Subjects: All women who reported to the Obstetrics and Gynaecology out patient department of Post- graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India, from March 2007 to January 2008 were screened for inclusion in the study. Those who had undergone bilateral oophorectomy, were less than 55 yr of age had baseline thyroid stimulating hormone (TSH) values within the normal range and were willing to comply with the protocol were included in the study. They were excluded if any of the following criteria were present: (1) suffering from any thyroid disease, (2) suffering from any autoimmune disorder, (3) already on isoflavones, (4) taken HRT/estrogen replacement therapy (ERT) within previous 8 wk, (5) presence of renal and/or hepatic disease, (6) active major psychiatric disorders, (7) history of thrombophlebitis or thromboembolism or cerebrovascular disorders, (8) uncontrolled hypertension with blood pressure > 180/100 mm Hg, (9) uncontrolled diabetes, (10) were on any drug which can affect thyroid function, (11) presence of active infection or malignancy, and (12) present or past history of soy or nut related food allergies.
The study protocol was approved by the Institutional Ethics Committee and all the participating women signed a written informed consent prior to enrollment to the study. Eligible women were randomized so that they had an equal probability of assignment to either of the two groups. The randomization code was developed using random number table to select random permuted blocks of length four. The identity of the drugs was hidden by packing in numbered opaque envelopes to ensure concealment of the sequence until assigned. Randomization, allocation sequence generation and packing of envelopes were done by investigators not directly involved in dispensing and evaluating the treatments so that it was double blind - both the patient and evaluating clinician not aware of the assigned treatment. The participants remained on the same allocation throughout the study period if they continued. The randomization code was revealed to the investigators once recruitment, data collection, and laboratory analyses were complete.
The participants attended the clinic at the time of randomization (baseline) and at three weekly intervals for 12 wk.
Study interventions: The participants were randomized to either placebo or study drug to be administered once a day orally for 12 wk. The study drug tablet contained 75 mg of soy isoflavones standardized to provide isoflavones 40 per cent (Genistein and Genistin 25%; Daidzein and Daidzin 15%) along with 200 mg elemental calcium, 100 mg elemental phosphorus, and 100 IU of vitamin D3. Placebo tablet matching in colour, size, shape and taste and containing identical quantity of calcium, phosphorus and vitamin D3 was used as control treatment. Patients were advised to take one tablet daily at bedtime with 150 ml of plain water for a total 12 wk period. All the patients were instructed to avoid dietary phytoestrogen consumption by avoiding soy, seeds and sprouts, beans and legumes during the study period. Other treatments which might interfere with thyroid function were also avoided during the study. However, concomitant treatments for other associated illnesses were allowed. Since the drug is available in the market at the required dose, drug product label instruction was followed for storing and dispensing of the drug. Appropriate treatment was provided for any observed adverse event. The patients were provided with a contact phone number to call in case of emergency. The patients were asked to bring the drug envelopes during subsequent visits and compliance was assessed by pill counting. More than 80 per cent compliance was considered as adequate.
In the present study the hypothesis tested was that isoflavones cause a significant alteration in thyroid economy and autoimmunity in comparison to placebo in euthyroid, surgical menopausal women. The primary outcome of the study was complete serum thyroid profile (TSH, free T3, free T4, Thyroid binding globulin and anti – TPO antibodies). The secondary outcomes included assessment of menopausal symptoms, serum follicle stimulating hormone (FSH) and estradiol levels.
For each patient, the serum thyroid profile was assessed at baseline, six wk and at study completion (12 wk) while presence of any goiter was ruled out by palpation of thyroid gland during each visit. The assessment for serum FSH and estradiol was done at baseline and 12 wk while menopausal symptoms were assessed during each three weekly visit.
For the assessment of various parameters, about 10 ml blood was withdrawn aseptically by venipuncture from each patient at baseline (0 wk), six and 12 wk after initiation of treatment. The blood samples were allowed to clot at room temperature and centrifuged at 1260 g for 10 min. The serum thus separated was stored at -20°C for further estimations. To reduce the effects of interassay variability, all samples from each subject were analysed in duplicate in the same batch. Free T3 and free T4 were determined by single antibody coated tube Radio-immuno assay (RIA) using 125I labelled hormone (Roche Diagnostics, Indianapolis, IN, USA). TSH was determined by double antibody RIA using 125I labelled hormone (Roche Diagnostics, Indianapolis, IN, USA). Thyroid binding globulin (TBG) and anti-TPO antibodies were determined by electrochemiluminescence immunoassay using specific antibodies labelled with a ruthenium complex (Roche Diagnostics). Serum FSH was determined quantitatively by chemiluminescence immunoassay in vitro which employs two different monoclonal antibodies specifically directed against human FSH. Radioimmunoassay was used to estimate serum estradiol levels (reference range in postmenopausal women 70-190 pmol/l).
The assessment of menopausal symptoms was done by calculating the menopause symptom score for each patient using Menopause rating scale (MRS)14
. The total score of MRS ranges between 0 (asymptomatic) to 44 (highest degree of severity).The score increases point by point with increasing severity of subjectively perceived complaints in each of the 11 items (severity expressed as 0-4 points in each). The various symptoms included in questionnaire were - psychological symptoms - 0 to 16 points (4 symptoms: depressed, irritable, anxious, exhausted); somato-vegetative symptoms – 0 to 16 points (4 symptoms: sweating/flash, cardiac complaints, sleeping disorders, joint and muscle complaints); and urogenital symptoms – 0 to 12 points (3 symptoms: sexual problems, urinary complaints, vaginal dryness). The composite scores for each of the dimensions (sub-scales) is based on adding up the scores of the items of the respective dimensions. The composite score (total score) is the sum of the dimension scores.
The participants were also assessed for general health status by routine clinical examination and appearance of any adverse events during each visit.
Sample size: The normal range of TSH is 0.5-5.5 micro IU/ml (3± 2.5). Considering a change of 2.5 micro IU/ml from baseline to the end of follow up to be clinically significant in isoflavone arm over placebo and assuming a SD of 2.5 at alpha of 5 per cent and power of 80 per cent, 16 patients were required in each group; thus needing a total of 32 patients. Expecting 60 per cent compliance, it was decided to include up to 54 patients in the study.
Statistical analysis: Data were expressed as mean ± SD unless specified, numbers (percentages) and median (interquartile range). Proportions were compared using chi-square tests with continuity correction or Fisher's exact test when appropriate. The between and within groups repeated measurements of outcomes were analysed using Generalized linear models- Repeat measures and Repeated measures analysis of variance, respectively. Also, mean change from baseline in various parameters between the two groups was compared using unpaired Student's t test with Welch correction for parametric data or Mann whitney U test for non parametric data. Two sided significance tests were used throughout. P<0.05 was considered significant.