Clomiphene citrate plus letrozole versus clomiphene citrate alone for ovulation induction in infertile women with ovulatory dysfunction: a randomized controlled trial

Study design and overview

This study employed a randomized controlled trial design. It was conducted at the Infertility and Reproductive Biology Unit of the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, from November 2020 to December 2021. Eligible participants were randomly allocated to a “combination group” or a “CC-alone group” after Institutional Review Board approval. Those assigned to the combination group received a daily dose of CC (50 mg) and letrozole (2.5 mg), whereas participants in the CC-alone group were administered a daily dose of CC alone (50 mg) [17]. Treatment medications were administered on days 3 through 7 of each participant’s menstrual cycle. The study enrolled infertile Thai women aged between 18 and 40 with ovulatory dysfunction (cycle length > 35 days or diagnosed with PCOS according to the modified Rotterdam criteria [18,19,20]). Participants were excluded if they had spontaneous pregnancy, uncorrected thyroid disease, hyperprolactinemia, allergy or contraindication to letrozole or CC, bilateral tubal occlusion, or a male partner with a total motile sperm count less than 10 × 10⁶ [21]. Before this research began, the Siriraj Institutional Review Board approved its protocol (Si-257/2020), and all participants provided written informed consent to participate. The study was registered at https://www.thaiclinicaltrials.org (TCTR20201108004) on 08/11/2020 and followed the CONSORT and IMPRINT guidelines [22]. The overview and timeline of the method are shown in Supplementary Fig. 1.

Randomization and blinding

The randomization scheme in this study was computer-generated using blocks of four, with group assignments concealed in sealed envelopes. The sonographer was blinded to the assignments. Participants were randomized in a 1:1 ratio to receive a daily dosage of CC (50 mg; Ovamit, Remedica Ltd, Limassol, Cyprus) in combination with letrozole (2.5 mg; Fresenius Kabi Oncology Ltd, Kolkata, India) or CC alone (50 mg) daily. Both treatment regimens were taken from days 3 through 7 of the menstrual cycle. Patients with long menstrual cycles were prescribed an oral progestogen to induce withdrawal bleeding. The progestogens administered were medroxyprogesterone acetate (10 mg daily; Provera; Pfizer, New York, NY, USA) or norethisterone acetate (5 mg daily; Primolut N; Bayer Thai Co Ltd, Bangkok, Thailand) for 7–10 days.

Study procedures

At the start of their menstrual cycle, participants were instructed to contact the investigator to arrange the ovulation induction schedule. The allocated treatment medication regimen was taken from days 3 through 7 of one menstrual cycle. Home urinary luteinizing hormone (LH) tests were performed twice daily, in the morning and at night, starting on cycle day 12 until a positive result was obtained or until cycle day 21 if the results continued to be negative. Patients were instructed to send pictures of the urinary LH test results to the researcher to confirm the results. Regular intercourse, performed two to three times per week, was recommended starting on cycle day 12 and on the day of the positive urinary LH test. Transvaginal ultrasound was performed on day 12 to day 14 of the cycle by a single operator throughout the project, and follicular growth and endometrial thickness were recorded. Serum progesterone levels were obtained 7 days after a positive urinary LH test or on cycle day 21 or day 22 in cases with negative urinary LH. A urine pregnancy test was performed 7 days after an ovulatory serum progesterone level or on cycle day 35 if there was no confirmation of ovulation and no menstrual period. Women with a positive urine pregnancy test were scheduled for a transvaginal ultrasound to confirm pregnancy 2 to 3 weeks later. A questionnaire was used to elicit information about any adverse effects of the study medications during the study period.

Outcome measures

The primary outcome was the ovulation rate, defined as a mid-luteal progesterone level greater than 3 ng/mL [23]. The secondary outcomes were the following: medically related side effects, including headache, dizziness, and hot flush, etc.; pregnancy complications or congenital anomalies; ovulation induction cycle characteristics, including endometrial thickness and the number of preovulatory mature follicles (defined as follicles with diameters ≥ 14 mm [24]); conception rate (diagnosed by a positive urine pregnancy test); clinical pregnancy rate (confirmed by positive fetal heartbeat on transvaginal ultrasonography); and live birth.

Sample size calculation and statistical analyses

The sample size calculation was informed by Meija et al. [10] and another study [16]. Meija et al. reported a 77% ovulation induction rate in women with PCOS treated with a combination of CC and letrozole. In contrast, the second study found a 48% rate for those treated with CC alone. A power analysis was conducted to determine the number of participants needed to detect a clinically meaningful difference in the ovulation rates of the 2 study groups. We calculated that 43 subjects per group would be required to achieve 80% statistical power (β) with a two-sided significance level (α) of 0.05. The sample size was increased to 50 participants per group to account for potential dropouts.

The statistical analyses were performed using PASW Statistics for Windows, version 18.0 (SPSS Inc, Chicago, IL, USA). The study conducted an intention-to-treat analysis involving all randomized participants and a per-protocol analysis restricted to those who followed the designated treatment. Descriptive statistics were used to describe patient and cycle characteristics. Continuous data were presented as the means ± standard deviations or medians (ranges) for normally distributed and nonnormally distributed data, respectively, whereas categorical data were expressed as numbers and percentages. For categorical data, Pearson chi-squared, Yates’ continuity correction, or Fisher’s exact test were performed to compare the proportions between two groups. The independent Student’s t-test was used for normally distributed continuous data, while the Mann-Whitney U-test was used for nonnormally distributed continuous data for comparing the mean or median, respectively, between two groups. Absolute differences were computed by substracting percentages of the combination group’s outcomes from percentages of the CC-alone group’s outcomes, while the rate ratio was computed by percentages of the combination group’s outcomes divided by percentages of the CC-alone group’s outcomes. The level of statistical significance was set at P < 0.05 for all tests.