P-489 Uterine fluid-immune cell analysis at the time of the embryo transfer
Authors: Strbo N , Rodriguez S , Ibrahim M , Padula L , Fisher E , Rivas K , Paidas M , Attia G 
Published: June 2023

Abstract

Study question
What are the subtypes of immune cells present in the upper and lower parts of the uterus at the time of frozen embryo transfer (FET)?

Summary answer
Our study is the first study confirming different immune cell subsets in the freshly collected uterine fluid at the time of the embryo transfer.

What is known already
Although endometrial receptivity is a key factor in influencing implantation in both naturally conceived and assisted reproductive technology (ART) cycles, very little is known about the endometrium milieu around the time of implantation. Previous studies have demonstrated the presence of several cytokines in the endometrium affect implantation. However, there is lacking data about the presence of immune cell subtypes within the endometrium and in the uterine cavity at the time of implantation.

Study design, size, duration
This study was approved by the Institutional Review Board (# 49174). The study was designed as a prospective observational cohort study between May 2021 and December 2022 at a single academic-based fertility center. All patients underwent at least one In Vitro Fertilization cycle (IVF) and have frozen embryos. Twenty-four participants were recruited for this study which was conducted during the frozen embryo transfer cycle (FET) regardless of the outcome of previous cycles.

Participants/materials, setting, methods
Trial transfer catheter was introduced under ultrasound guidance into lower uterine segment. Upon removal, the tip was rinsed in IMDN medium containing 10% FBS (lower). Embryo was then placed in upper uterus under ultrasound guidance. The tip of transfer catheter was rinsed in separate aliquot of the above media (upper). After centrifugation, pelleted cells were stained for the following surface markers: CD45, CD3, CD19, CD4, CD8, gamma delta TCR, CD25, CD127, CD66b, CD14, CD16, CD56.

Main results and the role of chance
Upon staining the pelleted cells, we were able to identify viable leukocytes from samples obtained from both, upper and lower uterus (0.1257 x 106 cells +/- SD 0.3167), (0.1230 x 106 cells +/- SD 0.1171), respectively. Among total viable cells, there was no significant difference in both the percent and number of CD45+ cells between the upper and lower uterus (9.880 +/- 6.983, 13.67 +/- 9.792, p = 0.1980) respectively. However, there was significantly higher expression of CD3 + (p = 0.006), CD19 + (p = 0.0319) and CD14 + (p = 0.0189) cells in samples collected from upper compared to lower uterus. Within all CD3+ cells, we found that gamma delta T cells (GDT) were the major population of T cells in both upper and lower uterus. In contrast, CD8+ T cells were significantly higher in the lower uterus when compared to the upper uterus (p = 0.009). There was no statistically significant difference in the expression of CD4+ T cells, T regulatory cells, NK cells (CD56+), neutrophils (CD66b+) and monocytes (CD14+) between upper and lower uterus.

Limitations, reasons for caution
Limitations of our study include number of participants as well as the number of parameters analyzed by flow cytometry. To maximize information from each sample, multicolor flow cytometry can give critical insight into immune cell composition of the uterine milieu and the difference in expression between upper and lower uterus.

Wider implications of the findings
We believe the immune milieu at the time of embryo transfer will affect implantation. Understanding the composition of immune cell will guide further research in identifying optimal immune milieus that favor implantation. Comprehensive analysis of endometrium is expected to lead to new diagnostic and therapeutic approaches to improve IVF outcomes.

Trial registration number
not applicable