{"paper_id":"3723a424-6c79-4df8-827c-3a056e685b17","body_text":"Successful implantation of an embryo requires a receptive endometrium, a good quality embryo, and embryoendometrial synchronization in all species ( 1 ).\nEndometrial receptivity during the implantation window is often assessed by ultrasonography markers such as\nendometrial thickness, echogenic pattern, blood flow, and\nbiochemical markers ( 2 ). An appropriately thickened endometrium is a crucial factor for embryo implantation and\ncan predict pregnancy outcome with high sensitivity and\nspecificity ( 3 ). Moreover, several studies have reported a significant positive correlation between a triple-layered\nthickened endometrium of 7 mm (preferably >9 mm) and\npregnancy rate ( 3 - 5 ).\nDespite significant advances in ovarian stimulation protocols, treatment of repeated, unresponsive thin endometrium is still a challenge in assisted reproductive cycles,\nwhich usually results in cycle cancellation or repeated implantation failures ( 6 ).\nThere is a growing body of evidence that the endometrial growth has a relationship with the state of uterine\nblood flow ( 2 ). Treatment options, such as low-dose aspirin, oestradiol administration and gonadotropin therapy,\nlow dose human chorionic gonadotropin (hCG), vitamin\nE, pentoxifylline, L-arginine, luteal phase support with\ngonadotropin-releasing hormone (GnRH) agonist, intrauterine granulocyte colony-stimulating factor (G-CSF),\nvaginal sildenafil, and recent application of stem cell\ntherapy are suggested for management of a thin lining endometrium ( 7 ).\nSildenafil citrate (Viagra®, Pfizer, NY, USA) is a 5-phosphodiestrase inhibitor that increases smooth muscle relaxation and vasodilation by preventing cGMP breakdown\n( 8 ). Sildenafil citrate potentiates uterine blood flow and,\nin conjunction with oestrogen, it leads to the oestrogeninduced proliferation of the endometrium ( 9 ).\nIt has been reported that vaginal sildenafil significantly\nreduced peripheral natural killer cell (NK-cell) activity\nand improved successful pregnancy rates in women with\nhistories of recurrent miscarriages. Although the mechanism of influence by sildenafil on natural killer cell activity is unclear, it seems that enhancement of uterine artery\nflow has an effective influence on the local endometrial\nNK-cell population ( 10 ). However, adverse effects that\ninclude myocardial infarctions and strokes have been associated with this drug in the normal healthy population\n( 11 ,  12 ).\nThis comparative pilot study evaluated the effect of\nvaginal sildenafil suppositories on endometrial proliferation and IVF outcome in infertile patients with a history\nof repeated IVF failure.\n\nThis phase II randomized, double-blind, placebo-controlled trial was performed at Royan Institute, Reproductive\nBiomedicine Research Center, Tehran, Iran, between February 14, 2014 and November 14, 2016 ( NCT03192709 ).\nThe assessors and patients were not aware of the treatment\nallocated. The Institutional Review Board and Ethics Committee of Royan Institute, Tehran, Iran reviewed and approved this study in compliance with the Declaration of\nHelsinki (EC/88/1045). Informed consent was obtained\nfrom all patients prior to their participation in the study.\nThe study population consisted of 66 infertile women\naged ≤38 years. The inclusion criteria was met when the\nwomen had normal ovarian reserve [blood anti-mullerian\nhormone (AMH) levels >1.5 ng/mL] with at least two\nprior cycles with follicle stimulating hormone (FSH) <10\nmIU/ml; a history of two prior consecutive failed IVF/\nICSI attempts with at least a transfer of two good quality fresh or frozen-thawed embryos; hCG day endometrial thickness <7 mm in all prior IVF/ICSI attempts; and\nnormal endometrial appearance according to either hysterosonography, hysterosalpingography, or hysteroscopy.\nWomen were excluded if they had a history of myomectomy or Asherman’s syndrome.\nA preliminary colour Doppler transvaginal sonography\nwith a 4-8 MHz probe (ProSound Alpha 10; Aloka, Japan) was performed by an expert radiologist on day 14\nof the patient’s prior menstrual cycle to investigate the\nuterine and adenexes for any abnormal findings. The endometrial parameters of endometrial thickness, endometrial pattern, pulsatility index (PI), and resistance index\n(RI) were measured. The uterine artery PI and RI were\nobtained through flow velocity waveforms from the ascending branch of the uterine artery at the point near to\nthe internal cervical orifice and calculated as previously\ndescribed ( 13 ). In the subsequent cycle, ovarian stimulation was performed with the long protocol using a GnRH\nagonist ( 14 ).\nIn the IVF treatment cycle, the patients were randomly\nassigned to three groups according to a random allocation sequence generated by a randomized block design.\nThe size of each block was 3. In group A, the sildenafil\n(vaginal suppositories, 100 mg/day, Parnian Daroo, Co.,\nTehran, Iran) were administered from the first day of the\nFSH injection until the day of oocyte retrieval. In group\nB, placebo (vaginal suppositories, Parnian Daroo, Co.,\nTehran, Iran) was initiated from the first day of the HMG\ninjection until 2 days before the hCG injection, after\nwhich sildenafil vaginal suppositories were initiated and\ncontinued until the day of oocyte retrieval. In group C, the\nplacebo was given from the first day of HMG injection\nuntil the day of oocyte retrieval. Participants received 10\n000 IU of hCG (Choriomon, IBSA, Switzerland) when\nat least two dominant follicles were 18 mm in diameter.\nEndometrial thickness, pattern, PI and RI were measured\non the day of hCG administration and compared with the\ndata obtained in the previous cycle without sildenafil citrate treatment. Oocytes were retrieved 36 hours later via\ntransvaginal ultrasound-guided needle aspiration.\nEmbryo transfer was performed after 48 hours of oocyte retrieval. Progesterone in oil (100 mg, IM daily) or\nintravaginal progesterone (400 mg, twice daily) was used\nfor luteal support and maintained until the pregnancy test\nwas conducted. Serum hCG levels were measured on the\n14 th  day following oocyte retrieval. Vaginal ultrasound\nconfirmation of pregnancy was performed at 4-6 weeks\nafter embryo transfer.\nA chemical pregnancy was determined by a positive\nß-hCG test result. A clinical pregnancy was confirmed\nby ultrasonography visualization of one or more gestational sacs or definitive clinical signs of pregnancy.\nThe spontaneous abortion was defined as a pregnancy loss of an intrauterine pregnancy before 22 weeks’ gestation ( 15 ).\nSuppositories containing 100 mg of sildenafil were\nprepared from the oral tablets by a local pharmacy (Parnian Daroo, Iran). We defined the endometrial thickness\nthreshold cut-off of <7 mm as a thin endometrium based\non other studies ( 8 ).\nData are expressed as mean ± standard error (SE)\nand proportion. Continuous and categorical outcome\nvariables were compared between three intervention\ngroups by one-way analysis of variance (ANOVA)\nand the chi-square test. All statistical analyses were\nperformed using SPSS version 22 for Windows 7\n(IBM Analytics, Armonk, NY). The significance level\nwas set at 0.05.\n\nDuring the recruitment process, we enrolled 66 patients and allocated 22 patients to each study group. A\ntotal of 10 patients were lost to follow up for measuring clinical pregnancy for the following reasons: all\nof the embryos of their treatment cycle were cryopreserved (n=3), they had no oocytes at retrieval (n=2),\nor no embryos to transfer (n=5). The flow diagram explicitly shows the number of participants at the beginning, intervention allocation, follow-up, and analysis\n( Fig .1 ).\nTable 1 shows the baseline characteristics of the three\nstudy groups. There were no significant differences\namong the study groups. The majority of patients had\nnormal endometrial patterns, which were similar between the three study groups ( Table 1 ). Uterine artery\nPI and RI in the left and right were not statistically significant among the study groups (P>0.05). Table 1 shows\nthe results of the ovulation stimulation cycle and include\ninitial endometrial thickness, number and type of ampoules used, time interval for ovulation stimulation, and\nnumbers of total and metaphase II (MII) oocytes. These\nparameters did not differ significantly among the three\nintervention groups.  Table 2  shows numbers of embryos\ntransferred in total and by grade (A, B and C). These values were not different among the groups. The generated\nembryos were graded as good (A and B) or poor (C) according to their morphological features, cleavage stage,\nmulti-nucleation, equal size blastomeres, and fragmentation rate ( 16 ).\nOf note, the embryo transfer days were similar between\nthe three groups. All of the embryos were transferred either two or three days after ovum pickup.\nThe endometrial thickness and patterns after the interventions were not statistically different between the\nstudy groups. Additionally, the three intervention groups\nwere not different in left and right uterine artery PI and\nRI. Implantation rate was not statistically significant\nover the three groups (P=0.290). Clinical pregnancy\nrates were 33.3 (sildenafil), 33.3 (sildenafil+placebo),\nand 17.6 (placebo) as seen in Table 2. Although the\nclinical pregnancy rates varied among the intervention\ngroups, they were not statistically different ( Table 3 ).\nVaginal administration of sildenafil had no adverse effects on the patients during the study.\nFlowchart of participants in this randomized controlled trials. IVF/ICSI;  In vitro  fertilization/intracytoplasmic sperm injection and ET; Embryo transfer\nComparison of baseline characteristics and cycle related factors between the sildenafil, sildenafil + placebo, and placebo groups prior to intervention\nBMI; Body mass index, PI; Pulsatility index, FSH; Follicle stimulating hormone, LH; Luteinizing hormone, RI; Resistance index, and MII; Mature metaphase II.\nComparison of treatment cycle outcomes between the sildenafil, sildenafil+placebo, and placebo groups after intervention\nPI; Pulsatility index, RI; Resistance index, and ET; Embryo transfer.\nComparison of reproductive outcomes between the sildenafil, sildenafil+placebo, and placebo groups after intervention\nData are presented as n (%).\n\nThe importance of the endometrial pattern as a predictor\nof treatment cycle outcome in IVF-treated patients is\nwell-documented ( 17 ). In addition, endometrial quality is\nalso an important factor for the successful implantation\nof the foetus ( 18 ). Endometrial thickness increases with\nhigh pregnancy rates; however, pregnancy rates are not\npredictable only based on endometrial thickness ( 19 ).\nUterine arterial blood flow seems to affect endometrial\ngrowth and the outcome of pregnancy. Studies have shown\nthat sildenafil widens the vasculature by its effects on the\nsmooth muscles of the arteries ( 10 ,  20 ). Sildenafil citrate\nis an inhibitor-5-phosphodiestrase type that, by preventing\nthe effect of cGMP, exacerbates the effect of NO on the\nsmooth muscle of the arteries ( 20 ,  21 ). According to the\nresults of some studies, the use of sildenafil during the\nproliferative phase of the cycle improves uterine blood\nflow and endometrial growth, and results in a higher\nlevel of implantation and pregnancy in patients with\nrepeated IVF failure and Ashermen syndrome ( 8 ,  22 ).\nAlso the administration of sildenafil (50 mg intravenous)\nin a sterilized animal model (sheep) has been shown to\nexacerbate uterine flow ( 23 ). Several studies have shown\na correlation between ‘‘thin endometrium’’ and low\nimplantation rates ( 9 ,  24 ). The results of current study\nshowed increased endometrial thickness in the three\ngroups on the day of hCG injection, but the increase\nwas not statistically significant. The results of previous\nstudies have shown that sildenafil citrate (vaginal or\noral alone or with oestradiol) is significantly effective in\nimproving endometrial thickness ( 25 - 27 ). Sher and Fisch\n( 22 ) reported that the use of sildenafil vaginal suppository\ncould reduce the adverse effects of headache and low\nblood pressure compared with oral sildenafil. In another\nstudy, they reported that vaginal sildenafil (25 mg, 4 times\nper day) improved endometrial thickness (≥9 mm) in\n70% of the patients ( 8 ). In a prospective study, Takasaki\net al. ( 25 ) compared the effects of vaginal vitamin E,\nL-arginine, and vaginal sildenafil citrate on endometrial\nthickness in patients with endometrial thickness less than\n8 mm and right arterial resistance in their radial vessels\n(RA-RI ≥0.81). The results showed that vitamin E,\nL-arginine, and sildenafil citrate significantly improved\nRA-RI and endometrial thickness in these patients;\nhowever, the improvement in endometrial thickness in\npatients treated with sildenafil citrate was more than the\nother two groups. Several studies ( 26 - 28 ) showed that\nvaginal sildenafil significantly improved endometrial\nthickness in patients with history of poor endometrial\nthickness in the previous cycles. Jerzak et al. ( 10 ) reported\nthat endometrial thickness significantly increased after\nadministration of oral sildenafil (25 mg, 4 times per day) in\nwomen with a history of abortion. Dehghani Firouzabadi\net al. ( 29 ) recommended oral sildenafil administration\nas an appropriate solution for improving endometrial\nadmission in patients with unsuccessful cycles from\nlow endometrial thickness. Their results showed that the\ntriple line endometrial pattern in the sildenafil citrate +\noestradiol group was significantly higher than in the\noestradiol-only group, while the intermediate pattern of\nthe endometrium was not significantly different between\nthe two groups. Fetih et al. ( 30 ) reported that sildenafil\nvaginal gel significantly increased endometrial thickness\nand uterine blood flow, and might improve pregnancy rate\nin patients with clomiphene citrate (CC) failure due to\nthin endometrium. The results reported by Chanona et al.\n( 31 ) showed that the use of vaginal sildenafil in patients\nwhose endometrial thickness was equal to or less than 7\nmm in the failed assisted reproductive technology (ART)\ncycles led to an increase in implantation and pregnancy.\nZinger et al. ( 32 ) reported that two patients with a history\nof curettage and secondary infertility were treated with\nsildenafil after removing adhesions by surgery (due to thin\nendometrial thickness in previous IVF cycles), and both\npatients became pregnant during the first cycle of sildenafil\nadministration. Increases in their endometrial thicknesses\nwas also shown in transvaginal ultrasonography.\nSeveral treatment modalities have been offered to\npatients with ‘‘thin’’ endometrium, including hormonal\nmanipulation by oestrogen and gonadotropin therapy,\nlow-dose hCG, tamoxifen, L-arginine or sildenafil,\nvitamin E, pentoxifylline, low-dose aspirin, hysteroscopic\nadhesiolysis, intrauterine infusion of growth factor such\nas G-CSF, and the recent application of regenerative\nmedicine. Despite the large variety of treatment,\nmost options lead to only minor modifications in the\nendometrium thickness and subsequent pregnancy,\nand when this modality fails, patients are eventually\ncandidates for surrogacy. Treatment of thin endometrium\nremains a challenge and future investigations are required\nto further clarify and ideally manage patients with thin\nendometrium ( 7 ).\nIn the present study, the clinical and chemical\npregnancy rates in the two groups of women who were\ntaking sildenafil alone and the women who took placebo and sildenafil showed a twofold increase compared to\nthe placebo-only-treated women, which according to the\nsample size, this increase is not significant. The results\nreported by Dehghani Firouzabadi et al. ( 29 ) in a study of\n80 patients showed that the chemical pregnancy rate was\nhigher but not statistically significant in the patients who\nused sildenafil; this finding was consistent with the results\nof our study. However, AbdelKader Fahmy et al. ( 33 ),\nwith a sample size of 70 patients, reported a significantly\ngreater chemical pregnancy rate in the sildenafil group.\nIn this study, the clinical pregnancy rate was higher in\nthe sildenafil and sildenafil+placebo groups than in the\nplacebo group. Although this increase was not statistically\nsignificant, it was clinically shown to be twofold. These\nresults were consistent with the findings of AbdelKader\nFahmy et al. ( 33 ) and Kim et al. ( 28 ). The study by\nAbdelKader Fahmy et al. ( 33 ) reported a 2.5-fold increase\nin pregnancy in the sildenafil group, but this difference\nwas not statistically significant. Kim et al. ( 28 ) reported\nthat the use of vaginal sildenafil plus oral oestrogen pills\nin the luteal phase of patients treated with an IVF-ET cycle\nincreased the pregnancy rate by two-fold, but this increase\nwas not statistically significant. Mangal and Mehirishi\n( 34 ) showed that the rate of pregnancy in the patients who\nused vaginal sildenafil in three successive intra uterine\ninsemination (IUI) cycles was significantly higher in the\nthird cycle than in the group who used oestradiol valerate,\nwhile this difference was not significant in the first and\nsecond cycles. The findings from a retrospective study by\nMargreiter et al. ( 35 ) showed a significant improvement\nin the rate of implantation, endometrial thickness and\npregnancy, and decreased abortion in the group of patients\ntreated with vaginal sildenafil.\nThe endometrial receptivity is an important stage\nin the ART cycles, the results of our study showed\na higher rate of implantation in the women who took\nsildenafil+placebo than in the other two groups, which\nwas consistent with the results of Dehghani Firouzabadi\net al. ( 29 ).\nIn the present study, 2 out of 7 cases of pregnancy\nended in abortion in the sildenafil group, 1 out of 6 cases\nof pregnancy in the sildenafil+placebo group, and 0 of\n3 cases of pregnancy in the placebo group. The rate of\nabortion was negligible in the sildenafil group. Follow-up\nevaluations showed a molar pregnancy in the sildenafil\ngroup. Dzieciol et al. ( 36 ) reported that sildenafil citrate\nincreased uterine tissue perforation during the uterus\npreparation for fertility.\nIn our study, higher numbers of MII oocytes were\nretrieved in the sildenafil groups compared to the\nplacebo group, which was clinically important; however,\none-way ANOVA results showed that this finding was\nnot statistically significant. To our knowledge, there\nis only one study (abstract available) by Vidal et al.\n( 37 ) that provided evidence of the benefit of sildenafil\nsupplementation in the first days of hyperstimulation\ninduction in terms of the numbers of mature and fertilized\noocytes. Therefore, more research on this topic needs to\nbe undertaken.\n\nVaginal sildenafil might conceivably improve chemical\nand clinical pregnancy rates in repeated IVF failure\npatients. Since this was a pilot study, we recommend that\nclinical trials should be conducted with vaginal or oral\nsildenafil on larger numbers of these patients.","source_license":"CC-BY-4.0","license_restricted":false}