P-399 Beyond chromosome screening: The impact of progesterone and in vitro oxidative stress on failed euploid blastocyst transfers
Authors: Cuadrado Torroglosa I , Quiñonero A , Garcia-Velasco J  A , Alecsandru D , Domínguez F 
Published: July 2024

Abstract

Study question
To assess the effect of serum progesterone levels on the production of radical oxygen species (ROS) by endometrial immune cells in patients with non-genetic miscarriages.

Summary answer
Elevated serum pre-transfer progesterone levels favour the development of an endometrial microenvironment that is not conducive to a healthy pregnancy, by increasing ROS production.

What is known already
Endometrial immune cells, especially uterine Natural Killer (uNK) cells, play an essential role in the proper development of pregnancy, promoting angiogenesis and adequate placentation. The maturation of these cells is indirectly determined by progesterone levels, which, during the secretory phase, enhance the synthesis of key cytokines for uNKs’ functionality.
Previous studies have already indicated that increases in progesterone levels during the follicular phase can alter the endometrial microenvironment, affecting its receptivity. However, the molecular effect that imbalances of this hormone may have on the endometrium on the day of embryo transfer and directly in relation to uNK is still unknown.

Study design, size, duration
We conducted a prospective exploratory study, between September 2022 and January 2024, including 15 patients with previous miscarriages after euploid single embryo transfer (SET), ruling out the genetic cause for such gestational losses. We obtained endometrial biopsies of all patients, after 5 days of progesterone, in a cycle with hormone replacement therapy. The same day of the biopsy, serum progesterone levels were measured in all the patients included.

Participants/materials, setting, methods
In vitro cultures enriched in uNK cells (CD56+CD16-) were established from the patients’ endometrial biopsies and cell enrichment was confirmed by flow cytometry. These cells were co-cultured with the JEG-3 cell line (placental cells), simulating the microenvironment of early pregnancy. Cellular ROS production was measured by fluorescence detection 40 hours post-culture using a commercial kit. A linear regression model was used to determine statistically significant correlations (p &lt; 0.05) between ROS and progesterone levels.

Main results and the role of chance
The mean age of the participating patients was 38.9 years and the BMI was 23.2 kg/m2. The mean progesterone level the day of the biopsy was 15.6 ng/ml. The cell cultures obtained from patients were successfully enriched in the desired cell population, with an average percentage of 89.6% uNK cells (CD56+CD16-) of total leukocytes (CD45+) obtained by flow cytometry. After culturing the same number of cells from each patient with the same number of spheroids of the JEG-3 cell line, an average ROS production of 31341.2 arbitrary units (a.u.) was obtained. Taking into account the co-cultures stablished from the 15 patients, a positive correlation between cellular ROS production and progesterone level was observed (r = 0.6728, p = 0.006).

Limitations, reasons for caution
Although the results shown may provide clues about the molecular dynamics of the endometrium during early pregnancy, they should be taken with caution, as it is not yet known whether this progesterone elevation is the cause or rather the consequence of an unfavourable endometrial microenvironment with high oxidative stress.

Wider implications of the findings
This study provides new information on the endometrial microenvironment of patients suffering non-genetic miscarriages and, although much remains to be studied, this association between hormone levels and oxidative stress may point the way to future studies investigating a possible explanation for these undesirable reproductive outcomes.

Trial registration number
Not Applicable