Abstract
Purpose
Endometrial extracellular vesicles are essential in regulating trophoblasts’ function. This study aims to investigate whether endometrial extracellular vesicles (EVs) from recurrent implantation failure (RIF) patients inhibit the proliferation, invasion, and migration of HTR8/SVneo cells.
Methods
Eighteen RIF patients and thirteen fertile women were recruited for endometria collection. Endometrial cells isolated from the endometria were cultured and modulated by hormones, and the conditioned medium was used for EV isolation. EVs secreted by the endometrial cells of RIF patients (RIF-EVs) or fertile women (FER-EVs) were determined by Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Fluorescence-labeled EVs were used to visualize internalization by HTR8/SVneo cells. RIF-EVs and FER-EVs were co-cultured with HTR8/SVneo cells. Cell Counting Kit-8, transwell invasion, and wound closure assays were performed to determine cellular proliferation, invasion, and migration, respectively, in different treatments.
Results
RIF-EVs and FER-EVs were bilayer membrane vesicles, ranging from 100 to 150 nm in size, that expressed the classic EV markers Alix and CD9. RIF-EVs and FER-EVs were internalized by HTR8/SVneo cells within 2 h. The proliferation rate in the FER-EV group was significantly higher than that in the RIF-EV group at 20 μg/mL. Moreover, the invasion and migration capacity of trophoblast cells were decreased in the RIF-EV group relative to the FER-EV group at 20 μg/mL.
Conclusion
Endometrial EVs from RIF patients inhibited the functions of trophoblasts by decreasing their proliferation, migration, and invasive capacity. Such dysregulations induced by RIF-EVs may provide novel insights for better understanding the pathogenesis of implantation failure.
Keywords: Extracellular vesicle, Recurrent implantation failure, Implantation, Endometrium, Trophoblast
Introduction
Recurrent implantation failure (RIF) has emerged as a new challenge for assisted reproductive technology (ART). Although a universal definition has not been established, it is widely accepted that three or more consecutive transfers of good quality embryos without successful implantation can be regarded as RIF [1, 2]. Multiple failed cycles result in heavy emotional and financial stress for RIF patients. Therefore, further understanding of the etiologies and pathogenesis of RIF is needed to propose novel and beneficial solutions for this condition [3].
It has been postulated that many factors, such as endometrial receptivity, inflammation, immunological disorders, and embryonic conditions, are related to RIF [4–6]. However, the precise pathogenesis of RIF has not been elucidated yet. Previous studies have reported that endometrial factors are responsible for approximately two-thirds of implantation failures [7]. Therefore, many studies have focused on highlighting discrepancies between the endometria of RIF patients and fertile controls to reveal possible mechanisms. These studies found that the endometria of RIF patients exhibited altered protein and RNA profiles; moreover, several implantation-related pathways, including the Wnt signaling and cellular adhesion pathways, were greatly affected in RIF patients [2, 8, 9]. Such alterations in molecular repertoires indicate the aberrant nature of RIF patient endometria; however, the detailed mechanisms by which such endometria interfere with embryos and contribute to implantation failure have not been elucidated. Therefore, further studies are required to investigate the direct associations between the defective endometrium of RIF patients and embryonic implantation.
Extracellular vesicles (EVs) are small bilayer membrane vesicles that have recently been recognized as a vital form of intercellular communication [10, 11]. These membrane-enclosed complexes transfer a broad spectrum of molecules, including nucleic acids, lipids, and proteins [12], from the releasing cells to recipient cells, thereby modulating the activity of neighboring or distant recipient cells [13]. Recent studies have indicated that EVs are involved in embryo-maternal communication during implantation [14, 15]. EVs secreted by endometrial cells that are taken up by embryonic cells could improve the quality of embryos and modulate trophoblast adhesive capacity [16, 17]. Furthermore, EVs are determined by the parent cell type and status [18]. Given the distorted nature of endometria in RIF patients, it is fair to assume that endometrial EVs from RIF patients may exert different effects on implantation compared to those of fertile women.
In this study, we aimed to estimate the influence of endometrial EVs from RIF patients on embryonic cells, and HTR8/SVneo cells, a cell line widely used to investigate the implantation process [19], were used. We constructed a co-culture model using HTR8/SVneo cells and endometrial EVs from RIF patients or fertile women. After co-culture, cellular viability, invasion, and migration were compared in different groups.
Material and methods
Study population and tissue collection
All participants (n = 31) were under the age of 40 years and had a regular menstrual cycle. The RIF group (n = 18) included women who experienced three or more consecutive implantation failures after the transfer of good quality embryos (both fresh and frozen-thawed) [6, 20]. Besides, the quality of embryos was determined morphologically, based on previous graded methods [21–23]. Cleavage embryos that have the correct number of cells corresponding to the day of its development, evenness of cell division, and less than 20% fragmentation by volume were considered good embryos. Moreover, blastocysts graded at 4AA, 4AB, 4BA, or 4BB according to the previous grading system were considered good quality [21, 23]. Meanwhile, the fertile group (n = 13) included women who conceived naturally, delivered a live birth via cesarean section without associated comorbidities in the past two years, and had their fallopian tubes ligated during cesarean section. They came to the Reproductive Medicine Center of Tongji Hospital and expressed the desire to have another child with the assistance of in vitro fertilization and embryo transfer (IVF-ET). Patients were informed of the procedures, and those who intended and consented to undergo hysteroscopy and endometrial sampling were enrolled in the study. The exclusion criteria were uterine abnormalities, antiphospholipid antibody syndrome, endocrine disorders (including diabetes, insulin resistance, thyroid diseases, polycystic ovarian syndrome, hyperprolactinemia, diminished ovarian reserve, and premature ovarian failure), endometriosis, and abnormal karyotypes. For hysteroscopy, the women underwent gynecological examination, and those presenting with vaginitis, Chlamydia trachomatis infection, or Mycoplasma genitalium infection were excluded. Endometrial tissues were analyzed histologically, and patients with endometritis or endometrial polyps were also excluded from the study. Endometrial samples were obtained during hysteroscopy on days 9–11 of the menstrual cycle, stored in ice-cold sterile phosphate-buffered saline (PBS), and transferred to the lab immediately. Written informed consent was obtained from all participants. This study was approved by the Institutional Review Board of Tongji Hospital (TJ-IRB20190420).
Endometrial cell culture
Endometrial tissues were minced and digested in IV collagenase solution (1 mg/mL; Servicebio, Wuhan, China), and the cell suspension was filtered through a 70-μm aperture sieve to isolate endometrial cells. During the window of implantation, both endometrial epithelial cells and stromal cells were essential for preparing the receptive endometrium that interacted with the embryos [24, 25]; therefore, both the cell types were included in this study. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 with 10% fetal bovine serum (FBS) and 100 IU/mL streptomycin-penicillin. When the cells reached 80% confluence, the culture medium was discarded, and the confluent cells were washed thrice in PBS to remove the remaining FBS. The cells were replenished with FBS-free DMEM/F12, primed with 10-8 mol/L estrogen and 10-7 mol/L progesterone, to mimic the secretory phase following a previous protocol [17]. The cells were treated for 2 days, and the conditioned medium was collected every 24 h.
EV isolation
EVs secreted by the endometrial cells of RIF patients (RIF-EVs) and fertile women (FER-EVs) were isolated using a conventional ultra-centrifugation method [26]. In brief, the conditioned medium was centrifugated at 2000×g for 20 min at 4 °C to remove cells. The supernatant was centrifuged at 10,000×g for 30 min at 4 °C to remove debris, followed by ultra-centrifugation at 120,000×g for 80 min at 4 °C. The pellets were suspended in PBS and ultra-centrifugated at 120,000×g for 80 min at 4 °C to isolate EVs. The EVs were resuspended in 30–50 μL PBS and stored at -80 °C until use. The EVs secreted by endometrial cells from individuals were considered independent samples. Repeated freeze-thaw cycles were avoided.
Western blotting
EVs and cells were lysed by radioimmunoprecipitation assay (RIPA) buffer combined with a cocktail proteinase inhibitor (Servicebio, Wuhan, China). Bicinchoninic acid (BCA) assay was performed to determine the protein concentration. Proteins mixed with loading buffer were incubated in boiling water to prepare the loading samples. Proteins (5 μg) were separated via electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% skim milk and incubated with the following primary antibodies at 4 °C overnight: CD9 (AF5139; 1:1000; Affinity, Cincinnati, OH, USA), calnexin (#2433; 1:1000; Cell Signaling Technology, Danvers, MA, USA), and Alix (DF9027; 1:1000; Affinity, Cincinnati, OH, USA; 1:1,000). Following incubation with a secondary antibody (anti-rabbit IgG, HRP-linked antibody, 7074S; 1:1000; Cell Signaling Technology), the membranes were permeabilized in electrochemiluminescence (ECL) substrate (abs920; Absin Bioscience, Shanghai, China). Signals were detected using a chemiluminescence imaging system (Gene Gnome XRQ; Syngene, Frederick, MD, USA).
Transmission electron microscopy
EV samples were diluted with distilled water and placed on the surface of a carbon-coated electron microscopy grid. EVs were negatively stained with 2% uranyl acetate, and images were captured using a transmission electron microscope (HT7700; Hitachi, Tokyo, Japan).
Nanoparticle tracking analysis
EVs were diluted at a ratio of 1:500 to 1:2000 with distilled water to meet the concentration recommended by the instrument. The size and distribution of EVs were measured using a ZetaView nanoparticle tracking analyzer (Particle Metrix, Meerbusch, Germany) following the manufacturer’s instructions. Each sample was detected in triplicate.
EV labeling and internalization
One hundred microliters of EVs or the same volume of PBS (negative control) were incubated with 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO; Beyotime, Shanghai, China) at 37 °C for 30 min. The labeled EVs or PBS was suspended in 26-mL PBS and ultra-centrifugated at 120,000×g for 80 min to remove the dye solution. The labeled EVs (10 μg/mL) or PBS was co-cultured with HTR8/SVneo cells (at a density of 2000 cells per well in the 96-well plates) for 2 h at 37 °C. Then, the cells were washed thrice in PBS and fixed in 4% paraformaldehyde. After incubation with diamidino-2-phenylindole (DAPI) for nuclear staining, the cells were observed under a fluorescence microscope (Axio Observer A1; Carl Zeiss, Oberkochen, Germany).
Cell Counting Kit-8 assays
HTR8/SVneo cells were suspended in FBS-free DMEM/F12 and cultured in 96-well plates at a density of 2000 cells per well for 12 h. The concentration of EVs was determined by the BCA assay, and all EV samples were adjusted to the same concentration using sterile PBS. The cells were treated with different concentrations of RIF-EVs or FER-EVs for 24 h at 37 °C. Then, cell viability was assessed using CCK-8 assays (Beyotime, Shanghai, China) following the manufacturer’s instructions. The absorbance was measured at 450 nm by a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Experiments were performed in triplicates independently.
Transwell invasion assay
The invasive capacity of HTR8/SVneo cells was evaluated using a transwell invasion assay. Cultrex Basement Membrane Extract (BME; 3533-005-02; R&D Systems, Minneapolis, MN, USA) was diluted with serum-free DMEM/F12 at a ratio of 1:2, and every transwell insert (8 μm pores and 24-well plates) was pre-coated with 55 μL of the diluted BME. HTR8/SVneo cells were suspended in serum-free DMEM/F12 and loaded on the BME pre-coated inserts of 24-well plates at a density of 10,000 cells per insert. The lower chamber was filled with 400 μL DMEM/F12 containing 10% FBS. RIF-EVs or FER-EVs were co-cultured with HTR8/SVneo cells for 24 h at 37 °C. Following co-culture, the upper side of the inserts was erased by cotton swabs to remove the non-invaded cells. Then, the inserts were fixed in 4% paraformaldehyde and stained with crystal violet. Photos of the inserts were captured using a microscope (Axio Observer A1; Carl Zeiss) and its associated software. Finally, cell numbers were counted in five random fields. Experiments were performed in triplicates independently.
Wound healing assay
HTR8/SVneo cells were seeded in 96-well plates at a density of 10,000 cells per well and cultured in DMEM/F12 containing 10% FBS. When the cells attained 90–100% confluence, the culture medium was discarded, and the cells were starved in FBS-free DMEM/F12 for 12 h before treatments. A horizontal scratch was made in the middle of the well using a 10 μL tip, and the cells were washed in PBS to remove the debris. Then, the cells were cultured in FBS-free DMEM/F12, and RIF-EVs or FER-EVs were added into the cell cultures for a final concentration of 20 μg/mL. The cells were treated for 12 h, and images of the scratch in each well were captured using a microscope (Axio Observer A1; Carl Zeiss) every 6 h. The percentage of wound closure was calculated as follows:
Wound closure % = (wound at 0 hour - wound at T hour)/wound at 0 hour * 100
Experiments were performed in triplicates independently.
Statistical analysis
Quantitative variables were expressed as mean ± standard deviation (SD). Clinical characteristics were analyzed by SPSS Statistics 24.0 software (IBM, Chicago, IL, USA) using the Kruskal-Wallis test (Table 1). Data from the experiments were analyzed by GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA) using the one-way ANOVA test, followed by the Bonferroni test. p < 0.05 was considered to be statistically significant.
Table 1.
Clinical characteristics of the study participants
| Variables | RIF (n = 18) | Fertile (n = 13) | p Value |
|---|---|---|---|
| Age (years) | 32.94 ± 4.60 | 32.46 ± 4.62 | 0.584 |
| BMI (kg/m2) | 21.14 ± 1.64 | 21.66 ± 2.05 | 0.509 |
| Menstrual cycle (days) | 28.78 ± 1.35 | 29.00 ± 1.96 | 0.919 |
| FSH (mIU/mL) | 6.65 ± 1.33 | 7.39 ± 1.13 | 0.089 |
| LH (mIU/mL) | 4.38 ± 2.13 | 3.57 ± 1.46 | 0.357 |
| E2 (pg/mL) | 46.42 ± 15.3 | 46.47 ± 1.36 | 0.109 |
| Embryo transfer cycles | 3.00 (3–5) | / | / |
| Number of embryos | 4.50 (3–9) | / | / |
| Number of live births | / | 1.00 (1–2) | / |
RIF recurrent implantation failure, BMI body mass index, FSH follicle-stimulating hormone, LH luteinizing hormone, E2 estradiol. Data for age, BMI, menstrual cycle, and hormonal levels were presented as mean ± standard deviation (SD). Data for embryo transfer cycles, number of embryos, and number of live births were presented as media (range)
Results
Clinical characteristics of participants
Table 1 lists the baseline characteristics of RIF patients (n = 18) and fertile women (n = 13). Age, body mass index (BMI), menstrual cycle length, and hormone levels were comparable in the two groups. The median number of embryo transfer cycles in the RIF group was 3.00, and the median number of transferred embryos was 4.50. In the fertile group, the median number of live births was 1.00.
Characterization of RIF-EVs and FER-EVs
The EV protein markers CD9 and Alix were detected in RIF-EVs and FER-EVs. Endometrial cells of RIF patients served as controls. RIF-EVs and FER-EVs were positive for CD9 and Alix, while calnexin, which is specific to the endoplasmic reticulum, was undetectable in the EV samples (Fig. 1a). The distributions of RIF-EVs (Fig. 1b) and FER-EVs (Fig. 1c) were detected by NTA, and the predominant EV size was 100–150 nm. The representative TEM images showed that RIF-EVs (Fig. 1d) and FER-EVs (Fig. 1e) were bilayer membrane vesicles.
Fig. 1.
Characterization of RIF-EVs and FER-EVs. a RIF-EVs and FER-EVs were positive for classic EV protein markers CD9 and Alix but negative for calnexin, which was specific to the endoplasmic reticulum. ECs from RIF patients served as controls. The size and distribution of RIF-EVs (b) and FER-EVs (c) were displayed. TEM images revealed the typical bilayer membrane structures of RIF-EVs (d) and FER-EVs (e). EVs, extracellular vesicles; RIF, recurrent implantation failure; FER, fertile women; ECs, endometrial cells; TEM, transmission electron microscopy
Internalization of RIF-EVs and FER-EVs
To visualize the internalization of EVs, HTR8/SVneo cells were co-cultured with DIO-labeled RIF-EVs, FER-EVs, or PBS (serving as the control group). Images showed that DIO-labeled RIF-EVs and FER-EVs presented with green fluorescence (Fig. 2a), whereas no fluorescence signals were detected in the control group. The nuclei of the HTR8/SVneo cells were stained with DAPI. Fluorescence-labeled RIF-EVs and FER-EVs were clearly seen in the cytoplasm of HTR8/SVneo cells. Meanwhile, undetectable fluorescence in the control group excluded the possibility that excess DIO solution stained the membranes of HTR8/SVneo cells. These results indicated that both RIF-EVs and FER-EVs could be internalized by HTR8/SVneo cells.
Fig. 2.
EVs modulated the viability of HTR8/SVneo cells. a Representative images of DIO-stained RIF-EVs, FER-EVs, or PBS (control) internalization by HTR8/SVneo cells. Fluorescence-labeled EVs or PBS were added to the cultures and treated for 2 h. Then, the cells were fixed and stained with DAPI for nucleic staining. The left panel showed the green fluorescence signals of DIO-treated RIF-EVs, FER-EVs, and PBS; the middle panel showed the nuclei of HTR8/SVneo cells; and the right panel showed merged images. Scale bar = 50 μm. b RIF-EVs and FER-EVs were added to cultures of HTR8/SVneo cells for final concentrations of 10 μg/mL, 20 μg/mL, and 40 μg/mL. Meanwhile, the same volume of PBS (0 μg/mL) served as the negative control. The proliferation of HTR8/SVneo cells was evaluated by CCK-8 assay. Bars that do not share the same letters (superscripts) are significantly different from each other (p < 0.05). Data were significantly different between the two linked groups (*p < 0.05). EVs, extracellular vesicles; DIO, 3,3′-dioctadecyloxacarbocyanine perchlorate; RIF, recurrent implantation failure; FER, fertile women; PBS, phosphate-buffered saline; DAPI, diamidino-2-phenylindole; CCK-8, Cell Counting Kit-8
EVs modulated the proliferation of HTR8/SVneo cells
Following incubation with different concentrations of RIF-EVs or FER-EVs, the proliferation of HTR8/SVneo cells was evaluated by the CCK-8 proliferation assay. In the FER-EV groups, the proliferation rate increased as the FER-EV concentration increased, and there was a significant difference between the 20 and 0 μg/mL groups. On the other hand, in the RIF-EV groups, no significant differences were found. When the RIF-EV and FER-EV groups were compared, the proliferation rate was decreased significantly in the RIF-EV groups at a concentration of 20 μg/mL (Fig. 2b).
RIF-EVs attenuated the invasion capacity of HTR8/SVneo cells
A transwell invasion assay was performed to evaluate the invasion capacity of HTR8/SVneo cells in different treatments. Representative pictures from the RIF-EV and FER-EV groups are shown in Fig. 3a. In the FER-EV groups, the number of invaded cells increased as the concentration of FER-EVs increased; however, no significant difference was found (Fig. 3b). In the RIF-EV groups, the number of invaded cells was slightly decreased in the 20 μg/mL group compared to that of the 0 μg/mL or 10 μg/mL group, without statistical significance. When the RIF-EV and FER-EV groups were compared, the number of invaded cells was significantly decreased in the RIF-EV groups at a concentration of 20 μg/mL.
Fig. 3.
RIF-EVs inhibited the invasive capacity and migration of HTR8/SVneo cells. a Representative images of invaded HTR8/SVneo cells treated with different concentrations of RIF-EVs or FER-EVs. Scale bar = 100 μm. b The number of invaded cells was compared in different groups. c Representative images showed the migration of HTR8/SVneo cells in the two treatments. d The migration of HTR8/SVneo cells was compared between the RIF-EV and FER-EV groups. Bars that do not share the same letters (superscripts) are significantly different from each other (p < 0.05). Data were significantly different between the two linked groups (*p < 0.05). EVs, extracellular vesicles; RIF, recurrent implantation failure; FER, fertile women
RIF-EVs decreased the migration of HTR8/SVneo cells
To detect the migration of HTR8/SVneo cells, a wound healing assay was performed. The representative images of scratches are shown in Fig. 3c. The percentage of wound closure increased in a time-dependent manner in both groups (Fig. 3d). When all data were compared, the percentage of wound closure was significantly decreased in the RIF-EV groups compared to the FER-EV groups at 12 h.
Discussion
RIF remains a challenge in ART that is yet to be overcome [27]. Although the pathogenesis of RIF has not been well understood, endometrial factors have increasingly been considered potential causes for implantation failure. A large number of studies have indicated that RNA and protein profiles are altered in the endometria of RIF patients [2, 8, 28, 29]. However, the mechanism by which distorted endometria interfere with implantation has not been addressed. Therefore, to investigate the effects of distorted endometria on trophoblasts in RIF patients, a co-culture model was constructed using endometrial EVs and HTR8/SVneo cells. In this study, EVs were isolated from the conditioned medium of endometrial cells and characterized by Western blotting, NTA, and TEM. Our results indicated that both RIF-EVs and FER-EVs were positive for EV markers and negative for calnexin, which excluded the contamination of cellular components. Additionally, RIF-EVs and FER-EVs presented with classic bilayer membrane morphology. These results confirmed the purity of endometrial EVs. Furthermore, these data also indicated that endometrial cells were able to secret EVs in vitro, which was in agreement with previous reports [10, 30].
Implantation is a complicated process that occurs at the embryo-maternal interface and involves several critical biological events, including apposition, adhesion, and invasion. Therefore, to determine the effects of RIF-EVs on embryonic cells, HTR8/SVneo cells were treated with EVs, and cellular proliferation, migration, and invasive capacity were examined. First, fluorescence-labeled EVs were co-cultured with HTR8/SVneo cells to visualize the internalization of EVs. Both RIF-EVs and FER-EVs were taken up by trophoblasts. These results were consistent with those of a previous study showing the internalization of EVs by trophoblasts within 3 h [31]. After co-culture, the proliferation of HTR8/SVneo cells was increased with the increasing concentration of FER-EVs in the FER-EV group, indicating that EVs from normal endometria promoted the growth of trophoblasts. Furthermore, this phenomenon was in agreement with a previous study indicating that endometria-derived EVs accelerated blastomere proliferation of murine embryos [32]. Interestingly, the proliferation rate was similar between the RIF-EV groups, suggesting that RIF-EVs had no stimulating effects on the proliferation of trophoblasts. During the window of implantation, after blastocysts hatched from the zona pellucida, the rapid proliferation of trophoblasts enabled the formation of large finger-like villous projections that connected and penetrated maternal tissues [33]. Therefore, due to a lack of stimulation by endometrial EVs, the embryos of RIF patients may display reduced implantation capacity.
Next, the invasion capacities of HTR8/SVneo cells that received different treatments were compared. Data showed that the invasive capacity of trophoblast cells increased with the increasing concentration of FER-EVs in the FER-EV groups. Although endometrial EV modulation of trophoblast invasion has seldom been investigated, several studies have revealed that endometrial EVs contained specific proteins, such as EMMPRIN and matrix metalloproteinases, which could be delivered to embryos and facilitate implantation [17, 34, 35]. Therefore, the upregulation of invasive capacity in the FER-EV-treated cells was reasonable. On the other hand, the invasion and migration were significantly decreased in the RIF-EV-treated cells. Given the crucial roles of migration and invasion during the implantation process, the endometrial-EV-induced trophoblast dysfunction in RIF patients may increase the risk of implantation failure.
EVs have been found to transfer information to recipient cells and modulate their functions [35]. Therefore, identifying the contents of RIF-EVs and FER-EVs may aid our understanding of the different effects of EVs on trophoblasts. EVs contain multiple types of molecules, including DNA, RNA, and proteins [36]. Given that EVs are capable of rapidly modulating cellular invasion and migration (within 6 h), we assumed that proteins were responsible for these regulations. The protein signature of EVs was unique and specific to their originating cells [37, 38]. Under normal conditions, endometrial EVs contain embryotrophic proteins that facilitate embryonic development and trophoblast differentiation [39]. Given the altered molecular repertoires in the endometria of RIF patients, it is reasonable to speculate that RIF-EVs may deliver an aberrant profile of proteins to embryos, thereby inhibiting the implantation process. To date, the protein composition of endometrial EVs from RIF patients is not available in the literature. Kasvandik et al. identified the proteome in uterine fluid from RIF patients and fertile women and found that the fluid was enriched in exosomal and extracellular membrane proteins [40]; they proposed that the detected proteins may be derived from endometrial EVs. Moreover, several proteins, such as NNMT, which promotes cellular invasion and migration, were significantly downregulated in the uterine fluid of RIF patients. According to our hypothesis, the decreased expression of these invasion-related proteins in the endometria may reduce the possibility of transferring them to embryonic cells via the EV pathway, and this could be related to implantation failure. Therefore, our study connected the alterations of molecular repertoires in the endometria with the dynamic implantation process via EV pathways and provided direct evidence indicating the association between distorted endometria and embryonic cells.
Conclusion
In summary, this study proposed that endometrial EVs from RIF patients inhibited trophoblast cells by decreasing their proliferation, migration, and invasive capacity. Such dysregulations may be related to the pathogenesis of implantation failure and can provide novel insights to improve our understanding of RIF. However, the aforementioned theory is still limited. It is important to note that EVs contain a broad spectrum of molecule categories and their contents are variable. Aside from proteins, other bioactive molecules like RNAs, which function to directly regulate or serve as templates for protein production in recipient cells, may also be involved in the regulation of trophoblast cells. Therefore, further studies are required to investigate the precise mechanisms underlying these potential regulatory processes.
Authors’ contributions
C.L. designed the experiments. C.L., L.L., H.Y., and S.S. collected the endometrial samples. C.L., S.S., and M.W. performed the experiments. C.L. wrote the manuscript, which was revised by H.Z. and C.S.
Funding
The current study was supported by grants from the National Natural Science Foundation of China (NSFC 81771582 and NSFC 81701450).
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.