(247) Circulating Small Extracellular Vesicles Isolated from Recurrent Pregnant Loss (RPL) Women are Different from Those of Normal Pregnant Women

Surabhi Gupta¹, Kanika Aggarwal¹, Shivi Khurana¹, Unnati Sharma¹, Jaganmoy Choudhury¹, Bodhana Dhole¹, Pradeep K Chaturvedi¹, K Aparna Sharma²

1. Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi, India.


2. Department of Obstetrics & Gynaecology, All India Institute of Medical Sciences, New Delhi, India.

Abstract Text:

Extensive communication between trophoblast cells of the blastocyst and endometrial cells of the uterus is required for proper implantation and a subsequent successful pregnancy. Any deficiency in this communication can lead to implantation defects resulting in pregnancy complications including pregnancy loss.

Many molecules involved in this communication have been identified including specific cytokines, growth factors, cell adhesion molecules, proteases and even hormones. Small extracellular vesicles (sEVs), which were once considered as “garbage disposal machinery”, are now known to carry a cargo of miRNAs, proteins and lipids between cells at a distance and hence, represent a new form of “ACTIVE cell-to-cell communicators”. Shedding of sEVs is now a well-recognized, important method of cell-cell communication in a number of different cell types. However, their importance in mediating embryo-maternal interactions during pregnancy has only recently been recognized. Since one important component of the fetal-maternal signalling are the sEVs which are released by the trophoblast cells as well as the maternal endometrium, we hypothesized that sEVs isolated from healthy pregnant women and women with recurrent pregnancy loss may be different in terms of their size, abundance, cargo or functional effect. Hence, this study aimed to explore these possible differences.

Circulating sEVs were isolated from pregnant women with RPL history or normal pregnancy. Nanoparticle tracking analysis and western blotting were used to characterize the sEVs.  MicroRNA cargo of sEVs isolated from normal pregnant and RPL women was characterized by next generation sequencing. Effect of the sEVs on the various cellular functions of trophoblast cells and on the decidualization of endometrial stromal cells was studied using HTR8/SVneo and THESC cell lines respectively. Migration and invasion capacity of the trophoblast cells was measured using scratch assay and Matrigel invasion assay. Effect on in vitro decidualization was evaluated by measuring the decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP1) at both transcript and protein levels.

sEVs isolated from the serum samples had size in the range of 30 – 150 nm and were positive for the sEV markers, CD9, CD81 and TSG101. NGS analysis of the sEV cargo for small RNAs identified 61 differentially expressed miRNAs (DEMs) out of which 36 were upregulated and 25 were downregulated in RPL samples. Though no differential effect on migration and invasion of HTR8/SVneo cells was observed but sEVs induced decidualization of THESCs. Further, it was found that transfection of THESCs with one of the miRs upregulated in the RPL, namely miR-423-3p, resulted in reduced levels of decidualization markers.

In conclusion, our findings suggest that circulating maternal sEVs isolated from normal pregnant and RPL women have different miRNA cargo which may modulate the in vitro decidualization of THESCs differently.

Funding: This work has been supported by research funds from Science and Engineering Research Board, Department of Science and Technology, Government of India [Grant no. EMR/2017/005430].