Document Type : Original Article
1 Department of Obstetrics and Gynecology, IVF Unit, Yas Hospital, Tehran University of Medical Sciences, Tehran, Iran
2 Non-communicable Diseases Research Center, Alborz University of Medical Sciences, Karaj, Iran;Department of Community Medicine, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran
3 4Department of Epidemiology and Reproductive Health, Reproductive Epidemiology Research Center, Royan Institute for Reproduc- tive Biomedicine, ACECR, Tehran, Iran
As one of the most common endocrine disorders, polycystic
ovary syndrome (PCOS), has complex pathophysiological
characteristics, which have not yet been understood
completely. PCOS involves about 5-10% of women of reproductive
age and it seems to be an important cause of infertility
Oxidative stress as a potential cause of poor oocyte quality
can influence female reproduction (
Consequently, the aim of the present study was to investigate the effect of melatonin on the rate of chemical pregnancy among PCOS patients undergoing intrauterine insemination (IUI).
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 (
Flowchart of the study
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 (
Basic and clinical features of the patients in the intervention and control groups
|Variables||Intervention group (n=94)||Control group (n=100)||P value|
|Age (Y)||28.4 ± 5.5||29.3 ± 5.6||0.241|
|BMI (Kg/m2)||27.6 ± 4.0||28.1 ± 3.7||0.056|
|Infertility duration (Month)||35.1 ± 21.7||43.5 ± 25.7||0.015|
|Primary endometrial thickness (mm)||4.6 ± 0.56||4.4 ± 0.52||0.093|
|Estradiol concentration (pg/ml)||1730 ± 281||1850 ± 534||0.001|
|IUI cycle duration (Day)||17.2 ± 2.6||16.8 ± 2.1||0.371|
|Total follicle count (n)||24.6 ± 4.7||23.9 ± 4.6||0.323|
|Primary infertility (%)||86 (91.5)||94 (94)|
|Secondary infertility (%)||8 (8.5)||6 (6)|
Data are presented as mean ± SD or n (%)
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.
Comparison of IUI outcomes between the intervention and control groups
|Variables||Intervention group (n=94)||Control group (n=100)||P value|
|Melatonin concentration (pg/ml)||190.7 ± 34.1||74.5 ± 17.1||<0.001a|
|Mature follicle (n)||2 (2-3)b||2 (1-3)||0.002c|
|Endometrial thickness after the treatment (mm)||9.2 ± 1.33||8.5 ± 0.87||<0.001d|
|Chemical pregnancy (%)||30 (32)||18 (18)||0.012e|
|Clinical pregnancy (%)||26 (27.6)||15 (15)||0.013e|
Data are presented as mean ± SD or n (%). IUI; Intrauterine insemination, a; Independent sample t test, b; Median and IQR, c; Mann-Whitney test, d; ANCOVA test with adjusting baseline Endometrial thickness, and e; Chi-square test.
The results of the present study suggest that following IUI, melatonin treatment has a favorable effect on mature follicles, ET, as well as chemical and clinical pregnancies in infertile PCOS women.
In recent studies, it has been shown that oxidative stress
has an adverse effect on infertility treatments, so researchers
are trying to find possible mechanisms of preventing
these unfavorable effects (
Studies at the molecular level have discovered that in
pregnant rats, melatonin supplementation improves serum
17ß-estradiol levels. Also, in rat uterine tissue, it enhances
the expression of MT(
In the present study, the results suggested that adding
melatonin to the IUI treatment cycle could significantly
improve the quality of follicles during the cycle in PCOS
cases. Along with our findings, Eryilmaz et al. (
In a recent study, Jahromi et al. (
The results of the current study also revealed that the
mean of ET in the melatonin-treatment group increased
more than that in the control group. Therefore, melatonin
has a favorable effect on ET in infertile PCOS patients following
IUI. Unfortunately, there are not many studies on
the relationship between melatonin and ET, but based on
an animal study on rats, it is suggested that melatonin can
affect the endometrial morphology and increase embryo
In the present study, chemical and clinical pregnancy
rates were significantly higher in the melatonin-treated
group compared to the placebo group. In a systematic review
and meta-analysis of five published randomized controlled
trials conducted by Seko et al. (
In a relatively large randomized controlled trial in PCOS
infertile cases undergoing intracytoplasmic sperm injection,
researchers compared outcomes in two groups including
myo-inositol 4g, folic acid 400mcg and melatonin
3mg per day (n=178) and myo-inositol and folic acid alone
(n=180). The results showed that patients in the first group
had greater numbers of mature oocytes and grade 1 embryos.
These results, therefore, support the positive effect of
melatonin in the treatment of PCOS infertile women (
In the present study, which was an attempt to provide novel information on the effects of melatonin on PCOS patients of a certain age group, some limitations existed, including the difficulty of frequent ultrasound scanning for patients until the right size of follicle was seen, and also patients’ poor cooperation with the treatment due to their unfamiliarity with melatonin prescription. Besides, the study was conducted on PCOS cases with the mean age of 28.9 years old and just IUI cycles were included in this study. Therefore, the obtained results are generalizable to infertile women suffering from PCOS in a relatively young age group. It is suggested to conduct a multi-center RCT to obtain more generalizable and valid results.
The results of this study demonstrated that the treatment of PCOS patients undergoing IUI with melatonin can significantly improve the quality of follicles and, as a consequence, the rate of chemical pregnancies.