O-143 Biopsy-free profiling of the uterine immune system in patients with recurrent pregnancy loss and unexplained infertility
Authors: Vomstein K , Egerup P , Kolte A M , Behrendt-Møller I , Dyhrberg Boje A , Bertelsen M L , Eiken C , Raupelyté Reiersen M , Toth B , La Cour Freiesleben N , Nielsen H S 
Published: June 2023

Abstract

Study question
What are the differences in menstrual blood lymphocytes between controls, patients with recurrent pregnancy loss (RPL) or with unexplained infertility (uINF)?

Summary answer
Compared with controls, RPL- and uINF patients had a different menstrual blood immune profile, including altered NK-subtypes indicating an altered cytotoxicity in these patients.

What is known already
T-cells, particularly regulatory T-cells, and uterine natural killer cells play a vital role as they are the main immunological regulators at the feto-maternal interface. However, an endometrial biopsy (EB) is required for the examination of these cells, necessitating an invasive and uncomfortable procedure. Moreover, the small tissue samples are mainly analyzed by immunohistochemistry, limiting the amount of analysable markers and ultimately results in controversial data. Menstrual blood (MB) has been used in proof-of-concept studies to analyse lymphocyte populations. So far, no study has investigated MB lymphocytes in a well-defined cohort of RPL- and uINF patients in comparison to healthy controls.

Study design, size, duration
In this prospective study, 46 healthy controls, 28 RPL patients and 11 uINF patients were included between 03/2021 and 02/2022. RPL was defined as ≥ 3 consecutive pregnancy losses. For being diagnosed with uINF, all standard-diagnostic procedures for infertility had to provide negative results in couples trying to conceive for &gt;12 months.

Participants/materials, setting, methods
To establish the procedure, lymphocyte compositions in EB in 7 controls were compared with those in MB during 48h. The first 24h and the second 24h were collected separately. In all other RPL- and uINF-patients, as well as controls, peripheral blood (PB) and MB was collected. Density gradient centrifugation was performed to isolate PB and MB mononuclear cells. Cells were analyzed by flow cytometry focusing on the main lymphocyte populations and NK-cell subsets.

Main results and the role of chance
The first 24h of MB closely resembled the uterine lymphocyte composition in EB, with were CD3+ T-cells and CD56+ NK-cells being the predominant lymphocyte populations in EB and MB. We therefore based further analyses on the first 24h of menstrual shedding. In comparison to controls, RPL-patients showed significantly higher MB-CD56+-NK-cell numbers (mean±SD: 31.13±7.52 vs. 36.73±5.4; p = 0.002). Furthermore, MB-CD56dimCD16bright NK-cells within the CD56+-NK-cell population were decreased in individuals with RPL and uINF compared to controls (mean±SD: 20.4±11.53;16.34±14.65;15.7±5.91; Ctrl vs. RPL p = 0.011; Ctrl vs. uINF p = 0.02). Further, compared to controls and RPL patients, uINF patients had lower CD3+ T-cell counts (mean±SD Ctrl. vs. uINF: 47.93±11.11 vs. 38.81±5.04, p = 0.013), with decreased CD4+ (mean±SD Ctrl. vs. uINF: 25.78±7.91 vs. 15.66±4.88, p = 0.001) and CD8+ T-cells (mean±SD Ctrl. vs. uINF: 15.32±4.14 vs. 8.43±2.85, p &lt; 0.001). Interestingly, uINF-patients showed higher CD25highCD127dim/neg regulatory T-cell counts than controls (mean±SD Ctrl. vs. uINF: 1.34±0.49 vs. 1.84±0.51, p = 0.009). The cytotoxicity receptors NKp46 and NKG2D were more present in uINF and RPL patients. Furthermore, RPL and uINF patients had significantly higher peripheral CD56+-NK-cell counts as compared to controls (mean±SD: 8.4±3.5;11.42±4.05;12.86±4.29; Ctrl vs. RPL p = 0.021; Ctrl vs. uINF p = 0.009).

Limitations, reasons for caution
The small sample size of our study limits our ability to extrapolate the findings to other populations, as well as perform further subgroup analyses in controls, RPL and uINF. Future research must concentrate on NK cell subpopulations in larger cohorts, potentially in a multi-center setting.

Wider implications of the findings
Strengths of this study include well phenotyped participants and that the immunological milieu of the endometrium is directly compared for EB and MB. In future studies, this non-invasive analysis might enable to identify and monitor patients who could profit from (immunomodulatory) medications, and thereby improve live birth rates.

Trial registration number
not applicable