Intrauterine administration of autologous peripheral blood mononuclear cells regulates the endometrium estrogen and progesterone receptor expression: An RCT

Abstract Background Repeated implantation failure (RIF) affects 15% of women of reproductive age. There is a high endometrial expression of both estrogen receptors and progesterone receptors (PRs) during the window of implantation in women with RIF. Objective To evaluate the effects of intrauterine administration of human peripheral blood mononuclear cells (PBMC) on estrogen receptor α (ERα) and PRs expression in the endometrium of women with RIF during the implantation window. Materials and Methods This randomized clinical trial study was conducted on 22 women with RIF history from January 2018 to August 2019 in Erfan hospital, Tehran, Iran. Participantswere divided into 2 groups (PBMC-treated group [n = 11] and control group [n = 11]). Endometrial tissue samples were collected at the implantation window time, during the mid-secretory phase (luteinizing hormone surge +7 days) of each menstrual cycle. The quantitative real-time polymerase chain reaction technique was used to measure the mRNA levels of ERα and PRs isoforms (PR-A and PR-B) in endometrial tissues. Furthermore, the protein expression of ERα and PRs was investigated using immunohistochemical staining. Results PBMC treatment significantly decreased the mRNA expression of endometrial ERα and PRs isoforms at the time of the implantation window (p < 0.001). Moreover, the endometrial ERα and PRs protein localization were significantly lower in PBMC-treated women compared with controls (p = 0.01, and p < 0.001 respectively). Conclusion The intrauterine administration of PBMC decreased the endometrial ERα and PRs expression during the window of implantation in women with RIF. This local response to PBMC therapy could promote endometrial receptivity and embryo implantation.


Introduction
Despite the improvements in assisted reproductive technologies, the rates of embryo implantation and pregnancy remain suboptimal.
Furthermore, a considerable number of couples have experienced repeated implantation failure (RIF). This situation affects 15% of women of reproductive age (1). In reproductive medicine, RIF refers to the failure of at least 3 consecutive fresh or frozen cycles in which 1 or 2 good-quality embryos were transferred (2). Among couples undergoing assisted reproduction treatment, RIF significantly increases the level of distress experienced by couples and also increases the cost of treatment (3). Embryo-uterine cross talk synchronizations are a critical factor for successful blastocyst implantation. The uterine endometrium is permissive to the implanting blastocyst only during the "implantation window". In humans, this timely event occurs at an interval of 4-6 days during the mid-luteal phase and is regulated by synergistic signals of the ovarian hormone's estrogen and progesterone (4). The function of progesterone and estrogen hormones is through their specific nuclear receptors that act as chromatin modifiers and ligand-activated transcription factors to control multiple gene expressions (4). Estrogen receptors (ERs) have 2 isoforms, ERα and β, derived from 2 different genes including estrogen receptor ESR1 and ESR2 (5). Studies using ESR1 and ESR2 knockout female mice have displayed that ERα but not ERβ, plays an important role in blastocyst attachment to the endometrium (6). There are 2 progesterone receptors (PRs) isoforms, PR-A and PR-B. In contrast to ERs, the PRs isoforms are derived from a single gene (progesterone receptor [PGR]) by alternate transcription and translation initiation sites (5). Both ER and PR are downregulated at the time of implantation. It has been reported that the downregulation of both ER and PR during the window of implantation is an extremely important event, which influences the endometrial receptivity (4). Recent evidence has shown that estrogen and progesterone interact with the immune system during menstrual cycle and pregnancy, and their receptors are identified on immune cells (7)(8)(9)(10)(11)(12)(13).
Many studies have investigated local immune cells involved in implantation and are actively engaged in embryo implantation (3,(14)(15)(16)(17). It has been shown that PBMC intrauterine administration increases several cytokines production, including interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF) α in the endometrium, which has an essential role in uterine receptivity (18). Besides, it has been shown that PBMC derived from women in early pregnancy enhances the BeWo cell and murine blastocyst invasion (19). In mice, intrauterine administration of PBMC during the implantation window increases endometrial vascular endothelial growth factor and leukemia inhibitory factor gene expressions and implantation rate (15).
Several studies have reported that intrauterine administration of PBMC could improve implantation and pregnancy outcomes in women with RIF (3,14,16,17,(20)(21)(22). The immunomodulation techniques could be a promising approach in the treatment of numerous RIF cases and in promoting in vitro fertilization (IVF) success rate (16). Therefore, this study was proposed to investigate the effects of intrauterine administration of human PBMC on ERs and PRs expression in the endometrium of women with RIF during the implantation window, to clarify the molecular mechanism of intrauterine administration of PBMC that can modulate endometrial receptivity and blastocyst implantation in women with RIF.

Study design
Participants consisted of infertile women in the natural cycle who underwent hysteroscopy as a diagnosis procedure before their infertility treatment.
Subjects were intended to the PBMC-treated group who received intrauterine administration of PBMC, 2 days before endometrial biopsy (n = 11) and the RIF control group undergoing endometrial biopsy without a previous intrauterine PBMC administration (n = 11).

PBMC treatment protocol
The human PBMC preparation was performed as described previously (9). Briefly, a peripheral blood specimen was collected 5 days before the mid-secretory endometrial biopsy.

Endometrial biopsy sampling
Endometrial biopsies were collected from all participants at the time of implantation window.
In the current study, implantation window is defined as 7 days after luteinizing hormone surge (luteinizing hormone surge +7 days) in mid-secretory phase of each menstrual cycle.
Endometrial tissue samples were obtained using a Pipelle sampler from the anterior wall of the uterine cavity, without cervical dilatation. The biopsy samples were divided into 2 portions.
One part was prepared for immunohistochemical analysis, and the other part was frozen in liquid nitrogen and stored at -80°C until used for RNA extraction and quantitative polymerase chain reaction.

RNA isolation and cDNA synthesis
The

qRT-PCR
qRT-PCR analyses of ESR1, PGR, and PGRB mRNA were carried out using ABI StepOne Plus

Immunohistochemistry
ERα and PR protein expression were evaluated using immunohistochemical staining. The endometrial samples were immediately fixed by 10% neutral buffered formalin overnight and next placed in paraffin wax. The endometrium samples were sectioned at a thickness of 5 μm and fixed with 4% paraformaldehyde in PBS for 20 min at 4°C. The PBS-washed sections were then incubated in 2N HCl at room temperature for 20 min. Then, 0.3% Triton X100 was applied for 30 min at room temperature for permeabilization. After

Results
A total of 22 women were defined into 2 groups: control (n = 11) and PBMC-treated group (n = 11). One participant in each group withdrew from the study due to personal reasons ( Figure 1 Figure   3A). Moreover, immunohistochemical analysis of endometrial sections identified that on comparison with control women, those in PBMC-treated cycles showed significant decreases in endometrial PR-A and PR-B protein levels (p < 0.001, Figure   3B). Thereby, these results showed that PBMC treatment promotes endometrial receptivity among women who suffer from RIF ( Figure   3C).

Discussion
In this study we determine the effect of  (14). It has been shown poor endometrial receptivity causes unsuccessful blastocyst attachment in IVF cycles (4). RIF is a limiting factor in the success of IVF programs (16).
It has been reported that endometrial receptivity decreased in women who suffered from RIF (25).
It is important to note that the dysregulation of It is also well-accepted that among some women with RIF, there is an inappropriate inflammatory environment in the uterine cavity (37). Clinical data have shown that the immunological environment of the blastocyst implantation site can be modulated by PBMC-derived cytokines (16). The association between intrauterine administration of PBMC and the subsequent increase in the success rate of embryo implantation in women with RIF is well established (37,38).

Conclusion
In conclusion, we provide new evidence demonstrating that the intrauterine administration of PBMC decreased the endometrial ERα and PR expression during the window of implantation in women with RIF. This local response to PBMC therapy could promote endometrial receptivity and embryo implantation.