Introduction

The good quality embryo, receptive endometrial, and perfect crosstalk between them is requiring for successful of in vitro fertilization and embryo transfer cycles. A weakening of any one of these issues may cause implantation failure (Harper & Harton, 2010).

Endometrial receptivity might be defined as a series of influences which prepare the endometrium to implantation of the embryo. The implantation window or “window of receptivity is a limited time during which interactions between the embryo and the uterus must have occurred. During this time, any breach in the communication between the endometrium and the embryo leads to implantation failure (Mišan & Severinski, 2016).

Endometrial receptivity depends on the several factors including morphological factors (endometrial thickness, pattern and sub endometrial blood supply) and biochemical factors (endometrial adhesion and anti-adhesion molecules, cytokines, growth factors like (Vascular Endothelial Growth factor(VEGF) and Insulin-like growth factor (IGF), endometrial immune factors, leukemia inhibitory factor (LIF) and endometrial glycodelin) (Bartosch, Lopes, Beires, & Sousa, 2011).

Vascular endothelial growth factor (VEGF), is belonging to proteins binding heparin that attach to endothelial cells, and which lead to proliferation and new blood vessel formation. The VEGF also stimulates the release of various cytokines by the endothelial cells leading to dilatation of blood vessels. It acts as an angiogenic factor to promote angiogenesis in different tissues. This explains the critical role of angiogenesis in a variety of women reproductive activities, like the growth of a dominant follicle, development of a corpus luteum, endometrial progress, and implantation (Nardo, 2005).

Ultrasonography evaluation of the endometrial morphology, including endometrial thickness and pattern and Doppler ultrasound of sub endometrial blood flow toward providing a good non-invasive tool for the evaluation of endometrial receptivity. Vascularization of the endometrium is regulated by the vascular endothelial growth factor, which is a strong angiogenic issue which was first described as a significant growth factor for endothelial cells of vessels (Rahmatullah, Al-Obaidi, Al-Saadi, & Selman, 2020).

Sildenafil is an effective and highly selective inhibitor of phosphodiesterase type 5 (PDE₅). It enhances the vasodilatory influences of NO on vascular smooth muscle by stopping the degradation of cGMP (Wareing et al., 2004).

Materials and Methods

Patients, materials and methods

The study was designed as a controlled randomized trial (interventional experimental study) conducted in High Institute of Infertility Diagnosis and Assisted Reproductive Technologies / AL-Nahrian University during the time from November 2018 till the end of April 2019.

Sixty infertile pairs have been included in this study who undergo ICSI cycle, they were told about the study and signed a printed informed agreement.

Inclusion Criteria

Patients at age group (19-40) years, infertility due to: tubal blockage, anovulatory cycles, unexplained infertility, cases of mild endometriosis whether diagnosed clinically or laparoscopically, due to male factors and couples with unexplained infertility

Exclusion Criteria

Patients with congenital uterine malformation, with extensive uterine pathology and medical contraindication of PDES-5 inhibitors.

Thirty (30) of patients had received sildenafil 25mg intravaginally four times daily form the day of stopping blood of menstrual cycle to the day of HCG injection.

IVF/ICSI procedures

Controlled ovarian hyperstimulation (COH), oocyte retrieval (OCR), fertilization and embryo culture, embryo quality, embryo transfer (ET).

Tow different types of controlled ovarian hyper-stimulation (COH) were used according to the demographic parameters of patients. First, a long agonist protocol started on cycle day 21 of the previous cycle, and patients were treated by GnRH agonist (Decapeptyl^® 0.1 mg, Ferring Co., Germany); then, on the cycle day 2, follicular stimulation began with an rFSH (follitropin alfa, Gonal F^®, Merck Serono) in a daily dose range of (150-450 IU).

Then secondly, the option used flexible antagonist protocol by starting with a rFSH (follitropin alfa, Gonal F^®, Merck Serono) in a daily dose range between (150-450 IU). When follicles reached 13-14 mm in size, a GnGH antagonist was started (Cetrotide^®, Merck Serono). Patients were monitored by transvaginal sonography (TVS). HCG (Ovitrelle^® 250 microgram, Merck Serono) was given once follicular size reached a diameter of 18 mm in three or more follicles.

Oocytes pick up was done using a transvaginal probe 34-36 hours following the HCG injection, immediately before the rupture of follicles. Oocytes were aspirated by transvaginal ultrasound-guided oocyte retrieval (TUGOR); oocytes at retrieval can be either the germinal vesicle (GV) or from metaphase I (MI). The absence of a polar body indicates it to be at the MI stage, which is an intermediate stage between the GV and MII (mature) stages, or metaphase II oocyte (MII), which is mature. Generally, before OCR, a semen sample is prepared. The patient's serum was obtained for measuring VEGF serum level also two-dimensional transvaginal ultrasound scan (two-dimensional) were done to evaluate endometrial width, regularity, and echogenicity, and sub-endometrial blood flow colour Doppler indices (RI, PI and Vs/Vd) were calculated.

At IVF laboratory, aspirated follicles were examined. Flushing was performed, and the follicles are kept one-to-two hours in a 37°C/CO2 incubator. Then, all oocytes underwent denudation and grading in a laminar flow cabinet. After that, a needle was carefully inserted through the shell of the egg into its cytoplasm, put in the CO2 incubator during waiting for the cell division. After insemination, zygotes were observed after 18-20 hours to examine for the occurrence of two pronuclei, then for 25-29 hours to confirm the presence of the first cleavage, which correlated with higher implantation rates. At day one, the presence of two pronuclei was considered a good prognostic sign. Embryo transfer was done at day two-or-three post-OCR, according to the number and grading of the embryos.

Results and Discussion

Demographic characteristics and baseline hormonal levels of infertile women

Our study showed no significant differences in the patient features and baseline hormonal levels in both study and control groups (Table 1). Our study was comparative study, so we selected females nearly comparable regarding the age, BMI, type, duration and causes of infertility to avoid the effect of these parameters on the outcome results.

Endometrial characteristics at the day of oocyte retrieval

The endometrial thickness on the day of ova pickup was higher in the study group but statically no significant than the control group (P = 0.599), 10.07 ±1.42 mm versus 9.84 ±1.91 mm, respectively, also no significant difference in endometrial pattern. However, (El-Maghrabi, El-Kasar, Elbaz, & Youssef, 2020) found that the endometrial thickness and trilaminar pattern of endometrium were comparable in both groups. (Firouzabadi, Davar, Hojjat, & Dhdavi, 2013) found that patients with previous unsuccessful ICSI cycles due to thin endometrial thickness showed good result after oral sildenafil treatment. (Fetih et al., 2017) recommended that uterine blood flow and endometrial thickness significantly increased after treatment with a vaginal gel of sildenafil, in women with low endometrium as a result of clomiphene citrate (CC). In our study, we didn't select the sildenafil group according to previous IVF failure due to poor endometrial thickness.

The mean pulsatility index in the study group was significantly lower than that in the control group (P = 0.042), 0.72 ±0.27 versus 0.99 ± 0.64., respectively. Also, no significant difference in resistant index (P = 0.350), 0.59 ±0.20 versus 0.54 ±0.15, respectively and in mean systolic to diastolic ratio between control and study groups P = 0.256), 2.99 ±2.59 versus 2.42 ±0.90, respectively (Table 2). (Yahia, Wafa, Moh, Alkhouly, & Reham, 2019) found (RI, PI and S/D ratio) was significantly lower in patients used sildenafil. (Malinova, Krasteva, & M, 2013) recommended that both PI and RI in patients managed with 25mg sildenafil citrate significant decrease. While (Moini et al., 2020) study found there were no differences in PI and RI between left and right uterine artery I in the three intervention groups (sildenafil, sildenafil+placebo and placebo group).

Embryological characteristics

Our study found no significant difference regarding the oocytes number, 8.57 ±5.31 versus 9.57 ±5.15, respectively (P = 0.642), metaphase II (MII) oocytes, 4.30 ±2.83 versus 5.47 ±2.99, respectively (P = 0.126), fertilization rate %, 74.36 ±23.03 versus 67.83 ±21.88, respectively (P = 0.256), in number of transferred embryos, 2.83 ±1.09 versus 2.87 ±0.97, respectively (P = 0.901), and number of grade I embryos between control and study group, 2.23 ±0.97 versus 2.33 ±1.06, respectively (P = 0.705), (Table 3). This agrees with results of (Ataalla, Elhamid, & Elhalwagy, 2016), they found no statistically significant difference between (Sildenafil group, Placebo group) in the total number of oocytes retrieved, of mature follicles, in the maturation and fertilization rate and a whole number of embryos obtained. The cause that the sildenafil does not improve ovarian parameters might be that several of these poor responding women have a diminished ovarian reserve and will not advance from improving ovarian blood supply. While (Yahia et al., 2019) found the women treated with sildenafil in ICSI cycle had higher total number of retrieved oocyte than the non-sildenafil group, these increase was statically significant. Sildenafil has vasodilator outcome augment the blood flow to the ovary and accordingly increase ovarian response to stimulation.

Serum level of Vascular Endothelial Growth Factor (VEGF) in study and control groups

Serum level of VEGF was significantly higher, (P < 0.001), in the study group, 126.48 (193.44) ng/L than in control group, 51.69 (52.96) ng/L, as shown in (Table 4). This finding in agreement with (Constantinescu, Bolfa, Constantinescu, Mironiuc, & Gherman, 2017) was found that expression of VEGFR-3 was highly significantly after sildenafil treatment compared to the control group. Another study described of sildenafil effect in patients with recurrent miscarriages; it has decreased natural killer cell activity as well as the facilitating effect on endometrial development. VEGF contributes to angiogenesis and improved blood vessels permeability during the mid-secretory phase, that is important for implantation (Jerzak, Kniotek, Mrozek, Górski, & Baranowski, 2008).

Serum level of Vascular Endothelial Growth Factor (VEGF) according to pregnancy outcome

The VEGF was highly significant in pregnant 277.76 (270.84) than non-pregnant women; 53.43 (57.77), (Table 5). The best VEGF cutoff value was > 123.453, with an accuracy level of 91.9 %, a sensitivity level of 87.5 % and a specificity level of 90.9 %, as shown in (Figure 2). (Sugino, Kashida, Karube-Harada, Takiguchi, & Kato, 2002) revealed that the appearance of VEGF and its receptor were higher during the mid‑secretory phase throughout regular menstrual cycles. Also, there was an elevated appearance of VEGF in decidual cells in early pregnancy. The authors concluded that VEGF enhances the successful implantation and preservation of pregnancy by improving vascular permeability or by building the vascular network in the decidua. (Gao, Zhao, Lin, Sun, & Zhang, 2012) were measured the concentrations of VEGF, endocrine gland derived vascular endothelial growth factor (EG-VEGF) and transforming growth factor-beta 1 (TGF-β1) in follicular fluid sample (FF) collected during oocyte retrieval (OR) and in serum sample collected two days after OR. There was a positive correlation between the serum concentration of the three growth factors and embryo quality. In contrast, only serum concentrations of EG-VEGF were positively associated with the pregnancy outcome. While (Asimakopoulos, Nikolettos, & Papachristou, 2005) compared serum VEGF levels in women entering the ICSI cycle according to the pregnancy outcome and found no significantly different between the pregnant and non-pregnant group.

The pregnancy rate in study and control groups

In the current study the pregnancy rate of the study group,11/30 (36.7 %) was higher than that of the control group, 5/30 (16.7) but, the difference is not significant, (Figure 1). This result in agreement (Moini et al., 2020) study showed no significant difference in the clinical pregnancy rate between study groups (sildenafil+placebo) and placebo group. In contrast to our study, Sher and Fisch (2002) study showed ongoing pregnancy rates were highly significant for sildenafil (45%) when compared with non-sildenafil groups.

Conclusion

Using vaginal sildenafil during fresh ICSI cycle improve endometrial receptivity by enhancing sub endometrial blood flow through reducing pulsatility index and increasing vascular endothelial growth factor.