Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency.

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies.

The Extracellular Vesicles Proteome of Endometrial Cells Simulating the Receptive Menstrual Phase Differs from That of Endometrial Cells Simulating the Non-Receptive Menstrual Phase.

Successful embryo implantation into a receptive endometrium requires mutual endometrial-embryo communication. Recently, the function of extracellular vehicles (EVs) in cell-to-cell interaction in embryo-maternal interactions has been investigated. We explored isolated endometrial-derived EVs, using RL95-2 cells as a model of a receptive endometrium, influenced by the menstrual cycle hormones estrogen (E2; proliferative phase), progesterone (P4; secretory phase), and estrogen plus progesterone (E2P4; the receptive phase). EV sized particles were isolated by differential centrifugation and size exclusion chromatography. Nanoparticle tracking analysis was used to examine the different concentrations and sizes of particles and EV proteomic analysis was performed using shotgun label-free mass spectrometry. Our results showed that although endometrial derived EVs were secreted in numbers independent of hormonal stimulation, EV sizes were statistically modified by it. Proteomics analysis showed that hormone treatment changes affect the endometrial EV's proteome, with proteins enhanced within the EV E2P4 group shown to be involved in different processes, such as embryo implantation, endometrial receptivity, and embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs.

The role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapeutics and diagnosis.

Extracellular vesicles (EVs) are small, nanometre sized, membrane-enclosed structures released by cells and are thought to be crucial in cellular communication. The cargo of these vesicles includes lipids, proteins, RNAs and DNA, and control various biological processes in their target tissues depending on the parental and receiver cell's origin and phenotype. Recently data has accumulated in the role of EVs in embryo implantation and pregnancy, with EVs identified in the uterine cavity of women, sheep, cows, horses, and mice, in which they aid blastocyst and endometrial preparation for implantation. Herein is a critical review to decipher the role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapies and diagnosis. The current knowledge of the function of embryo and endometrial derived EVs and their cargoes, with regards to their effect on implantation and receptivity are summarized and evaluated. The findings of the below review highlight that the combined knowledge on EVs deriving from the endometrium and embryo have the potential to be translated to various clinical applications including treatment, a diagnostic biomarker for diseases and a drug delivery tool to ultimately improve pregnancy rates.

Effects of the phytoestrogen genistein on the development of the reproductive system of Sprague Dawley rats.

OBJECTIVES: Genistein is known to influence reproductive system development through its binding affinity for estrogen receptors. The present study aimed to further explore the effect of Genistein on the development of the reproductive system of experimental rats. METHODS: Eighteen post-weaning female Sprague Dawley rats were divided into the following groups: (i) a control group that received vehicle (distilled water and Tween 80); (ii) a group treated with 10 mg/kg body weight (BW) of Genistein (Gen 10); and (iii) a group treated with a higher dose of Genistein (Gen 100). The rats were treated daily for three weeks from postnatal day 22 (P22) to P42. After the animals were sacrificed, blood samples were collected, and the uteri and ovaries were harvested and subjected to light microscopy and immunohistochemical study. RESULTS: A reduction of the mean weekly BW gain and organ weights (uteri and ovaries) were observed in the Gen 10 group compared to the control group; these findings were reversed in the Gen 100 group. Follicle stimulating hormone and estrogen levels were increased in the Gen 10 group and reduced in the Gen 100 group. Luteinizing hormone was reduced in both groups of Genistein-treated animals, and there was a significant difference between the Gen 10 and control groups (p<0.05). These findings were consistent with increased atretic follicular count, a decreased number of corpus luteum and down-regulation of estrogen receptors-a in the uterine tissues of the Genistein-treated animals compared to the control animals. CONCLUSION: Post-weaning exposure to Genistein could affect the development of the reproductive system of ovarian-intact experimental rats because of its action on the hypothalamic-pituitary-gonadal axis by regulating hormones and estrogen receptors.

Effects of the phytoestrogen genistein on the development of the reproductive system of Sprague Dawley rats

OBJECTIVES: Genistein is known to influence reproductive system development through its binding affinity for estrogen receptors. The present study aimed to further explore the effect of Genistein on the development of the reproductive system of experimental rats. METHODS: Eighteen post-weaning female Sprague Dawley rats were divided into the following groups: (i) a control group that received vehicle (distilled water and Tween 80); (ii) a group treated with 10 mg/kg body weight (BW) of Genistein (Gen 10); and (iii) a group treated with a higher dose of Genistein (Gen 100). The rats were treated daily for three weeks from postnatal day 22 (P22) to P42. After the animals were sacrificed, blood samples were collected, and the uteri and ovaries were harvested and subjected to light microscopy and immunohistochemical study. RESULTS: A reduction of the mean weekly BW gain and organ weights (uteri and ovaries) were observed in the Gen 10 group compared to the control group; these findings were reversed in the Gen 100 group. Follicle stimulating hormone and estrogen levels were increased in the Gen 10 group and reduced in the Gen 100 group. Luteinizing hormone was reduced in both groups of Genistein-treated animals, and there was a significant difference between the Gen 10 and control groups (p<0.05). These findings were consistent with increased atretic follicular count, a decreased number of corpus luteum and down-regulation of estrogen receptors-a in the uterine tissues of the Genistein-treated animals compared to the control animals. CONCLUSION: Post-weaning exposure to Genistein could affect the development of the reproductive system of ovarian-intact experimental rats because of its action on the hypothalamic-pituitary-gonadal axis by regulating hormones and estrogen receptors.