Effects of Melatonin Administration on Chemical Pregnancy Rates of Polycystic Ovary Syndrome Patients Undergoing Intrauterine Insemination: A Randomized Clinical Trial

This study was performed as a randomized, double-blinded clinical trial study (RCT) with a parallel-groups design. It was carried out using a 1:1 allocation ratio for the intervention group receiving melatonin and the controls receiving placebo at Yas Hospital in Tehran, Iran. The study population consisted of the PCOS-diagnosed patients who had been referred to the hospital due to infertility problems from March 2017 to September 2017. The sample of this study contained 198 patients with PCOS, meeting our inclusion criteria to participate in the study, and being recommended to undergo an IUI treatment by their physicians (Fig .1). The written informed consent was obtained from all individual participants included in the study. The proposal of this research has been approved in the Ethics Committee of Tehran University of Medical Sciences (Ethics committee code: 25667).

The inclusion criteria were the following: i. Being aged between 20 to 40 years, ii. Having husbands with normal spermograms, iii. Having normal hysterosalpingography, iv. Having the Rotterdam diagnostic criteria for PCOS, v. Having no underlying endocrine diseases, and vi. Using no hormonal drugs within the past three months. Furthermore, the exclusion criteria included the following: i. Being deficient in an adequate ovarian response, ii. Suffering from ovarian hyperstimulation syndrome, and iii. No history of treatment for infertility.

In the present study, a random allocation was performed, and the participants were divided into an intervention group receiving melatonin and a control group receiving placebo using a balanced block randomization technique. Considering blocks of 4 in this study, the Stata software was used to generate random-number sequences from 1 to 6 until the desired sample size was achieved. Since the total number of modes to set two people in the blocks of 4 was 6 modes, if the generated number exceeded 6, the next number was regenerated regardless of the previous number. Preparing the random allocation sequences of the participants, putting them in sealed airtight envelopes, and numbering them with a five-digit serial number were all performed by a third person who was not involved in the study design. All the envelopes (n=188) having a random 5-digit serial number were opened immediately after the completion of basic information and examination of the participants. Then, the participants were assigned to the intervention or control groups.

In this study, the primary outcome was the determination of the rate of chemical pregnancies during the IUI treatment cycle and the secondary outcome was the determination of endometrial thickness (ET) on the day of IUI.

Considering a type 1 error of 5%, a study power of 80%, and a difference of 20% between the chemical rates in the intervention and control groups, the sample size was estimated to be 93 patients in each group.

After initial evaluations by the physician, a basal vaginal ultrasound of the uterus and ovaries was performed during the days 1 to 3 of the menstrual cycle, using a Siemens ultrasound equipment (ACUSON X600, Germany), and the numbers of both antral follicles (AFC) and ET were recorded. Then, to induce ovulation two clomiphene citrate 50-mg tablets (Iran Hormone Company, Iran) were administered from cycle days 3 to 7. Furthermore, on the 3rd day of menstruation, a 3-mg melatonin tablet (Nature Made, USA) or its placebo (made by Faculty of Pharmacy, Tehran University of Medical Sciences) was given to the patients according to the randomized study protocol, and this prescription was continued until the day of hCG administration On the 10th day of menstrual cycle, another ultrasound was performed for both groups, and the thickness of the endometrium, AFC, and follicle sizes were recorded in millimeters. Subsequently, based on the information obtained at this stage, the need for 75 IU of human menopausal gonadotropin (HMG 75, Menogon, Germany) injection was estimated and it was injected intramuscularly by a trained nurse.

Vaginal ultrasound was performed periodically according to the needs of each patient by the physician until a suitable follicle size (i.e., greater than or equal to 18 mm) was seen. If the appropriate size of the follicle was observed, at the same time, estradiol was measured using Monobine kits and two hCG 500 ampoules (Choriomon, Swiss) were injected intramuscularly.

After the above steps, which took about 36-40 hours, the patients referred to the clinic and underwent IUI. Six days later, blood progesterone levels were measured by DRG Progesterone ELISA Kit (Marburg, Germany). Then, 14 days after IUI, hCG-B blood test (GenWay Biotech. Inc, USA) was performed using an antibody kit to determine the rate of chemical pregnancy.

The normality assumption was assessed by the Kolmogorov- Smirnov test. Mean ± standard deviation (SD) and median (inter-quintile range) were used for presenting data with normal and non-normal distributions, respectively. Differences between the two groups of participants were assessed using independent Student’s t tests. We used the Mann-Whitney test for continuous variables and chi-square tests for categorical variables. To evaluate the differences between the means of endometrial diameters in the intervention and control groups, an analysis of covariance with the adjustment of baseline scores was used. The Stata software (StataCorp LLC, version 13MP) was utilized to perform all the statistical analyses. Data with P<0.05 were considered statistically significant.

In the present study, the information on the clinical infertility treatments of 198 infertile patients referring to Yas Hospital in Tehran, Iran, were used (Fig .1). The mean age of the participants in the study was 28.9 ± 5.5 years. In this study, 94 patients received melatonin as the intervention group and 100 patients received placebo as the control group. The results of comparing basic and clinical features of the participants in the study revealed that these two groups did not show any significant differences in the basic features at the time of entering the study. Table 1 summarizes their basic and clinical information.

Regarding the evaluation of the serum concentration of estradiol in both intervention and control groups, the results represented the mean of this hormone concentration in the control group as 1850 ± 534 pg/ml and in the intervention group as 1730 ± 281 pg/ml. An independent t test indicated that the two groups had significantly different levels of serum estradiol concentration (P=0.001).

The IUI cycle in the intervention group lasted for 17.2 ± 2.6 days and in the control group lasted for 16.8 ± 2.1 days. The result showed that there was no significant difference between the two groups (P=0.371). Furthermore, the results of Mann-Whitney test confirmed that there were no statistically significant differences between the rates of HMG doses used within the two groups (P=0.970).

In Table 2, the results of the ET, the number of mature follicles, and the chemical and clinical pregnancy rates are compared between the two groups. As demonstrated in Table 2, the chemical pregnancy rate in the group receiving melatonin was about 30%, while this value was 18% in the control group. The chi-square test indicated that the difference between these two values was statistically significant (P=0.011). Also, the addition of melatonin to the treatment cycles of PCOS individuals significantly improved the sizes of follicles during the IUI cycles (P=0.002). Regarding the mean ET of the patients, our covariance analysis showed that there was a significant difference between the intervention and control groups (P<0.001). The mean ET of the patients in the intervention group increased more than that in the controls.