Embryo implantation is a complex and delicate process, which requires high-quality embryos, well-tolerated endometrium and appropriate immune privilege, in order to achieve successful pregnancy. In our study, the implantation and clinical pregnancy in rhG-CSF group were significantly improved compared with control group, and no differences was observed between the two group regarding early abortion and multiple pregnancy rate. The study shows that rhG-CSF is beneficial to the pregnancy outcome of patients with RIF. which provides reliable clinical evidence for the use of rhG-CSF in patients with RIF and are basically consistent with the related research results abroad.
Comparison with previous studies
In recent years, pregnancy outcomes have been reported to improve in these trials in which G-CSF was used in patients with RIF. Fatemeh Davari-tanha and colleaguespublished a randomized double blind placebo control trial including 80 patients who allocated to three group(G-CSF, saline and placebo group) indicated that intrauterine G-CSF may increase chemical pregnancy and implantation rate in patients with recurrent implantation failure. This was in accordance with our results in implantation rate. And meanwhile, the study of Maryam Eftekharin women with repeated IVF failure involving 89 patients who allocated to two group(G-CSF and control group) that indicated higher implantation and clinical pregnancy in RIF who had received rhG-CSF compared with the control group. The conclusion is completely consistent with ours. However, in recent study by Ziya Kalem and colleaguesused intrauterine G-CSF administration before ART in the RIF group had an effect on clinical pregnancy and live birth rates. Although the results of this study were no statistically significant, the G-CSF group had better implantation and pregnancy rate than the control group. In addition, different routes of G-CSF administration have also been studied in IVF. In a study on IVF cycles with received subcutaneous G-CSF before implantation by Ashraf Aleyasin and colleagues, they concluded administration of single-dose systemic subcutaneous G-CSF increases the implantation and pregnancy rates in infertile women with repeated IVF failure. In another study by Soheila Arefi and colleagues, demonstrates the possibility that pregnancy outcome is better in women with repeated unexplained IVF who are subcutaneously treated with G-CSF but no significant between two groups. And in a randomized clinical trial study in 2019, A. Eapen and colleagues investigated 150 women with unexplained recurrent pregnancy loss, they reported that no significant increase in clinical pregnancy with the subcutaneous injection rhG-CSF. They have an equal conclusions compared the previous studies in implantation and pregnancy rates.
Different conclusions were reached in these studies. The differences in administration route, participants’ age, endometrial thickness, and sample sizes could have contributed to the observed differences in the results. In the study of Maryam Eftekhar, Aleyasin Aand our study, the sample size was larger, rhG-CSF was administered systemically through Intrauterine perfusion pathway, the study populations were younger, and had normal proliferating function. The difference is that saline group was set as the control in Maryam Eftekhar’s study, and the effect of mechanical stimulation on endometrium was avoided. Moreover, our Binary logistic regression analysis concluded that intrauterine perfusion rhG-CSF was an independent risk factor for pregnancy outcomes in RIF patients. The route of administration of G-CSF is also controversial. A meta-analyses published by Ling Zhang et al.concluded that both intrauterine G-CSF and systemic administration treatment had good clinical outcomes after embryo transfer in the patients with RIF. Another, a consistent conclusion has been reached by Ying Jiang et al., in which subcutaneous injection was better. Nevertheless, a retrospective study in women undergoing IVF/ICSI treatments by Hulusi Bulent Zeyneloglu showed that dual administration of G-CSF was significantly more effective than the only method. Combined with our study, the positive effect of G-CSF on pregnancy outcomes in patients with RIF women should be affirmed, but the route of administration needs further study. An article on the correlation between G-CSF dose and pregnancy outcome has no consensus yet.. Using a single dose of 300 ug before the implantation was positive for the pregnancy outcome, and 150ug as an intrauterine infusion on day of oocyte recovery did not show a benefit in improving pregnancy outcomes. In our findings, we demonstrate that the predictive effect of intrauterine perfusion on clinical pregnancy rate and it provides a statistical basis for clinical treatment. Live birth rate was no significant differences between the two groups. Could be that whether treatment with rhG-CSSF is no longer a necessary condition for for their continued pregnancy, but it can affect the embryo implantation through its different physiological function before the embryo successfully implantation.
The role of GCSF in implantation and pregnancy
Our results fully illustrates that G-CSF might play an important role in the implantation and pregnancy process. As we all know, G-CSF can improve endometrial receptivity via increasing endometrial thickness, in order to make the embryo implantation and clinical pregnancy rate go up. At present, there is no unified view of the specific mechanism of rhG-CSF in assisted reproduction. Salmassi et al. proposed that G-CSF level in serum and follicular fluid was a predictor of IVF outcomeand its participation in the mechanism of ovulation as described above is quite clear. The authors concluded that G-CSF could have an important function in
achieving positive pregnancy. In another, G-CSF is an integral part of the utero-placental cytokine network needed to establish and maintain pregnancy, at the maternal-foetal interface. Rahmat et al. indicated that the expression of G-CSF receptor was increased in endometrium, at the highest dose of rhG-CSF stimulation. What's more, G-CSF and its receptor have been widely expressed in germ cells, such as placenta, endometrial epithelial cells, stromal cells and decidualized tissues, they play their biological roles by binding to G-CSF receptors.
With the increasing understanding of G-CSF in reproduction, the majority of researches on the mechanism of G-CSF began to emerge. The study indicated G-CSF seems able to influence endometrial expressions in implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. And G-CSF was reported to have positive effects on oocyte maturation and embryonic development. Decidualization of endometrial stromal cells is one of the key factors for successful implantation. T.Tanaka and coworkers demonstrates that G-CSF enhances the decidualization process in ESCs. Also G-CSF contributes to the improvement of the implantation by adding G-CSF into the cultural medium. The present results suggest that G-CSF may play a significant role in follicular developmentaid to embryo implantation.
For many decades, mounting evidence from studies in mice and humans supports the idea that G-CSF have positive effect in embryo implantation and pregnancy development. However, the specific target of G-CSF on endometrial cells is not clear. Clinicians must consider the patient's condition comprehensively and choose the appropriate time and dose to make the patients with RIF obtain better pregnancy outcomes.
The limitation of our study is that it is a retrospective clinical analysis, and the control group is lack of strict matching(No saline infusion as control); And the number of patients aged between 20 and 29 in the study was very small, and no further hierarchical analysis was performed. Among the subjects we included ,2 cases were perfused 4 times and all obtained clinical pregnancy,.and there were 12 cases with 3 times of perfusion ,9 of which obtained clinical pregnancy. Although this study established that the number of perfusion times has a high degree of specificity in predicting the outcome of clinical pregnancy, the sample size of the intervention group was small, We need to be further confirmed by large sample, multi-center randomized controlled trials.