The Efficiency of Introducing Intrauterine Infusion of Autologous Platelet-Rich Plasma versus Granulocyte Colony-Stimulating Factor in Repeated Implantation Failure Patients: An Unblinded Randomised Clinical Trial.

Repeated embryo implantation failure (RIF) is a highly challenging problem in the field of infertility that causes considerable distress for both patients and doctors. RIF pertains to the recurring failure of an embryo to successfully implant after transfer. Typically, RIF is defined as two or three unsuccessful attempts of in vitro fertilisation (IVF)-embryo transfer (ET) in which one or two high-quality embryos are transferred ( 1 ). Currently, the treatment options available for patients with RIF include various interventions that target either the uterus (e.g., hysteroscopy and endometrial scratch) or embryonic transfer (e.g., assisted hatching, sequential ET, and blastocyst transfer). Additionally, immunomodulation techniques may be employed that use intravenous immunoglobulin, subcutaneous granulocyte colonystimulating factor (G-CSF), or intrauterine infusions of G-CSF, peripheral blood mononuclear cell, autologous platelet-rich plasma (PRP) and injecting human chorionic gonadotropin directly into the uterus ( 2 - 10 ). Recently, there is a significant interest in using G-CSF to treat patients with RIF ( 8 , 11 , 12 ). G-CSF, which is present in various tissues, is produced by different types of cells such as fibroblasts and immune cells. Particularly, in the uterus, it is produced by natural killer cells. G-CSF has important roles in maturation of sperm and eggs, as well as in endometrial receptivity and implantation. Its effects may also extend to foetal development ( 13 ). It is also widely used in regenerative medicine ( 14 ). At the same time, PRP injections are a new therapeutic approach that show promising results in RIF patients. It offers a noninvasive treatment option with comparable safety. The positive effects of PRP in these patients are believed to be related to its ability to facilitate embryonal implantation by promoting endometrial development ( 15 ). The lack of agreement on whether PRP therapy or systemic/intrauterine G-CSF therapy is better for patients with RIF has led to differing opinions on which treatment produces better pregnancy outcomes ( 16 - 18 ). The objective of this study is to compare the effect of intrauterine G-CSF and PRP on the rate of ongoing pregnancy in RIF patients.

As indicated in Table 1, there were no significant differences in demographic characteristics and baseline reproductive hormone levels between the two groups. Similarly, no significant differences were found between the groups in terms of the dosage of gonadotropin, endometrial thickness, quality of embryos, total and number of retrieved eggs, mature eggs, total embryos, and number of transferred embryos, as well as the fertilisation rate, which is presented in Table 2. Baseline characteristics of the participants Data are presented as mean ± standard deviation (SD). All data were analysed by the student’s t test. PRP; Platelet-rich plasma, G-CSF; Granulocyte colony-stimulating factor, BMI; Body mass index, LH; Luteinising hormone, FSH; Follicle stimulating hormone, and AMH; Anti-Müllerian hormone. Table 3 shows the occurrence rates of different outcomes. The implantation rate was significantly higher in patients who received PRP (P=0.014). Chemical pregnancy occurred in 36 patients (36.7%) in the PRP group, which was significantly higher than the 16 patients (17.4%) in the G-CSF group (P=0.003). Moreover, both clinical pregnancy and ongoing pregnancy rates were significantly higher in the PRP group compared to the G-CSF group (P=0.001 and P=0.020 respectively). Comparison of stimulation characteristics between groups Data are presented as mean ± standard deviation (SD). All data were analysed by the student’s t test. PRP; Platelet-rich plasma and G-CSF; Granulocyte colony-stimulating factor. Comparison of pregnancy rates between the groups Data are presented as mean ± SD or n (%). G-CSF; Granulocyte colony-stimulating factor and PRP; Platelet-rich plasma.

The use of intrauterine PRP is a beneficial treatment option for RIF patients. In this study, the intrauterine infusion of PRP 48 hours before ET led to a significant increase in pregnancy rate in women with RIF. Even though the use of intrauterine PRP for RIF patients has shown potential, it remains a topic of controversy ( 20 - 24 ). Some studies have demonstrated positive outcomes, such as increased endometrial thickness and receptivity, as well as improved implantation and pregnancy rates ( 25 - 28 ). However, there are other reports that have not found significant benefits in using PRP as an IVF adjuvant for RIF treatment, especially in women with normal endometrial thickness ( 29 , 30 ). It is believed that PRP, which contains various growth factors, may have beneficial effects on endometrial health, as decreased growth factor expression has been implicated in RIF ( 31 - 34 ).The discrepancy observed in various studies regarding the effectiveness of using PRP for patients with RIF may be attributed to inconsistent inclusion of participants, differing definitions of RIF, or variations in the protocols for producing PRP. G-CSF infusion has shown encouraging results in RIF patients. Aleyasin et al. ( 35 ) demonstrated that the administration of a single-dose subcutaneous G-CSF injection prior to ET can raise the IVF success as well as implantation and pregnancy rates in patients with repeated IVF failure. Additionally, Zeyneloglu et al. ( 36 , 37 ) observed that G-CSF may be an effective and safe therapeutic option for RIF patients, especially if administered through both intrauterine and subcutaneous routes. G-CSF is a specific cytokine involved in the production of blood cells. It is released by various cell types including monocytes, macrophages, fibroblasts, endothelial cells, and stromal cells. Its main function occurs in the bone marrow where it promotes the production and maturation of neutrophils. In addition to its effects on blood cells, G-CSF receptors have also been found in certain reproductive system cells like trophoblasts, granulosa-luteinised cells, and endothelial cells ( 38 ). Research has shown that administering G-CSF via subcutaneous or intrauterine routes during FET and fresh ET cycles improves pregnancy rates in patients with RIF ( 39 , 40 ). However, it may not provide any benefits for patients with a normal endometrium ( 12 ). To determine the best treatment option for patients with repeated IVF failures, the decision remains difficult despite the promising outcomes seen with both PRP and G-CSF. In a retrospective observational study, information was gathered on a group of 225 patients who had an endometrial thickness greater than 7 mm. These patients were then divided into three categories: intrauterine infusion of G-CSF, intrauterine infusion of PRP, and no intervention (control group). The study found that the intrauterine infusion of G-CSF in women undergoing fresh ET with normal endometrial thickness resulted in an increase in endometrial thickness and implantation rate. However, it had no effect on other endometrial parameters or the cumulative pregnancy rate ( 18 ). Another study compared the effectiveness of intrauterine lympho-PRP and systemic G-CSF on the outcomes of ICSI in patients with repeated IVF failures. The findings revealed that patients who received PRP had significantly higher rates of clinical pregnancy ( 16 ). We discovered that intrauterine infusion of PRP directly into the uterus resulted in significantly higher rates of implantation, chemical pregnancy, clinical pregnancy and ongoing pregnancy rates for patients with RIF compared to infusions of G-CSF into the uterus. The chance of ongoing pregnancy in the PRP group was 2.6 times higher than G-CSF. However, the exact reason for the higher pregnancy rate in PRP infusion compared to G-CSF has not yet been fully determined. Nonetheless, research has shown that PRP may enhance tissue growth and repair and induce further improvement in the uterine lining, potentially leading to a higher pregnancy rate in these patients. Therefore, the true effect of each of these treatments is unclear and requires larger clinical trials. The primary drawback of this study was a failure to measure the thickness of the endometrium on the day of ET. Consequently, we were unable to determine which patients would benefit the most from this treatment and whether the effects of PRP on pregnancy rates are solely due to endometrial expansion.

We demonstrated that intrauterine infusion of PRP is significantly more effective than G-CSF in improving pregnancy outcomes in patients with RIF. As a result, it can be considered as a viable treatment option for these patients. However, further research that includes costeffectiveness studies and randomised controlled trials with larger groups of participants is necessary to support its regular use in clinical practice.

The study included women,<41 years of age, who had experienced more than two unsuccessful attempts at ET, despite having high-quality embryos. Patients who were unable to receive G-CSF and PRP, had allergies to G-CSF, a poor ovarian response based on Bologna criteria ( 19 ), previous preimplantation genetic diagnosis/ screening cycles, evidence of genetic disorders, previous use of ovum donors or surrogacy, diagnosis of severe endometriosis, severe male factor infertility, or did not undergo embryo cycles were excluded from the study. The study was conducted at the IVF Centre of Mehr Medical Institute in Rasht, Iran, from 2020 to 2022. The Research Ethics Committee of Guilan University of Medical Sciences, Rasht, Iran approved this study (IR. GUMS.REC.1400.017) and it is registered at the Iranian Registry of Clinical Trials (IRCT20180528039878N3). Written informed consent was obtained from all patients. The study included 200 patients with recurrent implantation failure who underwent ICSI-frozen ET (FET) cycles. These patients were randomised into two groups: intrauterine infusion of 1 ml of G-CSF (n=100) and intrauterine infusion of 1 ml autologous PRP (n=100) ( Fig .1 ). This was an unblinded study because of the specific traits of the intervention. CONSORT flow diagram. The findings from our previous study ( 16 ) showed an 18.9% difference in clinical pregnancy rates between G-CSF and PRP therapy; therefore, we calculated a sample size for each group of 90 with a 95% confidence interval. The final sample size for each group was determined to be 100 after taking into account a potential 10% dropout rate. Sealed Envelope® software (London, UK) was used for block randomisation. The researchers created six blocks, each consisting of four members, with different patterns (AABB, ABAB, ABBA, BBAA, BABA, BAAB) and numbered them from 1 to 6. To allocate patients, random numbers between one and six were generated, which determined the sequence in which the blocks were randomised. Each patient underwent an FET cycle. All participants received high-quality embryos, which were transferred under ultrasound guidance by an experienced gynaecologist. To prepare the uterus, the patient was given hormone replacement therapy that consisted of oestradiol valerate (Aburaihan Pharmaceutical Co., Iran) at a dose of 4-6 mg per day starting from day two or three of their menstrual cycle. The dose was increased to 6-8 mg per day if the endometrial thickness did not reach at least 7 mm. Once the thickness exceeded 7 mm, the patients received progesterone suppositories (Cyclogest ®, Actavis, UK) at a dose of 400 mg twice daily. Hormone replacement therapy was continued until the 12th week of pregnancy. G-CSF was administered on the first day of progesterone treatment. A volume of 1 ml of G-CSF (300 µg, Filgrastim 30 mIU/mL, DEM Medical, Dong-A, South Korea) was slowly infused into the uterine cavity using an intrauterine insemination (IUI) catheter (Salamatyar Hakim, Iran). Autologous PRP was created in two steps according to the manufacturer’s instructions (Rooyagen, Iran). In the first step, 8.5 ml of blood was taken from a peripheral vein using a syringe that contained 1.5 ml of Anticoagulant Acid Citrate Dextrose solution (ACD-A). The syringe was immediately centrifuged at 1200 rpm for 10 minutes to separate the red blood cells. Then, the remaining liquid was centrifuged again at 3300 rpm for 5 minutes to concentrate the PRP to approximately 4-5 times the normal platelet concentration. Finally, the PRP (1 ml) was infused into the endometrial cavity 48 hours before ET using an IUI catheter under ultrasound guidance. The main results of our study were clinical pregnancy and ongoing pregnancy rates. Clinical pregnancy was defined as the presence of a gestational sac with a beating heart observed by ultrasound. Ongoing pregnancy referred to the continuation of an intrauterine pregnancy for at least 12 weeks. Additionally, we also evaluated the secondary outcome of measuring positive pregnancy tests by biochemical methods. The data were analysed using Windows SPSS ver. 21 (SPSS Inc., Chicago, IL, US). The descriptive results were presented as mean ± standard deviation (SD) for numerical variables and percentages for categorical variables. The data were checked for normality using the Kolmogorov-Smirnov test, and then appropriate parametric or nonparametric statistical tests were used. Chi-square and Fisher exact tests were used to analyse the relationship between categorical variables. Per protocol analysis was conducted in this study. P<0.05 were considered statistically significant.