P–408 Inadequate increase in Tim–3 on peripheral NK cells after blastocyst transfer is associated with early miscarriage
Authors: Zhang T , Zhao Y , Wing C C , Chen X , Wang C C , Li T C 
Published: August 2021

Abstract

Study question
Whether the changing peripheral levels of Tim–3/Galectin–9 (Gal–9) and PD–1/PL–1 over 4 weeks after ET in ongoing pregnancies is different from pregnancies destined to miscarry.

Summary answer
A significant and sustained increase of Tim–3 in pNK cells was observed in pregnancies which were ongoing but not in pregnancies which later miscarried.

What is known already
The importance of maternal immune adaptation and tolerance to the implanting embryo, an allograft, has been extensively investigated for decades. Immune checkpoint molecules, like T-cell immunoglobulin mucin–3 (Tim–3) and programed cell death–1 (PD–1), are co-stimulatory receptors negatively regulating immune responses. During pregnancy, Tim–3 and PD–1 are expressed by several immune cells in the decidua and participate in the maternal-fetal immune interactions to mediate maternal immune tolerance through binding to their ligands Gal–9 and progressed death-ligand 1 (PD-L1) produced by trophoblast and immune cells. In addition to the implantation site, Tim–3 and PD–1 expressions in peripheral lymphocytes are modified during pregnancy.

Study design, size, duration
A prospective observational study includes 81 women who achieved ongoing pregnancy and 17 women who suffered from miscarriage after single day–5 blastocyst transfer. All the subjects were recruited from November 2018 to January 2020 in a university teaching hospital.

Participants/materials, setting, methods
Women undergoing single blastocyst transfer after in-vitro-fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment were recruited on the day of ET following informed, written consent. They had serial blood samples taken on the day of ET, and 4, 5, 6 and 7 weeks of gestation for measurement of (1) membranous Tim–3 and PD–1 expression on various peripheral lymphocytes by flow cytometry; and (2) serum concentrations of ligands Gal–9 and PD-L1 by ELISA.

Main results and the role of chance
The comparisons between two groups showed there was no significant difference between the 2 groups in baseline levels among all the parameters measured. In women who achieved ongoing pregnancy, a significant and sustained increase of Tim–3 in either peripheral NK (pNK) subsets was observed at 4-week, 5-week, 6-week and 7-week gestations compared to the baseline (Tim–3+CD56dimNK 39.14±1.51%, 41.14±1.62%, 41.34±1.94%, and 41.69±2.12% vs. 30.27±1.49%; Tim–3+CD56brihgtNK cells, 24.54±1.71%, 25.43±1.54%, 27.26±1.88% and 24.70±1.64% vs. 19.08±1.13%), and the concentration of serum PD-L1 was significantly increased at 6-week and 7-week gestations (48.33±17.78 pg/ml, 52.53±20.60 pg/ml) when compared to the day of blastocyst transfer (41.40±16.01 pg/ml). The expressions of Tim–3 in T, NKT cells and PD–1 in NK, T, NKT cells were not significantly changed across the 5 time points. In women who conceived but later miscarried, all the parameters examined from 4–7 weeks of gestation were not significantly different when compared with the baseline measurement. The only measurement which showed a significant difference between the 2 groups and across all time points after ET was the proportion of Tim–3+CD56dimNK cells which was significantly higher in women who achieved ongoing pregnancies compared with women who destined to miscarry from 4 to 7 weeks of gestation.

Limitations, reasons for caution
It is uncertain if the observation would be different between miscarriage associated with aneuploid embryo or euploid embryo as we had not been able to obtain karyotyping result in most of the miscarriage cases.
Wider implications of the findings: Our preliminary observation suggests that the proportion of Tim–3+pNK cells as early as 4-week gestation could be a potential immuno-bio-marker to predict if a pregnancy is likely to progress normally or result in a miscarriage. Clearly, the finding in this study needs to be confirmed in a larger cohort study

Trial registration number
not applicable.