a cytokine that acts as both a pro-inflammatory cytokine and an anti-
inflammatory myokine. It is in humans encoded by the IL6 gene (5). 
INTERLEUKIN-8: is a chemokine produced by macrophages and other cell types such as 
epithelial cells, airway smooth muscle cells (6) and endothelial cells. 
 
5. Materials and Methods: 
a. STUDY DESIGN: Two-Arm Randomized Clinical trial. 
b. STUDY SETTING: OPD patients in Mardan Medical Complex, Khyber Medical University   
c. STUDY DURATION: Six Months after approval of proposal 
d. SAMPLE SIZE:  
The sample size was calculated using OpenEpi software. With an expected reduction in 
interleukin-6 and interleukin-8 values of 15% in the Metformin arm, we would need 102 
women with PCOS to demonstrate a 40% reduction in interleukin-6 and interleukin-8 in 
the Metformin and Pioglitazone arm with a power of at least 80% and a ratio of 1 in the two 
arms. 
Arm#1: 51 women with PCOS will be selected based on the inclusion and exclusion 
criteria. They will receive metformin according to the body weight with maximum dose of 
1000 mg (BD) daily.  
Arm#2: 51 women with PCOS will be selected based on the inclusion and exclusion 
criteria. They will receive metformin and pioglitazone combination according to the body 
weight with maximum dose of 1000 mg and 30mg (BD) daily. 
 
e. SAMPLING TECHNIQUES: Blood samples will be taken from ante cubital vein after an 
overnight fast.  Samples will be taken from all groups at the initiation and at the end of 3 

months. Serum levels LH, FSH, prolactin, testosterone, interleukin-6, interleukin-8 and 
insulin will be analysed by ELISA method. Fasting glucose will be performed by chemistry 
analyzer. Evaluation of insulin resistance will be performed by using HOMA-IR Index 
(Homeostasis model assessment for insulin resistance) with following formula. :   
HOMA-IR= fasting plasma insulin (µIU/mL) x fasting plasma glucose (mmol/L) /22.5  
 
6. Subject Population: 
6.1 Target population 
 
Women with PCOS visiting the outpatient department of participating hospitals. 
 
  6.2 SAMPLE SELECTION: 
   Inclusion criteria: 
PCOS women will have the following inclusion criteria: age group 20 - 40 years, oligo-
anovulation, hirsutism, elevated serum testosterone levels, menstrual cycle irregulary. 
Diagnosis of PCOS on ultrasound will be made on the basis of European Society of 
Human Reproducion and Embryo/ American Society of Reproductive Medicine 
(ESHRE/ASRM criteria, 2003) according to which 10 or more follicular cysts ranging in 
size from 2-9 mm or increased ovarian volume of 10 cm in maximal diameter. 
 
 
    Exclusion criteria: 
Women with previous history of cushing syndrome, hyperprolactinemia, late onset 
congenital adrenal hyperplasia, androgen-producing tumors, pregnancy or those taking 
medications that alters the biochemical or hormonal profile will be excluded by clinical 
and medical examination. 
 
6.3 Screening:  
Before the study-specific screening and investigations, patient will sign a written informed 
consent and the investigator will also sign the consent with date.  
 

6.4 Registration:  
 
Subjects will be registered before starting study treatment and a registration number will 
be allotted. Requests for registration will only be accepted from authorized investigators at 
sites that have received ethical approval. Treatment would be planned to start after 
registration. Registration would be done according to the instructions in the protocol only 
after all screening assessments have been performed and the responsible investigator has 
both verified the subject’s eligibility and signed the completed registration form. 
Once the registration process has completed as per the instructions in the protocol, the 
subject will be assigned a subject, study number, and written confirmation of registration 
will be provided to the site. Individuals must be registered once in this trial. 
6.5 Randomization:  
Randomization will be performed using Block Randomization technique using online 
software Sealed Envelope. 
Appendix 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Schema 
 

7. Data Collection Procedure: 
7.1 PATIENT RECUIREMENT: 
Approval of the study will be obtained from the Ethical Review Committee of the Khyber Medical 
University Peshawar. Potential participants will be identified in the participatory center. Purpose 
and components of the study will be explained to each potential participant. Once the participant 
has agreed to take part, she will be screened for eligibility criteria. All eligible participants will be 
asked to give a written informed consent. Data regarding age, weight, height, menstural 
irregularity, hirsutism, past medical and surgical history, serum testosterone and fasting insulin 
will be taken. Each participant will undergo ultrasound for assessment of ovarian changes. The 
study participants will be grouped in the following two arms. 
Arm#1: 51 women with PCOS will be selected based on the inclusion and exclusion criteria. They 
will receive metformin according to the body weight with maximum dose of 1000 mg (BD) daily.  
Arm#2: 51 women with PCOS will be selected based on the inclusion and exclusion criteria. They 
will receive metformin and pioglitazone according to the body weight with maximum dose of 
1000 mg and 30 mg (BD) daily.  
  
Arm 1 
METFORMIN (1000 mg) 
Arm 2 
METFORMIN (1000 mg) 
AND  PIOGLITAZONE (30 mg) 
 
 
7.2 Concomitant medication reporting and treatment: 
Concomitant medications will not be recorded during the study. The following medications and 
treatment are recommended during the study  
7.2.1 Permitted 
 
The following medications are permitted during the study: NSAIDS, multivitamins, antibiotics and 
PPIs. 
7.2.2 Use with caution 
There are no medications that should specifically be used with caution in this study. 
7.2.3 Prohibited 

There are no medications and treatments that are specifically prohibited in this study. 
7.2.4 Compliance 
Subject medication compliance will be determined at each visit after 1 month and the patient will 
be counseled appropriately if significant non-compliance is determined. 
7.2.5 Treatment discontinuation 
The treatment of the patient or study on the subject will be discontinued in case of pregnancy or 
any disease given in exclusion criteria and in patient with hypersensitivity to drugs. There is no 
serious adverse effect observed after treatment discontinuation. 
                                         
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

8. Assessment Plan:   
HISTORY RELATED QUESTIONS AND CLINICAL ASSESSMENT 
Name:                                 Age:                          Date:                         
Address:                              Marital status:                         Phone No: 
 
 
 
• 
Menstrual irregularity:  ____________________ 
• 
Age of menarche: ____________________ 
• 
Hirsutism: ____________________ 
• 
 Acne:  ____________________ 
• 
 Daily physical exercise:   ____________________ 
• 
Infertility: ____________________ 
• 
Height (m) ___________________ 
• 
Weight (kg) __________________ 
        BODY MASS INDEX (kg/m2):  __________________ 
• 
Drug history (current and past medications):  ____________________ 
        Surgical history:  ____________________ 
• 
BP: ____________________ 
• 
Pulse: ____________________ 
 
 
 

Schedule of assessments 
 
Screening 
Baseline 
investigations 
On treatment 
End of 
treatment 
 
14-21 days 
prior to 
registration 
Within 7 days 
prior to 
registration 
Within 3 days 
prior to first 
dose 
After 3 months 
Informed consent 
 
 
 
 
Clinical 
assessment 
(mentioned 
above) 
 
 
 
 
Pelvic ultrasound 
right ovary and 
left ovary 
(10 or more 
follicles. 2 – 9 mm 
in diameter) 
 
 
 
 
Other imaging 
 
 
 
 
FBS 
RBS 
 
 
 
 
LH 
FSH 
Testosterone 
 
 
 
 
IL-6 
IL-8 
 
 
 
 
HOME. IR  
 
 
 
 
Concomitant 
medications 
 
 
 
 
Adverse events 
 
 
 
 
Outcome events 
 
 
 
 
 

ASSESSMENT PHASE DEFINITONS AND SPECIAL CIRCUMTANCES 
SCREENING 
All screening will be done before registration of the patients. 
 
BASELINE 
All baseline investigations including (but not limited to) levels of interleukin-6 and 
interleukin-8, insulin resistance and pelvic ultrasound will be performed before giving 
treatment to the patient. 
ON TREATMENT 
 
Assessment during the treatment will be performed. 
END OF TREATMENT 
All baseline investigations including (but not limited to) levels of interleukin-6 and 
interleukin-8, insulin resistance and pelvic ultrasound will be performed 12 weeks after 
treatment. 
FOLLOW UP AFTER TREATMENT 
No follow up will be performed. 
9. Outcomes and Endpoints: 
The outcomes of the study is the change in the levels of interleukin-6 and interleukin-8 
with the use of antidiabetic drugs (metformin alone and combination of metformin and 
pioglitazone). Other symptoms like hirsutism, polycystic ovaries and serum testosterone 
levels may decrease and menstrual cycles might become become regular. 
10. Safety Reporting:  
PREGNANCY 
Follow up in the treatment will be discontinued with pregnancy.  
 

REPORTING OF SERIOUS ADVERSE EVENTS  
Any adverse effects that occur during the treatment will be reported. 
11. Treatment Information: 
Metformin with the trade name of Glucophage is manufactured by Merck International 
Pharmaceutical Company Limited. Metformin plus Pioglitazone combination with the trade 
name of Zolid plus is manufactured by Getz Pharmaceutical Company Limited. We would 
consistently use these two brands. The dosage will be according to according to body 
weight. 
Cost bearing: The cost of the medicines and investigations will be provided to the study 
participants by the project managers (Dr Durre Shehwar and Dr Mohsin Shah) 
 
12. Data Analysis Procedure: 
 
Mean ± SD will be used for numeric data generated from our work. In each arm the mean 
difference in the interleukin-6 and interleukin-8 values will be calculated using paired 
sample t test. For between arms analysis, mean difference in the interleukin-6 and 
interleukin-8 values will be calculated using independent sample t test. Non-parametric 
statistics will be used if the data is skewed. A cut-off will be calculated for interleukin-6 and 
interleukin-8 separately using ROC curve analysis. Based on the cut-off, a binary logistic 
regression analysis will be performed to evaluate the predictive significance of the two 
treatment arms in reducing the levels of inflammatory markers interleukin-6 and 
interleukin-8. P-value of < 0.05 will be considered significant. Results will be presented in 
form of tables & figures. SPSS version 21 will be used for all the statistical analysis. 
 
 
 
 
 

 
13. Study committees: 
The study will be managed with the help of Graduate Study Committee, Clinical Trial Unit 
and ORIC of Khyber Medical University, Peshawar, Pakistan. 
14. Administrative aspects: 
The administrative aspects will be managed with the help of Office of Research, Innovation 
and Commercialisation (ORIC) of Khyber Medical University, Peshawar, Pakistan. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

15. References: 
1.Xu X, Du C, Zheng Q, Peng L, Sun Y. Effect of metformin on serum interleukin-6 levels in 
polycystic ovary syndrome: a systematic review. BMC women's health. 2014;14(1):1. 
2. Velazquez E, Mendoza S, Hamer T, Sosa F, Glueck C. Metformin therapy in polycystic 
ovary syndrome reduces hyperinsulinemia, insulin resistance, hyperandrogenemia, and 
systolic blood pressure, while facilitating normal menses and pregnancy. Metabolism. 
1994;43(5):647-54 
3. Sangeeta S. Metformin and pioglitazone in polycystic ovarian syndrome: a comparative 
study. The Journal of Obstetrics and Gynecology of India. 2012;62(5):551-6. 
4.Peng Z, Sun Y, Lv X, Zhang H, Liu C, Dai S. Interleukin-6 Levels in Women with Polycystic 
Ovary Syndrome: A Systematic Review and Meta-Analysis. PloS one. 2016;11(2):e0148531. 
5. Ferguson-Smith AC, Chen YF, Newman MS, May LT, Sehgal PB, Ruddle FH ;. "Regional 
localization of the interferon-beta 2/B-cell stimulatory factor 2/hepatocyte stimulating 
factor gene to human chromosome 7p15-p21". Genomics. 2010;2 (3): 203–8. 
doi:10.1016/0888-7543(88)90003-1. PMID 3294161.  
6. Hedges JC, Singer CA, Gerthoffer WT (2000). "Mitogen-activated protein kinases regulate 
cytokine gene expression in human airway myocytes". Am. J. Respir. Cell Mol. Biol.2008; 23 
(1): 86–94. doi:10.1165/ajrcmb.23.1.4014. PMID 10873157 
7. Palomba S, Falbo A, Chiossi G, Orio F, Tolino A, Colao A, La Sala GB, Zullo F. Low-grade 
chronic inflammation in pregnant women with polycystic ovary syndrome: a prospective 
controlled clinical study. The Journal of Clinical Endocrinology & Metabolism. 2014; 
29;99(8):2942-51. 
8. Diamanti-Kandarakis E, Paterakis T, Alexandraki K, Piperi C, Aessopos A, Katsikis I, 
Katsilambros N, Kreatsas G, Panidis D. Indices of low-grade chronic inflammation in 
polycystic ovary syndrome and the beneficial effect of metformin. Human Reproduction. 
2006 ;21(6):1426-31. 
9.Hwang KR, Choi YM, Kim JJ, Chae SJ, Park KE, Jeon HW, Ku SY, Kim SH, Kim JG, Moon SY. 
Effects of insulin-sensitizing agents and insulin resistance in women with polycystic ovary 
syndrome. Clinical and experimental reproductive medicine. 2013;40(2):100-5. 

10.Takemura Y, Osuga Y, Yoshino O, Hasegawa A, Hirata T, Hirota Y, Nose E, Morimoto C, 
Harada M, Koga K, Tajima T. Metformin suppresses interleukin (IL)-1β-induced IL-8 
production, aromatase activation, and proliferation of endometriotic stromal cells. The 
Journal of Clinical Endocrinology & Metabolism. 2007;92(8):3213-8. 
11.Ortega-Gonzalez C, Luna S, Hernandez L, Crespo G, Aguayo P, Arteaga-Troncoso G, Parra 
A. Responses of serum androgen and insulin resistance to metformin and pioglitazone in 
obese, insulin-resistant women with polycystic ovary syndrome. The Journal of Clinical 
Endocrinology & Metabolism. 2005 ;1;90(3):1360-5. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
STEPS/months 
1st 
2nd 
3rd 
4th 
5th 
6th 
Sample collection and 
recruitment 
  
  
 
  
  
  
Experimental 
and 
data tabulation 
  
  
  
 
  
  
Thesis writing 
  
  
  
  
  
  
Submission of thesis