Investigation of the Effect of Seminal Plasma Exosomes from the Normal and Oligoasthenoteratospermic Males in the Implantation Process

Abstract

Background: Seminal plasma exosomes are now recognized to play a complex role in the regulation of the female reproductive system infertility. The objective of this study was to assess the effect of exosomes derived from the sperm of men with oligoasthenoteratozoospermia on endometrial implantation-related genes.

Methods: To isolate the exosomes, we employed an ultracentrifugation method on samples derived from 10 fertile men with normal sperm parameters and 10 men with oligoasthenoteratozoospermia. The size distribution and ultrastructure of the exosomes were then characterized using transmission electron microscopy and dynamic light scattering. We detected an exosome marker using western blot analysis and confirmed the cytoplasmic localization of the exosomes by incubating them with DiI dye and visualizing them using fluorescence microscopy. After 6 hours of in vitro treatment of endometrial epithelial cells with 100 µg/ml seminal exosome, the endometrial receptivity genes were examined using qRT-PCR. To perform data analysis and quantification, we utilized Image J and Prism software. P< 0.05 were considered statistically significant.

Results: After 6 hours of treatment, the mRNA levels of MUC1, LIF, G-CSF, CX3CL1, and VEGF were significantly downregulated in the endometrial epithelial cells treated with oligoasthenoteratozoospermia exosomes compared to the normal group. Although changes were observed in the mean mRNA levels of IL8 and TGF-β genes in the oligoasthenoteratozoospermia group compared to the normal group, these differences did not reach statistical significance (p > 0.05).

Conclusions: Oligoasthenoteratozoospermia exosomes have a distinct effect on endometrial receptivity compared to normal exosomes, leading to reduced expression of implantation-related genes.

Keywords: Embryo implantation; Endometrium; Exosome; Infertility; Semen.

Fig. 1

The patients' sperm characteristics were evaluated, and significant differences were found in sperm count, motility, and morphology between the two populations. The results are presented as mean ± SD (n = 10), and **** indicates a statistically significant difference with a P< 0.0001.

Fig. 2

Images captured by transmission electron microscopy show exosomes. a) from men in the normal group. b) men in the OAT group. C) Western blots of exosomes from normal men (1) and exosomes from OAT men (2) show the presence of the CD9 exosome surface marker. Scale bars in a) and b) represent 100 nm.

Fig. 3

DLS validation of exosome size. a) Exosomes from men in Normal group. b) from OAT group. Mean SD is utilized to represent the results. c) Exosome average size is used for each group (n = 10) P> 0.05. Each group's exosome size is represented by a picture.

Fig. 4

Verification of EECs: a) Primary cell culture of EECs. b) Image of endometrium in the proliferative phase taken under a light microscope; scale bars: 100 micrometers. c) Results from flow cytometry demonstrate that EECs expressed cytokeratin-18.

Fig. 5

Exosomes internalization within EECs. After 6h, EECs internalized DiI-labeled exosomes (red). DAPI (blue) is used to mark the nuclei of EECs. 20 nm scale bars.

Fig. 6

Gene’s expression in EECs following normal and OAT exosome treatments for 6 h. The assessment of mRNA levels was done using qRT-PCR. GAPDH was used as an internal control (n = 10) and results were also reported as mean SD. *P< 0.05. two experimental groups are compared to the control groups. The *, **, ***, and **** indicate P< 0.05, P< 0.01, P< 0.001, and P< 0.0001, respectively.