Recurrent implantation failure (RIF) after embryo transfer remains an unresolved issue in reproductive medicine. The diagnosis of "RIF" is made, as a rule, after at least three to four transfers of good-quality embryos (Coughlan et al., 2014, Nardo et al., 2015, Bashiri et al., 2018, Shaulov et al., 2020, Mascarenhas et al., 2022, Wang et al., 2022), and according to ESHRE guideline (2023) even after the second transfer of euploid embryos (after preimplantation genetic testing for aneuploidy - PGT-A) (ESHRE Working Group on Recurrent Implantation Failure et al., 2023).

Many methods are used in clinical practice to increase endometrial receptivity, but there is a lack of evidence regarding their effectiveness (Bashiri et al., 2018, Cavalcante et al., 2020, Sfakianoudis et al., 2021, Stope et al., 2023, Kuroda, 2024). Systemic (subcutaneous or local - into the uterine cavity) administration of granulocyte colony-stimulating factor (G-CSF) is considered a promising means to improve implantation (Rutella et al., 2002, Cavalcante et al., 2015, Zeyneloglu et al., 2020, Hou et al., 2021, Kong et al., 2023, Su et al., 2024, Xie et al., 2024). G-CSF belongs to the family of colony-stimulating factors and is synthesized by many types of cells (endothelial cells, fibroblasts, macrophages, and lymphocytes), including the cells of the reproductive organs (ovary and endometrium). There is evidence that G-CSF regulates processes important for endometrial receptivity, including vascular remodeling, local immune changes, and cell adhesion (Rahmati et al., 2014).

G-CSF was first used in patients with RIF in assisted reproductive technology (ART) programs in 2000. Würfel, (2000)) showed that systemic administration of G-CSF substantially improved embryo implantation rates. Eleven years later, Gleicher et al. (2011) showed that intrauterine administration of G-CSF in four patients with thin hormone-resistant endometrium promoted its growth.

Since then, number of studies have been published on systemic or local administration of G-CSF in patients with RIF (Scarpellini and Sbracia, 2012, Barad et al., 2014, Singh et al., 2015, Aleyasin et al., 2016, Eftekhar et al., 2016, Obidniak et al., 2016, Su et al., 2024). The results of these studies are ambiguous; some show an improvement in the rates of implantation, pregnancy, and live births, whereas others do not.

In our opinion, this may be explained by the fact that the causes of implantation failure differ; therefore, there is no universal means of influence. Among the factors that can negatively affect implantation, disorders of the endometrial immune structure are prominent (Makrigiannakis et al., 2011, Wang et al., 2020, Kwak-Kim et al., 2021, Pantos et al., 2022, Von Woon et al., 2022, Chen et al., 2023, Mukherjee et al., 2023).

There is a shift from a pro-inflammatory to a regulatory phenotype in the endometrium during the implantation window, and the composition and functioning of many cells, including immune cells, change (Parham, 2004, Richman and Naftolin, 2006, Guerin et al., 2009, Saito et al., 2010, Arck and Hecher, 2013, Yang et al., 2019). One of the key cells involved in the creation of an environment tolerant to embryos, remodeling of the vascular bed, and recognition of trophoblasts are uterine natural killers (uNK) (King et al., 1989, Parham, 2004, Guerin et al., 2009, Arck and Hecher, 2013, Moffett et al., 2016, Díaz-Hernández et al., 2021, Fraser and Zenclussen, 2022). Several studies have shown that the quantity and ratio of uNK subtypes may differ between patients with RIF and healthy fertile women (Marron and Harrity, 2019, Marron et al., 2019, Von Woon et al., 2022).

We previously conducted a flow cytometry study on endometrial biopsy material from patients with RIF and fertile women who were gestational carriers. In some patients with RIF, the uNK population showed abnormally high expression of HLA-DR, CD16, CD158a, and CD335, which was not observed in gestation carriers. High expression of HLA-DR on T lymphocytes, HLA-DR, CD16, and uNK was almost always detected in the same patients (15 out of 42). We established this as a specific immune phenotype of a not transformed endometrium, that is, one in which the changes in uNK, typical for the implantation window, did not occur (Sudoma et al., 2023).

As a continuation of our previous study in the same group of women with RIF, we decided to analyze the effectiveness of intrauterine administration of G-CSF for improving embryo implantation depending on the endometrial immune phenotype and to study whether the immune endometrial profile changes under the influence of intrauterine administration of G-CSF.

The aims of our study were: 1) to investigate the pregnancy rate (PR) and live birth rate (LBR) depending on the endometrial immune phenotype in patients with RIF after intrauterine administration of G-CSF in an embryo transfer program; and 2) to determine how intrauterine administration of G-CSF affects the immune phenotype of the endometrium.