Effects of Isoflavone Supplementation on the Response to Medroxyprogesterone in Premenopausal Women with Nonatypical Endometrial Hyperplasia: A Randomized, Double-Blind, Placebo-Controlled Trial

Objective The purpose of this study was to evaluate the impact of isoflavone supplementation compared with placebo on endometrial histology and serum estradiol levels in premenopausal women with nonatypical endometrial hyperplasia. Materials and Methods The present double-blindplacebo-controlled clinical trial was conducted on 100 women with nonatypical endometrial hyperplasia in the age range of 30 to 45 years. Participants were randomly assigned to receive 50 mg of isoflavone (n = 50) or placebos (n = 50) daily for three months. Both groups received the standard treatment of nonatypical endometrial hyperplasia. Endometrial biopsy and blood samples were taken at the baseline and three months after the intervention. The incidence of drug side effects was assessed as well. Results After three months, 88.4% of isoflavone-administered subjects had a significant histological improvement compared to 68.9% subjects in the placebo group (P=0.02). There were no significant differences between the two groups in the changes of serum estradiol levels and the incidence of drug side effects. Conclusion The findings of the present study demonstrated that the coadministration of 50 mg of isoflavones and medroxyprogesterone acetate increases the treatment efficacy in women with nonatypical endometrial hyperplasia. Clinical Trial Registration. This trial was registered on the Iranian website for clinical trial registration (https://www.irct.ir/trial/53553).


Introduction
Endometrial cancer is the most prevalent malignancy of the female reproductive system in developed countries [1]. Endometrial hyperplasia is a precursor lesion for adenocarcinoma of the endometrium, which accounts for the majority of endometrial cancer [2]. Chronic exposure to unopposed estrogen is the main risk factor for the development of endometrial hyperplasia [3]. Abnormal endometrial histology such as endometrial hyperplasia is relatively common (about 14%) among premenopausal women with irregular menses [4]. Te risk of developing endometrial cancer following endometrial hyperplasia depends on the presence of atypia and its severity [5]. Endometrial hyperplasia without atypia regresses following curettage or progestin treatment and has a lower risk of progression to adenocarcinoma [6], but lesions with atypia often do not regress and may be resistant to repeated curettage or long-term treatment with progestin [7]. Previous studies have reported that surgery is the best strategy for treating endometrial hyperplasia with atypia, but the tendency to fnd alternative medical treatments for surgery with lower complications and higher efcacy is currently increasing [8]. Tere are many progesterone-containing medications for the treatment of endometrial hyperplasia such as medroxyprogesterone, megestrol, and norethindrone acetate [9].
Recently, herbal supplements such as soy-derived isofavone have been widely investigated for the treatment of nonatypical endometrial hyperplasia [10]. Genistein, a soyderived isofavone, was reported to be one of the new agents for the treatment of endometrial hyperplasia, which was more efective than norethindrone acetate [11]. Isofavone supplementation may prevent endometrial cancer due to its benefcial efects on cell proliferation, apoptosis, and progesterone receptors [12,13]. Isofavones have both estrogenic and antiestrogenic efects, which can be used for the treatment of breast and prostate cancers, symptomatic amelioration of postmenopausal women, and osteoporosis [14]. Shukla et al. [15] showed genistein at a daily dosage of 54 mg can decrease the local synthesis of estrogen and may inhibit progression of endometrial hyperplasia to endometrial cancer. Moreover, Steinberg et al. [16] revealed that isofavone supplementation for two years has a safety profle without any serious adverse efects in women.
Considering the high prevalence of endometrial hyperplasia and possible drug side efects of hormone-containing medications, administration of herbal medicines along with routine treatment may enhance treatment efcacy. Terefore, this study was performed to evaluate the efects of isofavone supplementation compared with placebo in the treatment of women with nonatypical endometrial hyperplasia.

Participants and Ethics Statement.
Te present investigation has been registered on the Iranian website for registration of clinical trials with the special ID of IRCT20200531047614N3 (https://www.irct.ir/trial/53553) after obtaining approval by the Research Ethics Committee of the Kashan University of Medical Sciences (KAUMS) and following the Declaration of Helsinki and Good Clinical Practice guidelines. Tis clinical trial was performed at the Gynecology Clinic of Shahid Beheshti Hospital (Kashan, Iran) between May 2020 and July 2021. After a face-to-face meeting, we explained the objective and methods of the study to patients, and written informed consent was obtained from all women. (1) Unwillingness to cooperate (2) Receiving hormone therapy within 6 months prior to enrollment in the trial (3) A history of atypical hyperplasia (4) Menopausal women (5) Hypersensitivity to soybean products (6) Patients with focal endometrial lesions (7) Women with congenital uterine anomalies 2.3. Sample Size Calculation. We used a randomized clinical trial sample size calculation formula, where type one (alpha) and type two (beta) errors were 0.05 and 0.10 (power � 90%), respectively. Te sample size was determined according to a study by Bitto et al. [11] to compare the efects of isofavones and placebos on endometrial hyperplasia. In this trial, 42% of isofavoneadministered participants (P 1 � 0.42) had a considerable improvement in symptoms compared to 12% of placeboreceived subjects (P 2 � 0.12). Using the formula, we needed 41 participants in each group; after allowing for 20% dropouts in each group, the fnal sample size was 50 subjects in each group.

Study Design.
In this randomized, double-blind, placebo-controlled trial, after balanced block randomization, participants were allocated into two intervention groups. Te randomization list was created from 1 to 100 by a random number generator website (https://stattrek.com/statistics/ random-number-generator.aspx), and women were randomly assigned into two groups to receive either isofavone (n � 50) or placebos (n � 50). Te medical team, participants, and data assessors were blinded to treatment allocation. Te patients in the experimental group received 50 mg/day isofavone supplements (Goldaru Pharmaceutical Company, Tehran, Iran), and subjects in the control group (n � 50) received daily one placebo tablet (Goldaru Pharmaceutical Company, Tehran, Iran) for three months. Isofavone supplements and placebos had similar packaging, color, shape, and size. Based on the information from the pharmaceutical company, an ultrasound-assisted extraction (UAE) method [17] was employed in the isolation of isofavone from soybeans. Qualitative and quantitative analyses of isofavones were determined by high-performance liquid chromatography (HPLC). Both groups received standard treatment for nonatypical endometrial hyperplasia with 10 mg of medroxyprogesterone acetate daily for two weeks per month for a three-month intervention period. To ensure that patients take tablets, while following up by phone, they were asked to bring the empty package of medication after the intervention.

Assessment of Outcomes.
Te primary outcome was the pathological response, but serum estradiol levels and drug side efects were considered secondary outcomes. After obtaining informed written consent, a checklist, including 2 International Journal of Clinical Practice demographic characteristics and disease-related variables, was flled out. At the baseline and after the 3-month intervention period, blood samples (10 mL) were collected from each patient at the Shahid Beheshti Gynecology Clinic. Estradiol concentration was quantifed using a solid-phase immunoassay (Roche Diagnostics, Milan, Italy). At the beginning and end of the third month, an endometrial biopsy was performed by a gynecologist (ZV). Samples were evaluated by the same pathologist initially and after the intervention. Women were instructed to report any probable side efects such as edema, weakness, anorexia, mastalgia, abdominal pain, and dizziness.
2.6. Statistical Analysis. Te normality of data was determined by the Kolmogorov-Smirnov test. To fnd out signifcant changes in continuous variables between the two groups, we applied independent t-tests. Pearson Chi-square tests were utilized to compare categorical variables. An analysis of covariance (ANCOVA) was used to evaluate the impact of isofavone supplementation compared with placebos on serum estradiol levels after adjusting for confounding parameters including age and baseline estradiol values. A P value less than 0.05 was considered statistically signifcant. All analyses were performed by using SPSS version 16 (Chicago, Illinois, USA).

Results
As shown in this study fow diagram (Figure 1), we enrolled 100 premenopausal women with nonatypical endometrial hyperplasia in this trial; however, 12 participants were excluded from the trial because of personal reasons. Finally, 88 participants (placebo (n � 45) and isofavone (n � 43)) completed the trial. Baseline characteristics of participants including height, age, weight, body mass index (BMI), systolic blood pressure and diastolic blood pressure, and the percentage of diabetes mellitus were not statistically diferent between the two groups (Table 1).
After three months, isofavone and medroxyprogesterone coadministration signifcantly improved endometrial hyperplasia compared with placebo plus medroxyprogesterone. Te efcacy of isofavone plus medroxyprogesterone in the treatment of nonatypical endometrial hyperplasia was 88.4%, while placebo plus medroxyprogesterone treated 68.9% of patients, and this diference was statistically signifcant (P � 0.02). Moreover, isofavone supplementation for 3 months had no signifcant efect on serum estradiol levels compared with the placebo ( Table 2).
Drug side efects while taking isofavones and placebos were investigated in two groups of women with nonatypical endometrial hyperplasia. According to the results of the study, the incidence of decreased appetite, dizziness, edema, mastalgia, abdominal pain, and weakness was not statistically signifcant between the two groups (Table 3).

Discussion
To the best of our knowledge, this trial is the frst investigation to evaluate the efects of isofavone supplementation compared with the placebo on endometrial histology and serum estradiol levels in premenopausal women with nonatypical endometrial hyperplasia. Our study indicated that administration of isofavone (50 mg/day for 3 months) to women with nonatypical endometrial International Journal of Clinical Practice hyperplasia signifcantly improved endometrial histology, but it had no efect on serum estradiol levels. In the present trial, 88.4% of isofavone-administered subjects had a signifcant histological improvement compared to 68.9% subjects in the placebo group.
Isofavone is a phytoestrogen that is found in plants such as soybeans [18]. Te human estrogen receptor (ER) exists as two subtypes (ER-alpha and ER-beta) [19]. ER-beta has an inhibitory efect on ER-alpha and is the dominant receptor type in the brain and cardiovascular system [4]. Te dominant receptor type in the uterus is ER-alpha [20]. Te afnity of phytoestrogen to bind to ER-beta is greater than ER-alpha [21]. On the other hand, isofavone (low dose) can bind to ER-alpha in the endometrium of the uterus and occupy the active site of the receptor and prevent the binding of endogenous estrogen, which is more efcient than isofavones, and thus acts as a partial agonist [22]. Furthermore, isofavone can prevent endometrial hyperplasia and cancer due to its efects on cell proliferation, apoptosis, and progesterone receptors [23]. Te prolonged use of high-dose isofavone (≥150 mg) can act as estrogen agonists, with a greater efect on the alpha receptor, and cause the proliferative efects of estrogen on the uterine endometrium [24]. Terefore, soybean-derived isofavones at a dose of 150 mg may cause endometrial hyperplasia, and at a dosage of 50 mg, they can reduce hyperplasia and exert protective efects on the endometrium and decrease the progression of lesions to endometrial cancer [25]. Various studies have evaluated isofavone supplementation with diferent doses and duration of intervention in women. Unfer et al. [26] investigated the efects of long-term (5 years) use of isofavone on endometrial histology in healthy postmenopausal women. In this study, women were assigned to two groups receiving 150 mg of isofavones or placebos daily, and the endometrial biopsy was performed at the baseline of the study and fve years later. Te fndings of this study revealed that the incidence rate of hyperplasia in the isofavone group (3.37%) was signifcantly higher than that in the placebo group (0%). In addition, endometrial atrophy was observed in 71% of women receiving isofavones and 80% of women taking placebos at the end of the intervention. No malignancy occurred in the two groups. Murray et al. [27] demonstrated that isofavone supplementation (120 mg daily) for six months compared with placebos in menopausal women treated with exogenous estrogen replacement therapy had no signifcant impact on the incidence rate of endometrial hyperplasia. Bitto et al. [11] compared the effects of isofavone and norethindrone acetate administration on endometrial histology, estrogen and progesterone receptor gene expression, and serum levels of sex hormones in 56 women with nonatypical endometrial hyperplasia. It has been shown that after 6 months, 42% of isofavone-   administered individuals (at a daily dosage of 54 mg) had a considerable improvement in symptoms compared to 12% subjects in the placebo group. Changes in hormone profles were not statistically diferent between the two groups. Te genes expression related to estrogen receptors (ER-alpha and ER-beta) signifcantly decreased in the isofavone group. In a study by Quaas et al. [28] on 350 postmenopausal women aged 45-92 years, it was found that daily administration of 154 mg isofavone for three years compared with placebos had no signifcant efect on the incidence rate of endometrial hyperplasia, endometrial thickness, and the incidence rate of malignancy. Te main possible mechanisms of isofavone in the recovery of nonatypical endometrial hyperplasia include decreasing mRNA expression of ER-alpha [29], matrix metalloproteinases-26 [30], increasing gene expression of the progesterone receptor [31], and being a partial agonist for ER-alpha [32]. Based on a recent meta-analysis study, low-dose isofavone (dose <100 mg/day) has a safety profle without any serious side efects. Only minimal adverse effects such as abdominal pain, diarrhea, myalgia, and headache have been reported [33]. Te limitations of this trial include the short period of intervention, relatively small sample size, and single-center design. Furthermore, due to inappropriate funding, there was no chance to evaluate other hormonal parameters such as serum progesterone levels, LH and FSH concentrations, and gene expression related to estrogen and progesterone receptors at the baseline and three months after the intervention.

Conclusion
Overall, the coadministration of 50 mg of isofavone and medroxyprogesterone acetate for three months increases the efcacy of treatment in premenopausal women with nonatypical endometrial hyperplasia.

Data Availability
Te datasets analyzed during the current study are available from the corresponding authors on reasonable request.

Ethical Approval
Tis study protocol was approved by the Ethics Committee of KAUMS "approval no. IR.KAUMS.MEDN-T.REC.1399.006." All procedures performed in the study involving human participants are in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments.

Consent
At the beginning of the study, the purpose of the present trial was explained to participants, and they were assured about the anonymity and confdentiality of their data. All participants gave their signed, written, informed consent letters.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
HB, ZV, and VO designed the study and conceptualized the experiments; ZV, ST-A, ZT, HM, HA, and HB conducted the study; VO supervised the study; VO and HB performed the statistical analyses; VO, ZV, ZT, HB, ST-A, HM, FR, and HA wrote the manuscript; VO and FR critically reviewed the manuscript. All authors provided critical conceptual input, analyzed and interpreted data, and critically revised the report. Te fnal version was read and confrmed by all authors for submission. ZV and HB contributed equally to this work.