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Subcutaneous Administration of Granulocyte- Colony Stimulating Factor Versus Local Infusion on IUI Outcomes in Women with Unexplained Infertility

Infertility is defined by the failure to achieve pregnancy after
12 months or more of regular intercourse without contraception
methods...

and NK cells [10]. G-CSF and its receptors are also expressed in reproductive tract, especially in follicular cells and at the maternalfetal interface during the time of implantation and early pregnancy [11]. This cytokine and other members of the CSF family are involved in preparing a local immune environment for a successful implantation. Further, G-CSF is associated with dendritic cell maturation, blastomere viability, and Th-2 cytokine secretion.
Finally, G-CSF plays an important role in tolerance maintenance at the utero-placental interface through regulatory T cell activation [11,10].
Based on the evidence that G-CSF contributed to implantation, several authors demonstrated that administration of G-CSF before implantation increased the IVF success, implantation, and pregnancy rate in women with unexplained recurrent miscarriage or implantation failure [7,9,12]. while, other studies did not find an improvement in implantation rate, or clinical pregnancy rate after ART with G-CSF administration [13,14]. However, large, randomized, controlled trials will help to overcome the discrepancies of G-CSF efficacy in infertile patients. The administration of G-CSF in patients with unexplained infertility undergoing IUI is less common and less extensively documented in literature. In addition, systemic administration of G-CSF is thought to be more effective than intrauterine infusion [13]. We conducted this investigation on patients with unexplained infertility to examine the efficacy of subcutaneous G-CSF administration versus local infusion on IUI success, biochemical, and clinical pregnancy rates.

Result
The Consolidated Standards of Reporting Trials (CONSORT) diagram of the participants was shown in Figure 1. In total, 200 women with unexplained infertility were evaluated for participation in this study. Of these, 61 patients were excluded because of non-eligibility for entering the study. The eligible women (139 patients) were randomly assigned to three groups including: subcutaneously G-CSF administration group (A, n=47), intrauterine G-CSF infusion (B, n=47), and control group (C, n=45). Two women from B group refused to receive allocated intervention due to personal reasons and two women in A group did not inform the pregnancy tests. Finally, the cycle outcomes of 135 patients in three groups were compared. The demographic characteristics are presented in Table 1. There were no significant differences among study groups regarding age, BMI, FSH, TSH, PRL, and number of follicles (p>0.05). The biochemical pregnancy and clinical pregnancy were significantly different among group (p=0.048, p= 0.044, respectively) ( Table 2).
In addition, Table 3 compares the pregnancy outcome of the two groups to whom G-CSF was administered as local infusion or subcutaneous injection. The chemical pregnancy rate was apparently higher in group with local infusion than subcutaneous group (24.4% vs 20%, p=0.460). The clinical pregnancy rate was also higher in the group with local infusion than subcutaneous group (24.4% vs 15.55%, p=0.322) but these differences were not statistically significant(p>0.05).

Discussion
Unexplained infertility refers to failure to achieve a pregnancy without an obvious cause [4]. Although the exact etiology underlying unexplained infertility is still unclear but inadequate endometrial receptivity is thought to be related to the etiology of unexplained infertility [15]. Therefore, identifying key factors that are involved in endometrial remodeling and implantation process could provide a promising tool in treatment of women with unexplained infertility or implantation failure. These observations that serum level of G-CSF continuously increases from the day of embryo transfer to the day of implantation and from the day of confirmation of pregnancy to gestation [16], and infertile women with implantation failure express the lower level of G-CSF receptors at the maternal-fetal interface [17] attracted the attentions toward this glycoprotein with growth factor and cytokine functions.
Evidence have illustrated that G-CSF is involved in the following events during implantation and pregnancy which include. G-CSF has also been found to promote maternal tolerance to the semi-allogenic embryo through shifting the inflammatory balance towards an anti-inflammatory Th2 response, T regulatory cells enhancement, and tolerogenic dendritic cell differentiation [18]. Due to the safety of G-CSF application in pregnancy [19] and no reported major side effects, Gleicher used the G-CSF for the first time in treatment of four unresponsive women with thin endometrium, and a pregnancy was achieved in all four patients [20]. since that, several clinical trials have conducted to evaluate the effect of G-CSF on IVF outcomes [9,12,7]. Some investigators have demonstrated the effectiveness of G-CSF administration in improving the reproductive outcomes in IVF / ICSI / FET cycles [9,21,22], even though, others found no treatment benefit of G-CSF in assisted reproductive technology (ART) [13,14,23]. To give a better answer to this topic under debate, a recent meta-analysis study in women with unexplained, repeated implantation failure (RIF) revealed the favorable effect of G-CSF on the implantation rate and clinical pregnancy rate, especially when administrated by subcutaneous injection [24]. However, other published systematic review that included 15 trials containing 1253 sub fertile women undergoing assisted reproduction declared the administration of G-CSF is effective only in women with two or more previous IVF failure history [25], and its effectiveness in other sub fertile women is unclear.
At present, scientific data regarding the application of G-CSF and the best route of its administration for infertile women trying to get pregnant through IUI is very limited. In present study, we found that G-CSF improves the biochemical and clinically pregnancy rates in patients with unexplained infertility after IUI compared to control. Our findings are in consistence with those from the only previous RCT in IUI cycle [26]  Additionally, we observed the local infusion of G-CSF seems to be more effective than subcutaneous administration, however our data was not statistically significant. In contrast, a most recently meta-analysis by Jiang et al. has concluded that both intrauterine and subcutaneous injection of G-CSF can improve implantation and clinical pregnancy rate, but the subcutaneous injection appears to be superior to intrauterine instillation for unexplained RIF patients.
We cannot accurately estimate the reasons behind our observation regarding the route of administration, but at least, we speculate intrauterine route can provide a desire environment around the implantation site by creating a higher local concentration.
However, it is important to notice that there is a lack of data on the straight comparison between different routes of administration in patients with normal endometrium undergoing IUI and this issue can be considered as a strength of current study. Further, the main limitations of our study were absence of blinding due to the nature of the study. Moreover, no placebo was involved in the research, therefore, we cannot omit the endometrial injury effect that could potentially influence the results of the study. In addition, we did not evaluate the live birth rate and miscarriage in all groups that could help to interpret the results with more validity and establishing the safety of G-CSF administration.

Conclusion
Our results provided further support for the beneficial effect of G-CSF on pregnancy outcomes in women with unexplained infertility undergoing IUI, especially when administrated intrauterine. However, no evidence favoring a specific route of G-CSF administration in women with unexplained fertility and normal endometrial thickness was found, and further trial studies with larger sample size are needed to elucidate this issue.