Clinical Drug Experience Knowledgebase

Material and methods

Study Population:

The data were collected from the Allied Hospital. All Females of ages 18 to 45 years, having PCOS attending the OPD of Allied Hospital was taken as the study population. Efficacy trials were conducted on the patients of PCOS, including females of age 18-45 years. They were divided into different groups (as per no. of selected treatments along with the control). Ten subjects left the study and were replaced. The project was approved by the ethical review committee.The feeding study was conducted with the cooperation of Allied Hospital. The study protocol was discussed with medical professionals and finalized after through technical suggestions.

Selection of subjects and Study Design

The patients were assorted randomly into four groups, 25 in each, namely control (T0),

A total 100 female subjects were included in the study as under A) Control: To: 25 B) Experimental groups (PCOS): 75

T1: 25
T2: 25
T3: 25

Inclusion Criteria:

All females of age 18 to 45 having PCOS

Women showing the presence of two out of three features (Oligo- or anovulation, hyper-androgenism, or poly cystic ovaries (Ovarian volume >10cc, 12 or more peripherally located follicles 2-9 mm in diameter or hypo-echoic enlarged ovaries with hyper-echoic central stroma in USG). Patients on Metformin (used for insulin resistance)

Exclusion Criteria:

Females under 18 and above 45 years of age
Those on any other medical therapy
Pregnant and lactating women
Those with a history of allergy to flax seed
Women on anti-platelet and fibrinolytic drugs
Women showing signs of PCOS but showing normal ovaries on ultrasound

Study Design

It was literature review based intervention study. The selected population for intervention purposes was studied twice, i.e. before and after the intervention of the selected nutraceutical. The same subjects were studied once again after one month of stopping the intake of selected nutraceutical. The change in results has confirmed the effects of the intervention.

This has been divided into four phases.

Phase-1:Assessment of the nutritional Status in PCOS
Phase -2:Development of nutraceutical (flaxseed sachets)
Phase -3:Intervention to study the effect of selected nutraceutical

Phase-4:Blood Analysis

The procedure adopted in each of the above phases is explained as follows:

Phase-1: Assessment of the nutritional status of selected patients

This was done in Allied Hospital, patients attending OPD of gynecology ward was assessed for nutritional status. Following parameters were studied for this purpose

Family History
Medical history
Physical signs (thyroid gland, height, weight, skin, hair, breasts, belly, and BMI)
Menstrual cycle detail (menarche, frequency, duration, and severity)
History of dysmenorrhea
Ferriman-Gall way scoring for hirsutism
Marital status, parity, and difficulty in conception

Phase -2: Development of Nutraceutical (flaxseed sachets)

Procurement of raw materials Preparation of flax seed powder Preparation of flax seed sachets

Phase-3: Intervention

An acceptable number of human volunteers were selected keeping in view all ethical considerations. (25 in each group)

Control group (To)
Flaxseed (5 g) xTTS = 15 g/day (T1)
Flaxseed (6.6 g) x TTS = 20 g/day (T2)
Flaxseed (8.3 g) xTTS = 25 g/day (T3)

The flaxseed powder from the selected treatments was given daily for a period of 12 weeks. The dietary history of the subjects was monitored with the help of a Food Diary on a weekly basis. Education/counselling were provided to the patients for necessary compliance with the restricted diet for PCOS.

Phase-4: Blood Analysis and Ultrasound

After three months of intervention blood tests: follicle stimulating hormone (FSH), Luteinizing hormone (LH) thyroid stimulating hormone (TSH), serum prolactin, and ultrasound will be repeated.

Biochemical assays

Follicles stimulating hormone

The blood sample was taken to determine the quantitative level of serum follicles stimulating hormone (FSH) in patients fed with flaxseed powder by following the Immuno-enzymatic calorimetric method (ELISA).

Luteinizing hormone

The blood sample was taken to determine the quantitative level of serum luteinizing hormone (LH) in patients fed with flaxseed powder by following the Immuno-enzymatic calorimetric method.

Sex hormone binding globulin

The serum sex hormone binding globulin hormone (SHBG) in patients fed with flaxseed powder was determined by following the Rosner's method.

Prolactin

The serum sex prolactin level in patients fed with flaxseed powder was determined by following the radio-immunological method.

Thyroid stimulating hormone

The thyroid stimulating hormone (TSH) level in patients fed with flaxseed powder was performed to determine the thyroid gland functioning by following the third-generation non-isotopic method.

Liver Function Test

Alkaline Phosphatase (ALP)

The activity of alanine phosphatase in the plasma was determined by using enzyme-linked immune-sorbent assay kit method.

Aspartate Aminotransferase (AST)

The activity alanine transferase in the plasma was determined by using di-nitrophenyl hydrazine (DNPH) method using Sigma kits 59-50 and 58-50 under the conditions.

Alanine Aminotransferase (ALT)

The activity alanine aminotransferase in the plasma was determined by using enzyme-linked immune-sorbent assay kit method.

Lipid Profile

Low-density lipoprotein (LDL)

Serum low-density lipoprotein (LDL) was estimated by following the protocol of Vinson.

High-density lipoprotein (HDL)

The high-density lipoprotein (HDL) was assessed by HDL-Cholesterol precipitation.

Triglyceride

The triglyceride in the collected serum was determined by liquid triglyceride (GPO-PAP) method.

Renal Function Tests

Urea

The flax seed fed patients were diagnosed with urea content by GLDH method with the help of commercial kit.

Creatinine

The blood creatinine of the patients fed on flax seed powder was determined by the Jaffe method using creatinine commercial kit.

Follicles present in ovaries

Ultrasound of the ovaries was carried out to check the number of follicles present in the ovaries.

Size of the ovaries

The size of the ovaries of patients fed with flax seed powder was also checked in ultrasound.

Fasting blood glucose level

The blood sample was taken to check the fasting blood glucose level. The sample was taken eight hours after eating. Fasting blood glucose level as determined by glucose dye oxido-reductase mediator reaction which is specific for glucose by glucometer Accuchek active.

Research instruments

Questionnaire: For screening/nutritional assessment of PCOS patients
Hormonal analysis: Follicle stimulating hormone (FSH), luteinizing hormone (LH) thyroid stimulating hormone (TSH), Sex Hormone binding globulin (SHBG), and serum prolactin.
Biochemical assay: ALP, ALT, AST, urea, Creatinine, Fasting blood glucose level, LDL, HDL, and triglyceride.

Ethical Considerations:Written ethical approval was taken from the management of the concerned hospital and HOD of Gynecology department of the selected hospital. The consent was taken from the subjects after explaining the purpose of the study and after getting their consent for their menstrual cycle history, blood tests and ultrasound.

Statistical analysis

The data were subjected to statistical analysis by using the SPSS statistical package, analysis of variance (ANOVA) and least significant difference (LSD) was used to determine any significant difference among the treatments at p<0.05.