This prospective clinical trial was conducted on 70 infertile women undergoing ARTs {In Vitro Fertilisation (IVF) and intracytoplasmic sperm injection} referred to the Infertility Clinic of Hamadan Fatemiyeh Hospital (Hamadan Endometriosis Centre). This study was begun in February 2016 and was ended in March 2017.

The protocol of the present study was registered as the IRCT201704119014N156 at the Iranian Registry of Clinical Trials. According to a study conducted in this field [10], the sample size was determined as 28 for each group according to a formula for comparing two ratios that were evaluated on the basis of the same ET, and to account for the dropout of 20% of the samples, the result was multiplied by 1.25, and therefore final sample size in each group was calculated as 35 people. Samples were entered into the study by census. The sampling was performed by randomised block consisting of 10 columns and 7 rows in which the treatment groups were randomly assigned the letters A and B, that were randomly allocated to the treatment groups by the prescribing person.

Inclusion criteria included age under 40 years, idiopathic infertility, ovulation problems that did not respond to several periods of ovulation induction and Intrauterine Insemination (IUI), fallopian tube and endometriosis problems, lack of using a Nitric Oxide (NO)-lowering drug, the presence of at least one embryo with grade A or B, and the lack of concomitant use of other ARTs. After the detection of the cause of infertility following examinations and analysis of the tests including hormone analysis, sperm analysis, and hysterosalpingography, the type of treatment for patients was selected and patients who had the indication for the use of ARTs were included in the study. The presence of hydrosalpinx in one or two fallopian tubes, having embryos with grade C or lower, and lack of providing consent to participate in the study were determined as the exclusion criteria.

The 70 patients who were included in the study were divided into two groups of 35 each; the patients in the group 1 received 25 mg vaginal sildenafil four times a day+6 mg E2 from the second or third day of the cycle, and the group 2 received only 6 mg E2; and on the seventh day, transvaginal ultrasound was performed, and according to the status of endometrium, we investigated whether ET had reached 8 mm or higher, and also the dosage of drugs was gradually increased until ET reached 8 mm or higher. The researcher who measured the ET was blind to the grouping of the patients.

Ovulation stimulation was performed by one of the appropriate protocols for the patient. After the egg was collected, IVF or microinjection was performed on oocytes and the fertilised embryos were transferred to the uterus at the appropriate time [11].

It should be noted that in order to eliminate the risk of excessive ovarian hyperstimulation syndrome, none of the patients whose number of follicles was more than 11 were included in the study. The causes of frozen embryo in our study included cervical stenosis, inappropriate endometrium, high number of foetuses, and systemic disorders [12]. The result of the treatment cycle was followed-up by performing Beta-Human Chorionic Gonadotropin (BHCG) on day 14 after embryo transfer. The data were then recorded in the checklist of the outcomes of the intervention.

After data collection, the SPSS version 16 was used to analyse the data. To describe the quantitative data, mean (±Standard Deviation) and quadrant were used and to describe qualitative data, frequency was used. To compare average ET between the two groups, parametric t-test was used.

In this study, 70 infertile women undergoing ARTs were assigned to two groups of 35 each, underwent treatment with E2 and sildenafil citrate, and then studied.

There was no statistically significant difference in the number of transferred embryos, number of previous pregnancies, number of abortions, number of cycles, number of retrieved eggs, and number of fertilised eggs between the E2 group and the E2+sildenafil citrate group (p>0.05).

There was also no significant difference in intervention type and ART between the average ET on the seventh day of the cycle (p=0.85). There was no significant difference in the duration to reach an ET of 8 mm between the two groups (p=0.83) [Table/Fig-1].

The mean duration (day) to reach an 8 mm ET in infertile women in the E2+sildenafil group (13.59±3.07) was lower than that in the E2 group (14.88±4.78), yet insignificant (p=0.21). The mean dose (mg) of E2 needed to reach an 8 mm ET in the E2+ sildenafil group (108.59±85.42) was lower than that (131.71±49.69) in the E2 group (p=0.2).

There was no significant difference between mean age of infertile women treated with E2 (31.54±5.41) and those treated with E2+ sildenafil citrate (31.34±5.26) (p=0.876) and the two groups were matched by age. There was no significant difference in the mean ET score between infertile women treated with E2 alone and those treated with E2+ sildenafil citrate (p>0.05) [Table/Fig-2].

For the cause of infertility in the group treated with E2, the most common cause was male factor {n: 18 (51.4%)} followed by uterus {n: 7 (20%)} and idiopathic {n: 10 (28.6%)}; and in the group treated with E2+sildenafil citrate, the most common cause was male factor {n: 21 (60%)} followed by uterus {n: 5 (14.3%)} and idiopathic {n: 9 (25.7%)}. The mean ET score on the seventh day was not significantly different between the E2+sildenafil citrate group and the E2 group (p=0.367) [Table/Fig-3].

There was no significant difference in the duration of infertility (year) between infertile women treated with E2 (7.83±4.18) and those treated with E2+sildenafil citrate (4.12±5.80) (p=0.117). There was also no significant difference in embryo grade between infertile women treated with E2 and those treated with E2+sildenafil citrate (p>0.05) [Table/Fig-4].

The number of BHCG positive cases in the sildenafil citrate+E2 group was 9 (25.7%) and in the E2 group 11 (31.4%) (χ²=0.28, p=0.396) [Table/Fig-5].

In the present study there are no effect on embryo grade and positive BHCG was observed after concomitant use of E2 and sildenafil citrate. In a study that evaluated the effect of sildenafil citrate on ET in the control and sildenafil groups, the results showed that the drug could significantly increase ET and triple-line patterns in the endometrium.

However, intermediate patterns did not differ between the two groups [13]. In another study by Malinova M et al., the results indicated that vaginal consumption of sildenafil citrate and serophene in infertile women, by increasing uterine blood flow, increased endometrial thickness and can be used as an effective treatment method for ovulation induction [14]. In another study, it was found that vaginal administration of sildenafil, in addition to a 70% increase in ET in the studied women, also caused pregnancy in infertile women to persist [15].

The study of Soliman et al., showed that thermosensitive sildenafil vaginal gel significantly improved ET and uterine blood flow (without any side effects). Sildenafil vaginal ointment also reduced the dose and duration of treatment [16]. Sildenafil also had positive outcomes with respect to increase in ET in two women with Asherman’s syndrome [17]. In infertile women with lower ET, treatment with vitamin E, l-arginine, or sildenafil citrate could increase ET [18]. Sildenafil can increase the ET by inducing the vasodilator property [19]. Recently, NO synthase isoforms have been identified in the uterus and the role of NO as a uterine blood flow modulator has been identified [17]. It has been shown that NO plays a role in relaxing vascular smooth muscle through the cyclic Guanyl Monophosphate (cGMP) mediated pathway.

This drug inhibits cGMP specific phosphodiesterase type 5 (PDE5) by inhibiting cGMP degradation, thereby enhancing the effect of NO and ultimately increasing blood flow and ET [13,15]. On the other hand, implantation success depends on the ability of the blastocyst to penetrate the endometrium and to create a source of blood that requires certain genes such as Plasminogen Activator Inhibitor 1 (PAI-1), tumour suppressor factor (p53), and Vascular Endothelial Growth Factor (VEGF) for the production of proteins that are required for digestion of the endometrial cell matrix, regulation of cell growth, and induction of angiogenesis; sildenafil can enhance the angiogenesis by increasing the expression of p53 and VEGF [20-22].

In a study, it was shown that Clomiphene Citrate (CC) could have adverse effects, including decreased ET and uterine volume growth at the follicular phase. E2 neutralised these malignant effects and increased uterine development at the follicular phase [23]. Other study also found that adding vaginal E2 to CC during the stimulation period could increase ET on ovulation day and the following day. However, this increase in ET did not lead to an increase in fertility [24,25].

For the foetus to be able to attach to the endometrial wall, specific molecular and cellular changes in the endometrium, especially the luminal epithelium and its glands, should be induced. The induction is mediated by steroid hormones (oestrogen and progesterone). Any disorder in these hormones can disrupt uterine acceptance and ultimately result in unsuccessful implantation of the foetus [26,27].

Lack of examination of the effects of the drugs that were used in the study on ovulation was the limitation of the present study.

In infertile women undergoing ARTs, co-administration of sildenafil citrate and E2 increased ET relatively and reduced the dose of E2 and the duration of its administration. However, after co-administration, no effect on the embryo grade and the number of positive BHCG cases were observed.