Comparing Ovulation Outcomes of Letrozole-Tamoxifen-Estradiolwith and without Vitamin E in Infertile Women with PolycysticOvary Syndrome: A Double-Blind, Randomized Clinical Trial.

Infertility is defined as failure to conceive after one year of unprotected sex ( 1 ). Infertility affects more than 186 million individuals and couples globally, with devastating social, psychological, and health consequences ( 2 , 3 ). Female disorders account for about 50% of the causes of infertility in couples ( 4 ). Polycystic ovary syndrome (PCOS), premature ovarian failure, hormonal problems, genital infections, fallopian tube blockage, congenital abnormalities of the uterus, endometriosis, and other reproductive disorders contribute significantly to female infertility ( 5 ). PCOS is the most common endocrine disorder in women of reproductive age, whose prevalence may reach 10-15% or even more ( 6 ). PCOS is the first cause of infertility in women without ovulation, and infertility is seen in 70-80% of sufferers ( 7 ). The high prevalence of PCOS, coupled with the complex and not fully understood etiology of the condition, makes it a critical area of research in reproductive medicine ( 8 ). According to common assumptions, people with genetic predisposition are ready to suffer from PCOS if they are exposed to certain environmental factors. Obesity and insulin resistance are mentioned as the most common environmental causes. Some hypotheses also consider fetal androgen exposure to be involved in this problem ( 9 ). As a result, PCOS can lead to infertility and other reproductive issues, making effective treatment strategies essential for improving fertility outcomes. Currently, clomiphene citrate (CC) and tamoxifen citrate (TMX) are the main drugs used to induce ovulation in women with PCOS, but many patients do not respond to these treatments ( 10 , 11 ). Additionally, letrozole, an aromatase inhibitor, has emerged as another treatment option that is particularly effective in women who have an endometrial thickness greater than 6.5 mm ( 12 , 13 ). Vitamin E is a potent antioxidant that plays a key role in neutralizing free radicals and maintaining normal physiological functions, including growth, development, and reproductive health ( 14 ). Vitamin E plays a crucial role in reproductive health through multiple cellular mechanisms. As a potent antioxidant, it protects cell membranes, particularly by incorporating α-tocopherol into membranes to maintain their integrity and fluidity, which is essential for proper hormone receptor signaling1. Vitamin E also influences gene expression related to oxidative stress response, inflammation, and lipid metabolism, impacting overall cellular health. In oocytes, it helps preserve mitochondrial function by reducing oxidative damage, which is critical for proper development and fertilization potential. Additionally, vitamin E may influence steroidogenesis by protecting steroid-producing cells and modulating enzyme activities involved in hormone synthesis. Its immune-modulating properties can also be beneficial in addressing inflammatory aspects of reproductive disorders like PCOS ( 15 ). Compared to other fat-soluble vitamins such as A, D, and K, vitamin E demonstrates more specific reproductive advantages, particularly due to its powerful antioxidant capabilities and direct effects on follicular development and endometrial receptivity. While vitamin A contributes to vision and immunity, vitamin D supports calcium metabolism, and vitamin K is crucial for coagulation, these vitamins have limited direct roles in reproductive enhancement. In contrast, vitamin E improves sperm motility, oocyte quality, cervical mucus production, and uterine lining thickness, all of which are essential for successful fertilization and implantation ( 16 , 17 ). Studies have shown that vitamin E deficiency can lead to miscarriage, premature birth, eclampsia, intrauterine growth restriction, and infertility in women ( 18 , 19 ). Recent studies have suggested that Vitamin E supplementation can improve lipid profiles, insulin resistance, and hormonal imbalances in women with PCOS, thus potentially enhancing fertility outcomes ( 20 ). Despite these promising findings, the precise impact of vitamin E supplementation on ovulation outcomes remains underexplored. This study aims to address this gap by comparing the therapeutic effects of a regimen consisting of letrozole, tamoxifen, estradiol, and vitamin E versus a standard regimen of letrozole, tamoxifen, and estradiol in women with PCOS.

The study included 90 participants, divided equally between two groups. The two groups were comparable in terms of mean age, BMI, and duration of infertility, with no statistically significant differences observed. Additionally, no significant differences were found between the groups regarding type of infertility, gravid status, number of abortions, occupation, or level of education ( Table 1 ). Comparing baseline characteristics in groups under study before intervention Data are presented as mean ± SD or n (%). BMI; Body mass index, NA; Not applicable, SD; Standard deviation, Independent t test, Chi-squared test, significant level <0.05. On day 3 of the menstrual cycle, there were no significant differences between the two groups in terms of follicular size, number of follicles, or endometrial thickness. By day 7, significant improvements were observed in the group receiving vitamin E as part of the treatment regimen. This group showed larger follicular size, higher follicle count, and increased endometrial thickness compared to the group without vitamin E. These differences remained significant on day 12, with the vitamin E group continuing to demonstrate superior outcomes in terms of follicular growth, number of mature follicles, and endometrial development ( Table 2 ). Effects of letrozole, tamoxifen, estradiol, and vitamin E on follicular size, number of follicles, and endometrial thickness during the menstrual cycle Data are presented as mean ± SD. Enlarged follicle, Mature follicle, and Independent t test, significant level<0.05. The pregnancy rate [frequent (percent)] was higher in the group receiving letrozole, tamoxifen, estradiol, and vitamin E (11.1%) compared to the group receiving only letrozole, tamoxifen, and estradiol (6.7%); however, this difference was not statistically significant (P=0.457). The incidence of OHSS was slightly lower in the vitamin E group (2.2%) than in the control group (4.4%), but this difference was also not significant (P=0.553). Irregular bleeding occurred in 4.4% of the letrozole, tamoxifen, and estradiol groups compared to 2.2% in the vitamin E group (P=0.603). Ovarian cysts were observed in 4.4% of the letrozole, tamoxifen, and estradiol groups and 8.9% of the vitamin E group. Overall, the majority of participants did not experience complications, with 91.1% in the letrozole, tamoxifen, and estradiol group and 88.9% in the vitamin E group reporting no adverse effects ( Table 3 ). Comparative analysis of pregnancy outcomes, OHSS incidence, and side effects between treatment groups Data are presented as n (%). OHSS; Ovarian hyperstimulation syndrome, Chi-square test, and Fisher’s exact test, significance level <0.05.

This study demonstrates that incorporating vitamin E into ovulation induction regimens may provide significant benefits, particularly in improving follicular development and endometrial conditions, ultimately enhancing fertility outcomes in women with PCOS. These results align with findings from Morsy et al. ( 24 ), which demonstrated that while vitamin E may not directly increase ovulation and pregnancy rates, it significantly improves endometrial thickness. This suggests that vitamin E could play a beneficial role in preparing the endometrium for potential implantation, thereby enhancing fertility outcomes in women with PCOS. Cicek et al. ( 25 ) showed that vitamin E administration can improve the endometrial response in unexplained infertile women through possible antioxidant and anticoagulant effects. Safiyeh et al. ( 26 ) reported that supplementation with selenium and vitamin E significantly increased AMH levels, antral follicle count (AFC), and mean ovarian volume (MOV) in women with occult premature ovarian insufficiency (OPOI). Vitamins can affect the reproductive system of men and women through the oxidative mechanism and the activity of antioxidants that reduce the excessive production of free radicals in infertile men and women ( 27 ). Vitamin E is one of the most important natural antioxidants that protect cells from damage caused by free radicals. Vitamin E deficiency causes sterility in male animals and reduced fertility or non-termination of pregnancy in mice ( 28 ). In addition to its antioxidant role, vitamin E has demonstrated the ability to improve menstrual-related symptoms in estrogen-dependent inflammatory conditions such as endometriosis. Amini et al. ( 29 ) conducted a triple-blind placebo-controlled clinical trial and found that supplementation with vitamin E (800 IU/day) and vitamin C (1000 mg/day) over 8 weeks significantly reduced oxidative stress markers such as malondialdehyde (MDA) and reactive oxygen species (ROS), and led to a notable decrease in the severity of dysmenorrhea, dyspareunia, and chronic pelvic pain. These findings support the potential role of vitamin E in modulating prostaglandin pathways and reducing inflammation-mediated hormonal dysregulation, which may also have relevance in PCOS where chronic low-grade inflammation and menstrual irregularities are common ( 29 ). According to one study, vitamin E supplementation, along with omega-3 or magnesium supplements, did not affect glycemic indices, hormonal profiles, other biomarkers of inflammation or oxidative stress, anthropometric measurements, and HDL-c levels ( 30 ). Therefore, it is thought that the positive effect of vitamin E in PCOS may be independent of weight loss, which logically highlights the potential anti-hyperlipidemic, antioxidant, and anti-inflammatory properties of vitamin E in PCOS. In this regard, Chen et al. ( 18 ) concluded that vitamin E supplementation can improve oxidative stress and reduce exogenous HMG dose, but does not change pregnancy rate. In a systematic review, Tefagh et al. ( 20 ) showed that vitamin E supplementation improves lipid profile, decreases insulin levels and HOMA-IR, and has positive effects on metabolic and hormonal parameters in women with PCOS. Studies have shown that oocytes collected from human follicles >15 mm had a higher chance of fertilization and pregnancy rate compared to oocytes collected from smaller follicles. The rate of single and multiple pregnancies increases in follicles larger than or equal to 18 mm ( 31 , 32 ). The study by Etezadi et al. ( 33 ) emphasized that an endometrial thickness between 9 and 14 mm is associated with the highest rates of implantation and pregnancy in ART cycles. In contrast, both thin (15 mm) endometrial linings have been associated with lower pregnancy outcomes. In our study, women receiving vitamin E achieved significantly higher endometrial thicknesses compared to controls, with mean values around 9.14 mm on day 12, which falls within this optimal range. Therefore, the observed improvement in ovulation induction parameters-including follicular maturation and enhanced endometrial receptivity-likely contributed synergistically to better fertility potential in this group. Recent literature increasingly highlights the complex role of micronutrients and nutraceuticals in female reproductive health. In particular, vitamin and antioxidant imbalances have been implicated in both ovarian dysfunction and treatment resistance in PCOS. Beyond vitamin E, emerging therapies involving compounds such as myoinositol, alpha-lipoic acid, selenium, and multivitamin regimens have shown potential in modulating ovulatory response, metabolic profiles, and endometrial receptivity ( 34 - 37 ). Furthermore, the involvement of thyroid dysfunction in the infertile pathway is a clinically relevant consideration. Subclinical or autoimmune thyroid abnormalities may impair ovulatory function and reduce the effectiveness of stimulation protocols. While this study excluded participants with overt thyroid disease, deeper phenotyping and routine thyroid screening should be considered in future research to enhance patient stratification and treatment personalization ( 38 ). This study suggests that adding vitamin E to ovulation induction treatments may improve follicular and endometrial development in women with PCOS, potentially enhancing fertility outcomes. Due to its low cost and accessibility, vitamin E could be a practical addition to fertility regimens, particularly in settings with limited resources. Clinicians may consider incorporating vitamin E for women undergoing ovulation induction. Further research is suggested, explore the mechanisms of vitamin E effects on ovarian and endometrial function, and evaluate its impact on pregnancy and live birth rates. Multi-center, long-term studies are required, as well as investigations into the optimal dosage and potential interactions with other fertility drugs. Future research should also focus on the broader role of antioxidants like vitamin E in fertility, particularly oxidative stress in reproductive disorders, and the potential synergistic effects with other micronutrients like selenium and omega-3 fatty acids. This study has several strengths. One of the key strengths is its randomized and double-blind design, which helps minimize selection and detection bias. Importantly, both participants and outcome assessors, including the sonographer, were blinded to group assignments. Additionally, the study employed a standardized protocol for ovulation induction, which enhanced the internal validity of the findings. However, certain limitations must be acknowledged. Although the trial was double-blinded, no placebo was used for vitamin E, which may have partially limited blinding among clinical staff administering the intervention. Moreover, the intervention period was relatively short (under one month). While significant improvements in intermediate outcomes such as endometrial thickness and follicular size were detected, the long-term effects of vitamin E on sustained ovulatory function, pregnancy rates, and live births remain uncertain. Since vitamin E primarily exerts its effects through antioxidant mechanisms, a longer duration of therapy may be needed to fully capture its reproductive benefits. Future studies should incorporate extended follow-up and evaluate live birth outcomes. The study team faced several challenges during the implementation phase. The most notable included difficulties in recruiting participants who met the strict eligibility criteria based on the Rotterdam criteria and hormonal evaluations. Additionally, ensuring strict adherence to blinding protocols while managing treatment logistics without the use of a physical placebo for vitamin E posed practical challenges. Despite these challenges, the study was completed successfully with full participant retention and data integrity maintained throughout.

This randomized clinical trial provides evidence that the addition of vitamin E to the letrozole, tamoxifen, and estradiol regimen significantly improves key fertility parameters in infertile women with PCOS. Specifically, vitamin E supplementation was associated with enhanced follicular size, follicle count, and endometrial thickness parameters that are critical for successful ovulation and implantation. The antioxidant properties of vitamin E likely mitigate oxidative stress, thereby improving oocyte quality and enhancing endometrial receptivity. While these findings indicate a potential therapeutic benefit, the study’s limitations-such as its single-center design and the absence of long-term follow-up on pregnancy outcomes-warrant caution in generalizing the results. To fully assess the clinical impact of vitamin E supplementation on fertility outcomes, larger-scale, multi-center trials with extended follow-up are necessary. Future research should also explore the optimal dosing regimen, as well as the potential synergistic effects of vitamin E with other fertility treatments. In conclusion, the addition of vitamin E to ovulation induction protocols appears promising for enhancing reproductive outcomes in women with PCOS. However, further investigation is required to confirm these findings and establish comprehensive clinical guidelines for its use in fertility management.

This study was designed as a double-blind, randomized clinical trial conducted from December 2023 to August 2024 at the Women’s Clinic in Jahrom, located in Fars Province, southern Iran. The trial adhered to the Consolidated Standards of Reporting Trials (CONSORT) guidelines to ensure methodological transparency and rigor. The protocol was registered on the Iranian Registry of Clinical Trials on December 14, 2023 (registration number: IRCT20150407021653N20), and ethical approval was obtained from the relevant institutional review board (IR.JUMS.REC.1401.135). Written informed consent was secured from all participants prior to enrollment. The study procedures adhered to the ethical standards set forth by the institutional and national research committees, by the 1964 Helsinki Declaration. The study included infertile women diagnosed with PCOS according to the Rotterdam criteria ( 21 ). These criteria require the presence of at least two of the following features: oligo-ovulation or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic ovaries observed via ultrasonography. Prior to diagnosis, other conditions that mimic PCOS-such as thyroid dysfunction, hyperprolactinemia, non-classic congenital adrenal hyperplasia, Cushing’s syndrome, and androgen-secreting tumors-were ruled out. Eligible participants were women under the age of 40 who had been unable to conceive after at least 12 months of unprotected intercourse. Exclusion criteria included known drug sensitivities to letrozole, tamoxifen, estradiol, or vitamin E; significant hepatic or renal dysfunction; type 1 or type 2 diabetes mellitus; thyroid disease; congenital adrenal hyperplasia; or abnormal findings on hysterosalpingography (HSG). Specifically, exclusionary HSG results included complete bilateral tubal occlusion, unilateral tubal occlusion with contralateral tubal damage, evidence of hydrosalpinx, and marked peritubal adhesions. Uterine abnormalities such as large submucous fibroids, endometrial polyps, intrauterine adhesions (Asherman’s syndrome), or congenital anomalies (e.g., septate or bicornuate uterus) also constituted grounds for exclusion. In addition to the eligibility of the female participants, specific criteria were applied to evaluate the sperm parameters of their male partners. Inclusion required semen volume of at least 1.5 mL, sperm concentration of 15 million/mL or more, total sperm count of 39 million/ejaculate or higher, total motility of at least 40%, progressive motility of 32% or greater, normal morphology of 4% or more, and sperm vitality of at least 58%. Male partners with azoospermia, leukocytospermia (defined as over 1×10 6 white blood cells per mL), or sperm concentrations below 15 million/mL were excluded. The sample size was calculated using G*Power software based on a moderate effect size (Cohen’s d=0.5), as defined by Cohen. Brown et al.’s systematic review ( 22 ) was used to determine the clinically relevant ovulatory outcome, rather than to extract a direct effect size. With a power of 80% and a type I error (α) of 5%, and allowing for a 10% dropout rate, a total of 90 participants (45 per group) were required. Participants were selected through convenience sampling based on predefined inclusion and exclusion criteria. A computer-generated block randomization method (12 blocks) was used with a 1:1 ratio. Random allocation sequences were created using Random Allocation Software. Allocation concealment was maintained using sequentially numbered, opaque, sealed envelopes opened only after participant enrollment and consent. In this study, blinding was performed according to the registered protocol using a double-blind design. Participants were provided with a general introduction to the study groups at enrollment, after which written informed consent was obtained. Random allocation was then conducted, and participants were assigned to treatment groups labeled A or B, without disclosure of the specific treatment components. Outcome assessment was performed by a blinded evaluator; specifically, the sonographer responsible for follicular monitoring and endometrial thickness assessment was unaware of group allocation ( Fig .1 ). Treatment adherence was assessed through patient selfreport at each follow-up visit, during which participants were asked about missed doses and medication use, and adherence was reviewed during routine ultrasound monitoring visits. Consort -flow-diagram. Patients in group A received daily doses of letrozole (5 mg), tamoxifen (20 mg divided into two 10 mg doses), and estradiol (2 mg) from the third to the seventh day of their menstrual cycle. In addition, they were prescribed vitamin E (100 mg daily) for 25 consecutive days ( 13 , 23 ). Patients in group B received the same regimen, excluding vitamin E. Both groups underwent transvaginal ultrasound examinations on the third, seventh, and twelfth days of their menstrual cycles. If ovarian and endometrial conditions were favorable, ovulation induction therapy was initiated. When the endometrial thickness reached 7 mm, estradiol was discontinued. If follicles matured to sizes between 18 to 24 mm and at least three to four mature follicles were observed, along with an endometrial thickness exceeding 7 mm with a triple-line appearance, 10,000 international units (IU) of human chorionic gonadotropin (hCG) were administered to trigger ovulation. In cases with only one or two mature follicles, a full 10,000 IU dose of hCG was given. When three or four dominant follicles were present, participants were counseled about the risks of multiple pregnancy and ovarian hyperstimulation syndrome (OHSS). Upon receiving informed consent, the hCG dose was reduced to 5,000 IU if the patient opted to proceed. Participants were advised to engage in intercourse every two days following the hCG trigger administration. Ovulation induction cycles were canceled under the following conditions: i. Poor ovarian response, defined as the absence of follicular growth during stimulation, ii. Premature ovulation, identified by the spontaneous rupture or disappearance of a dominant follicle prior to hCG administration, iii. Abnormal baseline hormonal profiles, including elevated day-3 follicle-stimulating hormone (FSH) or low anti-müllerian hormone (AMH) levels, indicative of diminished ovarian reserve, iv. Excessive follicular development, defined as more than four follicles measuring ≥15 mm in diameter; and ( 5 ) clinical signs suggestive of OHSS, such as significant ovarian enlargement, abdominal discomfort, vomiting, thromboembolic symptoms, or fluid imbalance. The primary outcome was the achievement of a clinical pregnancy, defined as a gestational sac with fetal heart activity visible on transvaginal ultrasound between the sixth and seventh week of gestation. Secondary outcomes included the number and size of mature follicles and endometrial thickness measured via ultrasound. Tertiary outcomes involved the assessment of adverse drug reactions in both treatment groups. Ultrasound examinations were performed on the third, seventh, and twelfth days of the menstrual cycle to monitor follicular development and endometrial changes. Data were collected using a structured form consisting of three sections. The first section gathered demographic and anthropometric data, including age, weight, height, body mass index (BMI), educational level, employment status, and duration of infertility. The second section included baseline and follow-up information on hormonal profiles, menstrual cycle patterns, hirsutism, ultrasound findings, and HSG results. The third section recorded treatment outcomes such as pregnancy type, maternal complications, and any side effects of the medications used. During follow-up, ovulation was assessed via ultrasound. Patients in both groups who responded to treatment, indicated by a follicle size greater than 18 mm and an endometrial thickness of at least 8 mm (three-layered and transparent), received a 10,000 IU injection of hCG to trigger ovulation. Statistical analysis was performed using Stata software version 14 (StataCorp, College Station, TX, USA). The normality of the data was assessed using the Kolmogorov-Smirnov test, which confirmed that all variables were normally distributed. Descriptive statistics, including means and standard deviations (SD), were reported for continuous variables. For comparisons between the two groups, the independent t test was used for all quantitative variables, as the data were normally distributed. Categorical variables were compared using the chi-square test, and Fisher’s exact test was used when the expected frequencies were less than five. A significance level of P<0.05 was considered statistically significant.