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Article

Effect of EVT-Derived Small Extracellular Vesicles on Normal and Impaired Human Implantation

by
Marina Alexandrova
1,
Mariela Ivanova
1,
Ivaylo Vangelov
1,
Iana Hristova
2 and
Tanya Dimova
1,*
1
Institute of Biology and Immunology of Reproduction “Acad. Kiril Bratanov”, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
University Obstetrics and Gynecology Hospital “Maichin Dom”, Medical University, 1431 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(16), 8866; https://doi.org/10.3390/app15168866
Submission received: 14 July 2025 / Revised: 7 August 2025 / Accepted: 8 August 2025 / Published: 11 August 2025
(This article belongs to the Special Issue Cell Biology: Latest Advances and Prospects)
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Abstract

Uncontrolled and excessive inflammation could negatively impact embryo implantation, potentially leading to implantation failure or miscarriage. Small extracellular vesicles (sEVs) secreted by extravillous trophoblasts (EVTs) play a significant role in mediating the homeostasis at the maternal–fetal interface. In the present work we assessed the role of EVT-derived sEVs in the protection of the human blastocyst’s integrity and function in a microenvironment with excessive Th1-induced inflammation using the Sw71 blastocyst-like surrogate (Sw71 BLS) as a model of implanting a human embryo. Conditioned media from primary trophoblast-derived EVT cells were used as the source for sEVs’ isolation by precipitation. sEVs were characterized by TEM, IEM, and protein content. To simulate Th1-induced inflammation, we performed TCR stimulation and polyclonal activation of isolated T cells, which preferentially led to Th1 cytokine production. The use of the Sw71 spheroid model allowed us to monitor directly the damaging effect of high levels of Th1 cytokines on the ability of trophoblast cells to self-organize and migrate. The addition of EVT-sEVs unlocked the absolute migration capacity of the trophoblast cells in a healthy microenvironment. However, EVT-sEV treatment could not counteract the adverse effects of excessive Th1-mediated inflammation. This study provides a platform for further elucidation of the EVT-sEV dosage and potency for trophoblast functional recovery.

1. Introduction

Human reproduction is an inefficient process, with only one in three pregnancies possibly resulting in a live birth [1,2,3,4]. Implantation is the first and most critical period of pregnancy. Indeed, early pregnancy loss due to implantation failure occurs in about 30% of conceptions in both natural and assisted reproduction. Another 30% of losses come in the immediate post-implantation stages, a significant proportion of which are due to ineffective placentation [1,2]. The main causes of implantation failure are the presence of a damaged embryo, an endometrium with insufficient receptivity, or a lack of synchronization [2,5,6], as well as a hostile environment in the uterus [7]. A successful pregnancy requires the acceptance and maintenance of the semi-allogeneic embryo. All of this is supported by ensuring the effective protection of the mother and fetus [8,9] against pathogens and neoplastic transformation. Efficient implantation requires mild inflammation as it helps the embryo attach to the uterine wall and triggers the angiogenesis to nourish the developing pregnancy [10,11]. However, the uncontrolled and excessive inflammation with differing etiology could negatively impact embryo implantation, potentially leading to implantation failure or miscarriage [7,12,13]. Small extracellular vesicles (sEVs) secreted by the placenta play a significant role in mediating the homeostasis at the maternal–fetal interface (MFI) and, in particular, in navigating the implantation [14,15,16,17]. They contribute to the regulation of female fertility and implantation, as well as the early stages of pregnancy. The sEVs transmit cellular signals between maternal and fetal tissues. During the first trimester of a healthy pregnancy, their number in maternal plasma increases significantly, but they have a significant role in maintaining homeostasis in the later stages of pregnancy as well [18,19]. Understanding the origin of these sEVs reveals further insights into their crucial roles. sEVs originate not only from sources such as follicular fluid and the endometrium, but also from trophoblast cells. Both endometrial and trophoblast sEVs are known to support the invasion of the embryo into the endometrium [20,21]. Extravillous trophoblast cells (EVTs) are a special population of trophoblasts cells that actively participate in implantation and the formation of a functional placenta. They invade deep into the endometrial wall to reconstruct the uterine spiral arteries and turn them into sinusoidal vessels with high conductivity, a guarantee of balanced blood flow to the site of implantation/placenta [22]. Moreover, EVTs have an atypical HLA profile. This unique characteristic allows them to interact with maternal immune cells at the site of contact with the embryo. Their goal is to induce immune tolerance and to remodel maternal spiral arteries. [15,23]. Impaired secretion of EVT-derived sEVs is considered a major factor in the pathogenesis of a number of complications during pregnancy [15,24]. For example, dysfunctional remodeling of uterine spiral arteries might be associated with preeclampsia [15,24]. We have already shown that first trimester EVTs secrete the immunosuppressive molecules HLA-G and HLA-C via sEVs [25]. In the present work we assessed the role of EVT-derived sEVs in the protection of the blastocyst’s integrity and function in a microenvironment with excessive Th1-induced inflammation using the Sw71 blastocyst-like surrogate (BLS) as a model of implanting a human embryo.

2. Materials and Methods

2.1. Cell Culture and Collection of Conditioned Media (CM)

2.1.1. Primary EVT Culture

First trimester chorionic tissues were obtained from healthy pregnant women directed to elective abortion (8–12 gestation weeks, gw) at the University Hospital of Obstetrics and Gynecology “Maichin Dom”, Sofia, Bulgaria. The tissue samples were processed within 2 hours (h) using established protocols for the isolation of primary villous trophoblasts combining mild enzymatic treatment and Percoll gradient centrifugation [26] and subsequent culture in DMEM/F12 complete medium for several weeks to obtain EVTs [27]. DMEM/F12 complete medium contained DMEM/F12 (Ref.: PM150312, Pricella, Elabscience, Houston, TX, USA), supplemented with 10% FBS (Ref.: F7524, Sigma -Aldrich, Taufkirchen, Germany), 100 U/mL penicillin/streptomycin (Ref.: 4458, Sigma-Aldrich, Taufkirchen, Germany), 1 mmol/L sodium pyruvate (Ref.: 11360-039, Gibco, Waltham, MA USA), 10 mmol/L HEPES buffer (Ref.: H0887, Sigma), and 0.1 mmol/L MEM essential amino acid mixture (Ref.: M7145, Sigma-Aldrich, Taufkirchen, Germany). To confirm the acquired EVT nature of cultured primary trophoblasts, the simultaneous expression of Vim/CK7/HLA-C/HLA-G was proved by FACS analysis [27]. Confluent EVT cells were used as the source of conditioned media (EVT-CM) for sEVs’ isolation (Figure 1A, up). Since the serum itself contains sEVs, 48 h before EVT-CM collection the monolayer cultures were washed with PBS and the culture medium was replaced with serum-free DMEM/F12 complete medium. The EVT-CM was collected after centrifugation at 2000× g for 30 min for debris removal and immediately processed for sEVs’ isolation via precipitation. Four independent EVT-CM collections were performed.

2.1.2. Sw71 EVT-Like Cell Culture

The Sw71 EVT-like cell line (CVCL-D855) is established from normal first trimester human placenta. It is a non-cancerous, trophoblastic cell line immortalized by ectopic expression of the telomerase catalytic subunit [28]. Sw71 cells resemble primary EVTs by their simultaneous expression of Vim/CK7/HLA-C/HLA-G, and their ability to migrate and degrade the extracellular matrix, invading between endometrial stromal cells [27,29,30]. The propagation of Sw71 EVT-like cells was in 5 mL DMEM/F12 complete medium using T-25 flasks (Ref.: 430639, Corning, Amsterdam, The Netherlands). For differentiation of 3D Sw71 BLS (spheroids) we used a green fluorescent protein (GFP)-expressing variant of the Sw71 EVT-like cells (Figure 1A, middle).

2.1.3. Effector T-Cells’ (Teff) Generation

Peripheral mononuclear cells (PBMCs) were isolated from fresh pooled blood samples, obtained from healthy donors in heparin-coated vacutainer tubes (BD Biosciences, Franklin Lakes, NJ, USA). Blood was diluted twice with PBS subjected to PBMC isolation using Lymphoprep (density: 1.077 g/mL; Axis-Shield PoCAS, Oslo, Norway). After centrifugation for 20 min at 800× g (brake off), the PBMC band was collected, washed with PBS, and assessed for viability with the trypan blue exclusion test. T cells were isolated from PBMCs with a commercial kit (Human T Lymphocyte Enrichment Set-DM, Ref.: 557874, BD). A T cell population with purity above 90% was subjected to specific TCR stimulation with antibodies against CD3 and CD28 receptors and IL-2 for 72 h, followed by resting/differentiation without stimuli for 48 h. Then polyclonal PMA/Ionomycin activation for 4 h was performed to obtain T effector cells (Teff, Figure 1A, down). Anti-CD3/anti-CD28 stimulation allows activation of over 80% of T cells without any need for antigen-presenting cells. PMA/Ionomycin activates the cells that are already primed to produce the cytokines. The culture medium for T cells was RPMI complete containing RPMI-1640 (Ref.: R8758, Sigma-Aldrich), supplemented with 10% FBS (Ref.: F7524, Sigma), 100 U/mL penicillin/streptomycin (Ref.: 4458, Sigma-Aldrich), 1 mmol/L sodium pyruvate (Ref.: 11360-039, Gibco), 10 mmol/L HEPES buffer (Ref.: H0887, Sigma), and 0.1 mmol/L MEM essential amino acid mixture (Ref.: M7145, Sigma). Teff cells were harvested for FACS analysis. Collected Teff-CM was centrifuged and stored at −80 °C for subsequent treatment of Sw71 spheroids to simulate Th1-induced inflammation. Four independent Teff-CM collections were performed.

2.2. FACS Analysis

The effective TCR stimulation, differentiation, and polyclonal activation were proved by FACS analysis of T cells. Flow cytometry was employed to analyze the proportions of CD4 and CD8 subpopulations within Teff as well as the expression of the activation markers CD69, CD25, and HLA-DR, differentiation markers CD27 and CD28, the functional markers such as Th1 cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα), and cytotoxic molecules Granzyme B and Perforin. In brief, cells adjusted to a concentration of 1 × 106 per sample were subjected to immunofluorescent staining with the following monoclonal antibodies in appropriate combinations: CD4 (Ref.: H10-041-02-H32, clone: OKT-4, SungenBiotech, Beijing, China), CD8 (Ref.: H10 081-09-G, clone: HIT8a, SungenBiotech, Beijing, China), CD69 (Ref.: 555531, clone: FN50, BD Pharmingen, San Jose, CA, USA), CD25 (Ref.: E-AB-F1194E, clone: BC96, Elabscience), HLA-DR (Ref.: 559866, clone: G46-6, BD Pharmingen), CD27 (Ref.: 48-0279-42, clone: 0323, Invitrogen Ebioscience, San Diego, USA), CD28 (Ref.: 25-0289-42, clone: CD28.2, Invitrogen Ebioscience), IFNγ (Ref.: E-AB-F1196E, clone: B27, Elabscience, San Diego, USA), TNFα (Ref.: 559321, clone: Mab11, BD Pharmingen, San Jose, CA, USA), Granzyme B (Ref.: 560211, clone: GB11, BD Pharmingen, San Jose, USA), and Perforin (Ref.: 51-65994X, clone: deltaG9, BD Pharmingen, San Jose, USA). Following incubation at 4 °C in the dark for 20 min, the cells were washed with ice-cold FACS buffer (PBS with 0.1% BSA) and fixed in 0.5% PFA in PBS.

2.3. Differentiation of 3D Sw71 BLS as Spheroid Trophoblast Model of an Implanting Human Blastocyst

As mentioned above, for differentiation of 3D Sw71 spheroids we used the GFP-expressing variant of Sw71 EVT-like cells (Figure 1A, middle) applying the established protocols [29,30]. Briefly, monolayered Sw71 trophoblasts were collected and counted and assessed for vitality with trypan blue dye (Ref.: 93595, Sigma-Aldrich). At over 90% viability of the culture after trypsinization, 4000 cells per well were seeded in a special ultra-low attachment 96-well U-shaped bottom plate (Ref.: 7007, ULA, Costar, Amsterdam, The Netherlands), centrifuged at 300× g and incubated at 37 °C for 48 h with 5% CO2 and 95% humidity. The generation of Sw71 BLS was monitored with a time-lapse live cell imaging system Omni (Axion Biosystems, Inc., Atlanta, GA, USA) and additionally captured in detail at 0, 24, and 48 h with an ECHO Revolve hybrid microscope (RVL-100-M, Echo, San Diego, CA, USA). During differentiation, Sw71 EVT-like cells gradually organize from a monolayer, collected at the bottom of the wells into a loose, spheroid structure, and for 48 h give a dense spheroid with a clearly defined periphery, stable for transfer and suitable for functional experiments (Figure 2). Fully differentiated Sw71 spheroids at 48 h (day 2) were assessed morphologically by measurement of the mean diameter, area, roundness, and volume. The mean diameter was determined as the mean of the two largest, perpendicular diameters. The area was calculated directly with ECHO Revolve Software (ECHO Pro, RVL-100-M, San Diego, CA, USA). The roundness is determined by the index from the spheroid’s larger diameter divided by the smaller diameter. The closer the index is to 1, the closer the Sw71 spheroid is to the shape of an ideal sphere. To determine the volume, we used the following formula: V = 0.52 × [larger diameter] 2 × smaller diameter. It is important to note that Sw71 BLSs are a relatively short-living culture and need to be generated de novo for each functional experiment [30].

2.4. Migration Assay of Sw71 Spheroids

Briefly, Sw71 spheroids were placed in a 96-well flat-bottom cell culture plate to allow the attachment and radial migration of EVT-like Sw71 cells. Migration was performed in Opti-MEM medium (Ref.: 51985-034, Gibco) for 48 h and monitored with the time-lapse imaging system Omni (Axion Biosystems, Inc., Atlanta, GA, USA) and ECHO Revolve microscope (RVL-100-M, Echo, San Diego, CA, USA). The trophoblast migration from the Sw71 spheroid periphery resembles the implanting EVT behavior. In the first few hours of culture, the spheroids are firmly attached to the bottom of the plate, as the embryo sinks steadily into the decidua. Gradually, single Sw71 EVT-like cells begin to detach from the periphery of the spheroid, similarly to the EVT, emerging from the anchoring villi of the chorion and invading the decidual stroma. This way, a so-called “migration zone” is formed around the spheroid. The number of migrating cells increases with time, and the peripheral zone of the spheroid loses its clear outline. Sometimes, empty spaces, called lacunae, form in the migration zone. Migration was evaluated by migration rate (migrating spheroids out of all tested) and area of the migration zone. The area of the migration zone was calculated using ECHO Revolve Software and the following formula:
M i g r a t i o n   z o n e = t o t a l   a r e a   o f   m i g r a t i o n − r e m a i n i n g   i n t a c t   s p h e r o i d   a r e a − l a c u n a e   a r e a   ( w h e n   p r e s e n t )

2.5. Isolation and Characterization of sEVs from EVT-CM

2.5.1. Isolation by Precipitation

sEVs were precipitated from EVT-CM with a commercial reagent “Total exosome isolation reagent” (Ref.: 4478359, Invitrogen) according to the manufacturer’s protocol. The collected, debris-free EVT-CM was distributed into seven 1.5 mL Eppendorf tubes. Half the volume of the reagent was added to each tube, mixed well, and sEVs allowed to precipitate overnight at 4 °C. Then the samples were centrifuged at 10,000× g for 1 h at 4 °C. The supernatant was carefully discarded, and the pellet containing the isolated sEVs was resuspended in PBS buffer (100 µL/tube). Fresh pooled sEV isolates were used for characterization and subsequent treatment of the Sw71 spheroids (Figure 1B). Four independent isolations of sEVs were performed.

2.5.2. Transmission Electron Microscopy (TEM)

For detailed TEM morphological analysis, sEVs were absorbed onto formvar/carbon-coated nickel grids (15 µL/grid, FCF400-NI, Electron Microscopy Sciences, Hatfield, PA, USA). After drying the sample, the grid was subjected to negative contrasting with a drop of 0.5% uranyl acetate. The dried preparations were observed and photographed with a transmission electron microscope (HR STEM JEOL JEM 2100, Freising, Germany). The mean diameter was calculated in nanometers as the average of the two largest, perpendicular diameters for the sEVs. A total of 114 sEVs were measured using the automatic/microscope scale bar to set the scale and the measure plug in Fiji software (Fiji 7.1.4) [31,32].

2.5.3. Immunogold Electron Microscopy (IEM)

To visualize and localize the specific sEV marker CD63 on the isolated sEVs at the ultrastructural level, we combined the specificity of antibodies with the high resolution of electron microscopy by using colloidal gold particles as electron-dense markers attached to antibodies. EVT-sEV-loaded grids were covered with a drop of PBS containing 1% BSA to block the non-specific staining and then were incubated with primary rabbit anti-human purified anti-CD63 antibody (Ref.: E-AB-63818, Elabscience) for 1 h at room temperature (RT). Negative control grids were incubated with 0.1% BSA/PBS only. After washing with PBS containing 0.1% BSA, the CD63-positive intact sEVs were revealed with secondary, colloidal gold-conjugated (12 nm), goat anti-rabbit IgG (H+L, Ref.:111-205-144, Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h at RT. Following a wash, the grids were negatively stained with uranyl acetate. Dried specimens were examined and documented with TEM (electron microscope HR STEM JEOL JEM 2100, Freising, Germany). Two independent experiments were performed.

2.5.4. Protein Concentration of EVT-sEV Isolates

We used a commercial BCA kit according to the manufacturer’s instructions (Ref.: 71285-3, Novagen, Darmstadt, Germany) to determine the protein content of the suspensions with sEVs. In brief, the method is based on the biuret reaction of the reduction of copper ions (Cu2+ to Cu+) from bicinchoninic acid (BCA) by proteins in an alkaline solution. In the presence of protein in the sample, a purple complex is formed, the color intensity increasing in accordance with the increasing protein concentrations [33]. We normalized the amount of protein in sEV suspensions used for the treatment.

2.6. EVT-sEVs’ Treatment of Sw71 BLSs During Their Differentiation and Migration in Optimal or in Th1-Inflamed Microenvironment (Figure 1C)

  • Experiment 1: EVT-sEV treatment of Sw71 cells during differentiation of Sw71 BLS
We defined 4 groups of Sw71 spheroids (in triplet) as follows: (1) Sw71 BLS differentiating in optimal conditions without EVT-sEV treatment; (2) Sw71 spheroids differentiating in optimal conditions with EVT-sEV treatment; (3) Sw71 spheroids differentiating in a Th1-inflamed microenvironment without EVT-sEV treatment, and (4) Sw71 spheroids differentiating in Th1-inflamed microenvironment with EVT-sEV treatment. To simulate Th1 inflammation (groups 3 and 4) we primed the spheroid cultures with Teff-CM. We added an equal volume of Teff-CM to the culture medium DMEM/F12 complete (50:50) during the differentiation of Sw71 BLSs (48 h). To assess the effect of EVT-sEVs we added 34 µg of sEV precipitate in the culture media of groups 2 and 4.
  • Experiment 2: EVT-sEV treatment of Sw71 cells during migration of Sw71 BLS
We defined 4 groups of Sw71 spheroids (in triplet) as follows: (1) Sw71 spheroids migrating in optimal conditions without EVT-sEV treatment; (2) Sw71 spheroids migrating in optimal conditions with EVT-sEV treatment; (3) Sw71 spheroids migrating in a Th1-inflamed microenvironment without EVT-sEV treatment, and (4) Sw71 spheroids migrating in a Th1-inflamed microenvironment with EVT-sEV treatment. To simulate Th1 inflammation (groups 3 and 4) we primed the spheroid cultures with Teff-CM. We added an equal volume of Teff-CM to OptiMEM culture medium during the migration of Sw71 BLSs (48 h). To assess the effect of EVT-sEVs we added 34 µg of sEV precipitate in the culture media of groups 2 and 4.
Three independent differentiation or migration assays were performed.

2.7. Statistical Analyses

Data were statistically processed with GraphPad Prism v.8.0 software using the unpaired non-parametric Mann–Whitney test for comparison between treated and non-treated groups. Data are shown as mean ± SD. Statistical significance is defined as p < 0.05 (*); p < 0.005 (**); p < 0.001 (***).

3. Results

3.1. Characterization of the Isolated EVT-sEVs

We visualized freshly precipitated EVT-sEVs by TEM and the analysis showed the presence of intact vesicles similar in shape and size between 30 and 77 nm. The average size was 47 ± 11 nm (Figure 3A,B). The precipitated sEVs were positive for the common sEV marker tetraspanin CD63 (Figure 3A). We found a similar amount of protein in all four obtained isolates with a mean concentration of 650 ± 43 µg/mL (Figure 3C). We normalized the amount of EVT-sEVs used for treatment as the content in 34 µg of protein.

3.2. Phenotype and Function of Teff Cells

Our results showed that TCR stimulation and subsequent polyclonal activation leads to the proliferation of mainly CD4 T cells, which are almost twice as many as CD8-positive T cells (Figure S1A,B). Half of the T stimulated and resting cells are activated (CD25-positive) with no significant difference between the CD4 and CD8 subsets. A similar trend is observed for the late activation marker HLA-DR. About 80% of Teff are activated (positive for CD69) with this percentage being valid for the CD4 and CD8 subsets separately (Figure S1B). Both the CD4 and CD8 subsets within Teff cells predominantly produced Th1 cytokines IFNγ and TNFα. The differentiation status of Teff cells shown by CD27/CD28 staining determined several subsets with different effector functions (Figure S1C). The functional activity of the Teff CD4 and Teff CD8 subsets differed significantly with respect to their differentiation profiles. Within the Teff CD4 subset, CD27+CD28+ cells (naive/memory) secreted mainly Th1 cytokines, TNFα and IFNγ, and had no cytotoxic activity. Memory/effector subsets (CD27+CD28− and CD27−CD28−) produced low levels of Th1 cytokines and also had no cytotoxic activity. Long-lived memory CD27-CD28+ cells produced high levels of Th1 cytokines. This functionality is consistent with the helper function of CD4 cells in general. Within the Teff CD8 subset, CD27+CD28+ cells secreted mainly Th1 cytokines, more IFNγ and less TNFα, and produced the cytotoxic molecule granzyme B, but no perforin was detected, which is an indication that these cells have no cytotoxic activity. Within the CD27+CD28− and CD27−CD28− subsets the production of IFNγ is enhanced, while less than 20% of CD27−CD28− cells produce TNFα and the cytotoxic molecules perforin and granzyme B. Nearly 80% of memory long-lived CD27-CD28 clones produce high levels of IFNγ, about 40% TNFα, about 20% granzyme B, and very low levels of perforin. The functional activity of both CD4 and CD8 Teff cells showed that the TCR stimulation/PMA/Ionomycin activation protocol specifically amplified Th1 effector cells.

3.3. Effect of EVT-sEVs on Normal and Th1-Compromised Human Implantation Using Sw71 BLS as a Model

3.3.1. EVT-sEV Treatment Failed to Limit the Th1-Induced Destructive Effect on Differentiating Sw71 BLS

Within 48 h in optimal conditions the Sw71 EVT-like cells were able to organize into a spherical, highly compacted 3D spheroid structure with an intact periphery and stable so as to be transferred for subsequent functional tests. When EVT-sEVs were added to Sw71 spheroids differentiating in optimal conditions, we did not observe a significant effect on their integrity and morphology shown by a lack of difference in their mean diameter (0.325 ± 0.041 mm), area (0.086 ± 0.020 mm2), and volume (0.021 ± 0.007 mm3) when compared to those differentiating in optimal conditions without EVT-sEVs (0.350 ± 0.027 mm, 0.097 ± 0.015 mm2, and 0.023 ± 0.006 mm3, respectively, n = 15, p > 0.05, Figure 4B). In addition, when tested for migration, these Sw71 spheroids were functional without significant difference to non-treated Sw71 spheroids in respect to the migration capacity (100% migrated in both groups) and efficacy (migration zone 1.095 ± 0.561 mm2 vs. 1.213 ± 0.603 mm2, p > 0.05, Figure 4C). However, all tested Sw71 spheroids (n = 20) differentiating in the Th1-inflamed microenvironment were fragmented and disintegrated in contrast to well-differentiated ones under optimal conditions (n = 15, Figure 4A). The EVT-sEV treatment of Sw71 spheroids differentiating in the Th1-inflamed microenvironment failed to limit the destructive effect on their integrity, compaction, and morphology (Figure 4A).

3.3.2. EVT-sEV Treatment Increased the Percentage of the Migrating Sw71 Spheroids

Our data showed that spheroids primed with Teff-CM initiated migration, which was blocked between 24 and 48 h. As shown in Figure 5, the excessive inflammation significantly inhibited Sw71 spheroid migration as shown by the migration rate and efficacy. Twenty-three percent of Sw71 BLSs did not migrate at all (Figure 5A). The migration zone of the rest (77%) was severely impaired and single EVTs only were able to migrate (0.316 ± 0.156 mm2, Figure 5B,C, up). Live and migrating Sw71 cells (Figure 5D) can be easily distinguished morphologically from dead cells flaking off the spheroid (Figure 5C). Interestingly, EVT-sEV treatment resulted in 100% of migrating spheroids either being Teff-CM primed or non-primed (Figure 5A). However, the inflammation-inhibited Sw71 migration efficacy did not improve significantly when EVT-sEVs were added. Although attempting to migrate with larger migration efficacy (0.476 ± 0.177 mm2 zone, p < 0.05, Figure 5B), Sw71 cells were not able to form an intact, clearly defined migration zone (Figure 5C, down). It is important to note that the addition of EVT-sEVs to Sw71 spheroids migrating in optimal conditions also stimulated the EVT migration capacity to 100%, but their migration zone did not increase significantly (Figure 5A,B). For Sw71 BLSs migrating in optimal conditions and EVT-sEV treated, it increased slightly, to 1.819 ± 0.728 mm2, as compared to those non-treated (1.643 ± 0.724 mm2, p > 0.05, Figure 5B).

4. Discussion

In this study we evaluated the potential of EVT-sEVs to confront and limit the destructive effect of excessive inflammation on the trophoblast function. Although mild inflammation is required for effective implantation [10], when the inflammation becomes excessive, the strong maternal immune response hits the semi-allogeneic implanting embryo. A high Th1/Th2 ratio with an irregular inflammatory uterine environment is amongst the immunologic disturbances leading to abnormal implantation/placentation or recurrent implantation failure [34]. Another cause of endometrial excessive inflammation might be the endometriosis that often causes chronic inflammation in the pelvic area leading to female infertility [35]. Bacterial, viral, or fungal infections in the reproductive tract can trigger such an inflammatory response as well [12,13].
To simulate Th1-induced excessive inflammation, we primed EVTs with Teff-CM, characterized by highly elevated levels of Th1 cytokines. T effectors have a strong proinflammatory potential, shown by their phenotype and high production of IFNγ and TNFα. In line with other authors, we found that high expression of CD45RA is a guarantee of the presence of effector T cells in the pool [36]. Additionally, we showed the effective specific TCR-induced proliferation and activation within the pool of Teff cells through the elevated expression of a number of activation markers such as CD25, CD69, HLA-DR [37]. It is not Th1 cytokines in general but their excessive levels and synergistic effect that may be a problem for the normal course of implantation. Although the indispensable role of IFNγ in the remodeling of decidual spiral arteries has been demonstrated [38], the high-dose IFNγ promotes abortion in mice [39] and miscarriage in women [40]. Hu et al., 2006 demonstrated that uNK cell supernatants inhibit EVT migration through an IFNγ-dependent mechanism [41]. While the TNFα-regulated endometrial stroma secretome promotes trophoblast invasion [42], the overproduction of TNFα can lead to harmful inflammation and tissue damage, and can significantly impair embryo implantation [43]. Recently published data also explain cytokine immunopathology with a synergistic effect of TNFα and IFNγ, which induces cell death (pyroptosis and necroptosis) and tissue damage as a result of excessive and uncontrolled inflammation [44].
The priming of the Sw71 spheroid model with Teff-CM allowed us to directly monitor the damaging effect of high levels of Th1 cytokines on the ability of EVT-like Sw71 cells to self-organize and migrate. The standardized and validated Sw71 BLS model is a suitable trophoblast surrogate for the implanting human blastocyst hatched from the zona pellucida [27,29,30,45,46]. Sw71 spheroids have been used to study trophoblast migration and invasion during implantation in normal and pathological conditions [42,47,48,49]. Similar 3D spheroid models, relevant to the study of human embryo implantation, have been constructed with other EVT-like cell lines such as HTR8/SVneo [50]. However, this cell line is a mixed population of trophoblasts and mesenchymal cells from the chorionic villi. In contrast, Sw71 cells have an EVT phenotype [27]. Here we confirmed the destructive effect of both IFNγ and TNFα on the compaction, integrity, and function of Sw71 BLS as demonstrated already in experiments with placental explants [51,52,53].
Given the outlined challenges with inflammation, our investigation extends to evaluating the potential of EVT-sEVs to mitigate the adverse effects on Sw71 EVTs. Although little is known about the mechanism by which packaging occurs, sEVs are exocytosed by the parent cell after fusion of multivesicular bodies with the cell membrane and contain a variety of signaling molecules such as miRNA, mRNA, and cytoplasmic proteins. Several studies suggested the immunomodulatory potential of sEVs released by early and term placenta [14,54,55,56]. Therefore, we hypothesized that sEVs with their high biocompatibility, minimal immunogenicity, and versatility [57] could contribute to the alleviation of Th1-induced inflammation.
We succeeded in the isolation of intact EVT-sEVs and visualized them as nano-sized vesicles by TEM, the gold standard for sEV morphological characterization. The positive signal for tetraspanin CD63 confirmed the nature of EVs. We found that in the absence of proinflammatory stimuli, EVT-sEVs did not influence the main structural characteristics of the Sw71 BLS model (integrity and morphology). Indeed, the addition of EVT-sEVs unlocked the absolute migration capacity of the Sw71 trophoblast cells, in line with published data about the ability of EVT-sEVs to promote the invasion and proliferation of trophoblasts [20,58]. Since sEVs containing TGF-b activity promote tumor cell epithelial–mesenchymal transition (EMT) in vitro [59], this could be a plausible mechanism for the stimulation of EVT migration. EVTs share many characteristics with tumor cells including EMT.
Our results showed, however, that the EVT-sEVs, at least in the applied dose, could not counteract the adverse effects of the excessive Th1-mediated inflammation, i.e., to prevent the disintegration of the Sw71 BLSs and the inhibition of their migration. It has been shown that in a healthy microenvironment, the promoting effect of trophoblast-derived exosomes on trophoblast invasion and proliferation happens when 100 µg exosomal protein per ml for up to 24 h is applied [58]. Our observation that the sEV treatment of Teff-CM-primed Sw71 BLSs reactivated some of the trophoblast cells to migrate, although with a small migration zone, suggests that an increase in the dose of EVT-sEVs might have a beneficial effect or could even restore the trophoblast differentiation and migration. The anti-inflammatory effect of sEVs containing TGF-b and IL-10, as well as specific miRNAs, has already been revealed as being able to reduce inflammation [60,61].
Although there is recognition of the role of EVs in intercellular signaling during implantation and pregnancy, it has been shown that a poor yield of EVs is a significant barrier to their implementation for treatment. As has been shown, the majority of preclinical experimental research uses cell culture to obtain EVs, where the EV protein production is limited to less than 1 µg per ml of culture, needing large-scale cell culture for clinical studies. Different methods to dose EVs are used such as reporting cell equivalents, protein concentration, and/or specialized quantitative analytical measurements by specific instruments studies [57]. We used protein concentration determination as a fast and low-cost method, but it may not reflect bioactive ingredients and may measure non-sEV-associated protein as well [57]. Therefore, the EVT-sEVs’ dosage and the efficacy of the isolation methods deserve further elucidation. Moreover, mass spectroscopic analyses for detailed sEV content would allow us to explore in depth the molecular mechanism behind sEVs promoting migration.

5. Conclusions

In summary, this study highlights the potential of EVT-derived sEVs to promote healthy trophoblast migration. Although the EVT-sEV treatment could not restore the impaired trophoblast migration induced by an excessive Th1-proinflammatory microenvironment, this study provides a platform for further elucidation of the EVT-sEV dosage and potency for trophoblast functional recovery.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15168866/s1, Figure S1: Phenotype and function of Teff cells used for Teff-CM collection.

Author Contributions

Conceptualization, M.A. and T.D.; methodology, M.A., M.I. and T.D.; software, M.A., M.I. and T.D.; validation, M.A., M.I. and T.D.; investigation, M.A., M.I., I.H., I.V. and T.D.; data curation, M.A., M.I. and T.D.; writing—original draft preparation, M.A. and M.I.; writing—review and editing, T.D.; visualization, M.A., M.I. and T.D.; supervision, T.D.; project administration, T.D.; funding acquisition, T.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Bulgarian National Science Fund within the National Science Program VIHREN, contract number KP-06-DV3.

Institutional Review Board Statement

This study was carried out in accordance with the Declaration of Helsinki and was approved by the Human Research Ethics Committee at the University Obstetrics and Gynecology Hospital “Maichin Dom”, Sofia, Bulgaria (250528/2022).

Informed Consent Statement

Informed consent was obtained from all women involved in the study.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The normal, immortalized, first-trimester human placenta Sw71 (#CVCL_D855) trophoblast cell line used in the study was kindly provided by Gil Mor.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BLSBlastocyst-like surrogate
EVTExtravillous trophoblast
sEVsSmall extracellular vesicles
MFIMaternal–fetal interface
IFNγInterferon gamma
TNFαTumor necrosis factor alpha
TEMTransmission electron microscopy
IEMImmunogold electron microscopy
BCABicinchoninic acid
TeffT effector cells
CMConditioned media

References

  1. Chard, T. Frequency of implantation and early pregnancy loss in natural cycles. Baillieres. Clin. Obstet. Gynaecol. 1991, 5, 179–189. [Google Scholar] [CrossRef] [PubMed]
  2. Macklon, N.S.; Geraedts, J.P.M.; Fauser, B.C.J.M. Conception to ongoing pregnancy: The “black box” of early pregnancy loss. Hum. Reprod. Update 2002, 8, 333–343. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, X.; Chen, C.; Wang, L.; Chen, D.; Guang, W.; French, J. Conception, early pregnancy loss, and time to clinical pregnancy: A population-based prospective study. Fertil. Steril. 2003, 79, 577–584. [Google Scholar] [CrossRef] [PubMed]
  4. Muter, J.; Lynch, V.J.; McCoy, R.C.; Brosens, J.J. Human embryo implantation. Development 2023, 150, dev201507. [Google Scholar] [CrossRef] [PubMed]
  5. Kennedy, T.G. Physiology of Implantation. In Proceedings of the 10th World Congress on In Vitro Fertilization And Assisted Reproduction, Vancouver, BC, Canada, 24–28 May 1997. [Google Scholar]
  6. Cakmak, H.; Taylor, H.S. Implantation failure: Molecular mechanisms and clinical treatment. Hum. Reprod. Update 2011, 17, 242–253. [Google Scholar] [CrossRef]
  7. Romero, R.; Espinoza, J.; Mazor, M. Can endometrial infection/inflammation explain implantation failure, spontaneous abortion, and preterm birth after in vitro fertilization? Fertil. Steril. 2004, 82, 799–804. [Google Scholar] [CrossRef] [PubMed]
  8. PrabhuDas, M.; Bonney, E.; Caron, K.; Dey, S.; Erlebacher, A.; Fazleabas, A.; Fisher, S.; Golos, T.; Matzuk, M.; McCune, J.M.; et al. Immune mechanisms at the maternal-fetal interface: Perspectives and challenges. Nat. Immunol. 2015, 16, 328–334. [Google Scholar] [CrossRef]
  9. Bai, K.; Li, X.; Zhong, J.; Ng, E.H.Y.; Yeung, W.S.B.; Lee, C.-L.; Chiu, P.C.N. Placenta-Derived Exosomes as a Modulator in Maternal Immune Tolerance During Pregnancy. Front. Immunol. 2021, 12, 671093. [Google Scholar] [CrossRef]
  10. Dekel, N.; Gnainsky, Y.; Granot, I.; Mor, G. Inflammation and implantation. Am. J. Reprod. Immunol. 2010, 63, 17–21. [Google Scholar] [CrossRef]
  11. Akopians, A.L.; Pisarska, M.D.; Wang, E.T. The Role of Inflammatory Pathways in Implantation Failure: Chronic Endometritis and Hydrosalpinges. Semin. Reprod. Med. 2015, 33, 298–304. [Google Scholar] [CrossRef]
  12. Rokhsartalab Azar, P.; Karimi, S.; Haghtalab, A.; Taram, S.; Hejazi, M.; Sadeghpour, S.; Pashaei, M.R.; Ghasemnejad-Berenji, H.; Taheri-Anganeh, M. The role of the endometrial microbiome in embryo implantation and recurrent implantation failure. J. Reprod. Immunol. 2024, 162, 104192. [Google Scholar] [CrossRef]
  13. Rezaei, M.; Moghoofei, M. The role of viral infection in implantation failure: Direct and indirect effects. Reprod. Biol. Endocrinol. 2024, 22, 142. [Google Scholar] [CrossRef]
  14. Mincheva-Nilsson, L. Placental exosome-mediated immune protection of the fetus: Feeling groovy in a cloud of exosomes. Expert. Rev. Obstet. Gynecol. 2010, 5, 619–634. [Google Scholar] [CrossRef]
  15. Salomon, C.; Yee, S.W.; Mitchell, M.D.; Rice, G.E. The Possible Role of Extravillous Trophoblast-Derived Exosomes on the Uterine Spiral Arterial Remodeling under Both Normal and Pathological Conditions. Biomed Res Int. 2014, 2014, 693157. [Google Scholar] [CrossRef]
  16. Jung, M.K.; Mun, J.Y. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. 2018, 131, e56482. [Google Scholar] [CrossRef]
  17. Czernek, L.; Düchler, M. Exosomes as Messengers Between Mother and Fetus in Pregnancy. Int. J. Mol. Sci. 2020, 21, 4264. [Google Scholar] [CrossRef] [PubMed]
  18. Ghafourian, M.; Mahdavi, R.; Akbari Jonoush, Z.; Sadeghi, M.; Ghadiri, N.; Farzaneh, M.; Mousavi Salehi, A. The implications of exosomes in pregnancy: Emerging as new diagnostic markers and therapeutics targets. Cell Commun. Signal. 2022, 20, 51. [Google Scholar] [CrossRef] [PubMed]
  19. Salomon, C.; Torres, M.J.; Illanes, S.; Kobayashi, M.; Sobrevia, L.; Ashman, K.; Mitchell, M.; Rice, G. Placental cell-derived exosomes increase in maternal circulation with gestational age. Placenta 2013, 34, A79–A80. [Google Scholar] [CrossRef]
  20. Atay, S.; Gercel-Taylor, C.; Kesimer, M.; Taylor, D.D. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells. Exp. Cell Res. 2011, 317, 1192–1202. [Google Scholar] [CrossRef]
  21. Guo, X.R.; Ma, Y.; Ma, Z.M.; Dai, T.S.; Wei, S.H.; Chu, Y.K.; Dan, X.G. Exosomes: The role in mammalian reproductive regulation and pregnancy-related diseases. Front. Physiol. 2023, 14, 1056905. [Google Scholar] [CrossRef] [PubMed]
  22. Burton, G.J.; Woods, A.W.; Jauniaux, E.; Kingdom, J.C.P. Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta 2009, 30, 473–482. [Google Scholar] [CrossRef] [PubMed]
  23. Tersigni, C.; Meli, F.; Neri, C.; Iacoangeli, A.; Franco, R.; Lanzone, A.; Scambia, G.; Di Simone, N. Role of human leukocyte antigens at the feto-maternal interface in normal and pathological pregnancy: An update. Int. J. Mol. Sci. 2020, 21, 4756. [Google Scholar] [CrossRef]
  24. Liu, H.; Chen, M.; Ning, F.; Ye, Y.; Lu, Q.; Lu, S.; Duan, Y.; Gan, X.; Zhao, M.; Guo, K.; et al. Extravillous trophoblast cell-derived exosomes induce vascular smooth muscle cell apoptosis via a mechanism associated with miR-143-3p. Mol. Hum. Reprod. 2023, 29, gaad026. [Google Scholar] [CrossRef]
  25. Alexandrova, M.; Manchorova, D.; Vangelov, I.; Terzieva, A.; Dimitrova, V.; Mor, G.; Dimova, T. First trimester extravillous trophoblast secretes HLA class I molecules via small extracellular vesicles. Placenta 2025, 167, 11–21. [Google Scholar] [CrossRef]
  26. Stenqvist, A.-C.; Chen, T.; Hedlund, M.; Dimova, T.; Nagaeva, O.; Kjellberg, L.; Innala, E.; Mincheva-Nilsson, L. An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies. Am. J. Reprod. Immunol. 2008, 60, 33–42. [Google Scholar] [CrossRef]
  27. Alexandrova, M.; Manchorova, D.; You, Y.; Terzieva, A.; Dimitrova, V.; Mor, G.; Dimova, T. Validation of the Sw71-spheroid model with primary trophoblast cells. Am. J. Reprod. Immunol. 2023, 90, e13800. [Google Scholar] [CrossRef]
  28. Straszewski-Chavez, S.L.; Abrahams, V.M.; Alvero, A.B.; Aldo, P.B.; Ma, Y.; Guller, S.; Romero, R.; Mor, G. The Isolation and Characterization of a Novel Telomerase Immortalized First Trimester Trophoblast Cell Line, Swan 71. Placenta 2009, 30, 939–948. [Google Scholar] [CrossRef] [PubMed]
  29. You, Y.; Stelzl, P.; Zhang, Y.; Porter, J.; Liu, H.; Liao, A.H.; Aldo, P.B.; Mor, G. Novel 3D in vitro models to evaluate trophoblast migration and invasion. Am. J. Reprod. Immunol. 2019, 81, e13076. [Google Scholar] [CrossRef] [PubMed]
  30. Alexandrova, M.; Manchorova, D.; You, Y.; Mor, G.; Dimitrova, V.; Dimova, T. Functional HLA-C expressing trophoblast spheroids as a model to study placental–maternal immune interactions during human implantation. Sci. Rep. 2022, 12, 10224. [Google Scholar] [CrossRef]
  31. Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676–682. [Google Scholar] [CrossRef]
  32. Kotrbová, A.; Štěpka, K.; Maška, M.; Pálenik, J.J.; Ilkovics, L.; Klemová, D.; Kravec, M.; Hubatka, F.; Dave, Z.; Hampl, A.; et al. TEM ExosomeAnalyzer: A computer-assisted software tool for quantitative evaluation of extracellular vesicles in transmission electron microscopy images. J. Extracell. Vesicles 2019, 8, 1560808. [Google Scholar] [CrossRef] [PubMed]
  33. Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76–85. [Google Scholar] [CrossRef] [PubMed]
  34. Lédée, N.; Petitbarat, M.; Chevrier, L.; Vitoux, D.; Vezmar, K.; Rahmati, M.; Dubanchet, S.; Gahéry, H.; Bensussan, A.; Chaouat, G. The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization. Am. J. Reprod. Immunol. 2016, 75, 388–401. [Google Scholar] [CrossRef]
  35. Boucher, A.; Brichant, G.; Gridelet, V.; Nisolle, M.; Ravet, S.; Timmermans, M.; Henry, L. Implantation Failure in Endometriosis Patients: Etiopathogenesis. J. Clin. Med. 2022, 11, 5366. [Google Scholar] [CrossRef] [PubMed]
  36. Maecker, H.T.; McCoy, J.P.; Nussenblatt, R. Standardizing immunophenotyping for the Human Immunology Project. Nat. Rev. Immunol. 2012, 12, 191–200. [Google Scholar] [CrossRef]
  37. Sainz, T.; Serrano-Villar, S.; Díaz, L.; González Tomé, M.I.; Gurbindo, M.D.; de José, M.I.; Mellado, M.J.; Ramos, J.T.; Zamora, J.; Moreno, S.; et al. The CD4/CD8 ratio as a marker T-cell activation, senescence and activation/exhaustion in treated HIV-infected children and young adults. AIDS 2013, 27, 1513–1516. [Google Scholar] [CrossRef] [PubMed]
  38. Ashkar, A.A.; Di Santo, J.P.; Croy, B.A. Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J. Exp. Med. 2000, 192, 259–270. [Google Scholar] [CrossRef]
  39. Liu, H.-Y.; Liu, Z.-K.; Chao, H.; Li, Z.; Song, Z.; Yang, Y.; Peng, J.-P. High-dose interferon-γ promotes abortion in mice by suppressing Treg and Th17 polarization. J. Interf. cytokine Res. Off. J. Int. Soc. Interf. Cytokine Res. 2014, 34, 394–403. [Google Scholar] [CrossRef] [PubMed]
  40. Jenkins, C.; Roberts, J.; Wilson, R.; MacLean, M.A.; Shilito, J.; Walker, J.J. Evidence of a T(H) 1 type response associated with recurrent miscarriage. Fertil. Steril. 2000, 73, 1206–1208. [Google Scholar] [CrossRef]
  41. Hu, Y.; Dutz, J.P.; MacCalman, C.D.; Yong, P.; Tan, R.; von Dadelszen, P. Decidual NK cells alter in vitro first trimester extravillous cytotrophoblast migration: A role for IFN-gamma. J. Immunol. 2006, 177, 8522–8530. [Google Scholar] [CrossRef]
  42. You, Y.; Stelzl, P.; Joseph, D.N.; Aldo, P.B.; Maxwell, A.J.; Dekel, N.; Liao, A.; Whirledge, S.; Mor, G. TNF-α Regulated Endometrial Stroma Secretome Promotes Trophoblast Invasion. Front. Immunol. 2021, 12, 737401. [Google Scholar] [CrossRef] [PubMed]
  43. Dai, F.-F.; Hu, M.; Zhang, Y.-W.; Zhu, R.-H.; Chen, L.-P.; Li, Z.-D.; Huang, Y.-J.; Hu, W.; Cheng, Y.-X. TNF-α/anti-TNF-α drugs and its effect on pregnancy outcomes. Expert. Rev. Mol. Med. 2022, 24, e26. [Google Scholar] [CrossRef] [PubMed]
  44. Kruse, B.; Buzzai, A.C.; Shridhar, N.; Braun, A.D.; Gellert, S.; Knauth, K.; Pozniak, J.; Peters, J.; Dittmann, P.; Mengoni, M.; et al. CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours. Nature 2023, 618, 1033–1040. [Google Scholar] [CrossRef] [PubMed]
  45. Holmberg, J.C.; Haddad, S.; Wünsche, V.; Yang, Y.; Aldo, P.B.; Gnainsky, Y.; Granot, I.; Dekel, N.; Mor, G. An in vitro model for the study of human implantation. Am. J. Reprod. Immunol. 2012, 67, 169–178. [Google Scholar] [CrossRef]
  46. Dimova, T.; Alexandrova, M.; Vangelov, I.; You, Y.; Mor, G. The modeling of human implantation and early placentation: Achievements and perspectives. Hum. Reprod. Update 2024, 31, 133–163. [Google Scholar] [CrossRef]
  47. Racicot, K.E.; Wünsche, V.; Auerbach, B.; Aldo, P.; Silasi, M.; Mor, G. Human chorionic gonadotropin enhances trophoblast-epithelial interaction in an in vitro model of human implantation. Reprod. Sci. 2014, 21, 1274–1280. [Google Scholar] [CrossRef]
  48. Grasso, E.; Gori, S.; Soczewski, E.; Fernández, L.; Gallino, L.; Vota, D.; Martínez, G.; Irigoyen, M.; Ruhlmann, C.; Lobo, T.F.; et al. Impact of the Reticular Stress and Unfolded Protein Response on the inflammatory response in endometrial stromal cells. Sci. Rep. 2018, 8, 12274. [Google Scholar] [CrossRef] [PubMed]
  49. Guzman-Genuino, R.M.; Dimova, T.; You, Y.; Aldo, P.; Hayball, J.D.; Mor, G.; Diener, K.R. Trophoblasts promote induction of a regulatory phenotype in B cells that can protect against detrimental T cell-mediated inflammation. Am. J. Reprod. Immunol. 2019, 82, e13187. [Google Scholar] [CrossRef]
  50. Wong, M.K.; Wahed, M.; Shawky, S.A.; Dvorkin-Gheva, A.; Raha, S. Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids. Sci. Rep. 2019, 9, 12607. [Google Scholar] [CrossRef] [PubMed]
  51. Otun, H.A.; Lash, G.E.; Innes, B.A.; Bulmer, J.N.; Naruse, K.; Hannon, T.; Searle, R.F.; Robson, S.C. Effect of tumour necrosis factor-α in combination with interferon-γ on first trimester extravillous trophoblast invasion. J. Reprod. Immunol. 2011, 88, 1–11. [Google Scholar] [CrossRef]
  52. Lash, G.E.; Otun, H.A.; Innes, B.A.; Bulmer, J.N.; Searle, R.F.; Robson, S.C. Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases. Biol. Reprod. 2005, 73, 374–381. [Google Scholar] [CrossRef] [PubMed]
  53. Lash, G.E.; Otun, H.A.; Innes, B.A.; Kirkley, M.; De Oliveira, L.; Searle, R.F.; Robson, S.C.; Bulmer, J.N. Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2006, 20, 2512–2518. [Google Scholar] [CrossRef]
  54. Kshirsagar, S.K.; Alam, S.M.; Jasti, S.; Hodes, H.; Nauser, T.; Gilliam, M.; Billstrand, C.; Hunt, J.S.; Petroff, M.G. Immunomodulatory molecules are released from the first trimester and term placenta via exosomes. Placenta 2012, 33, 982–990. [Google Scholar] [CrossRef]
  55. Hedlund, M.; Stenqvist, A.-C.; Nagaeva, O.; Kjellberg, L.; Wulff, M.; Baranov, V.; Mincheva-Nilsson, L. Human Placenta Expresses and Secretes NKG2D Ligands via Exosomes that Down-Modulate the Cognate Receptor Expression: Evidence for Immunosuppressive Function. J. Immunol. 2009, 183, 340–351. [Google Scholar] [CrossRef]
  56. Stenqvist, A.-C.; Nagaeva, O.; Baranov, V.; Mincheva-Nilsson, L. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus. J. Immunol. 2013, 191, 5515–5523. [Google Scholar] [CrossRef]
  57. Willis, G.R.; Kourembanas, S.; Mitsialis, S.A. Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency. Front. Cardiovasc. Med. 2017, 4, 63. [Google Scholar] [CrossRef]
  58. Salomon, C.; Kobayashi, M.; Ashman, K.; Sobrevia, L.; Mitchell, M.D.; Rice, G.E. Hypoxia-Induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes. PLoS ONE 2013, 8, e79636. [Google Scholar] [CrossRef] [PubMed]
  59. Li, W.; Zhang, X.; Wang, J.; Li, M.; Cao, C.; Tan, J.; Ma, D.; Gao, Q. TGFbeta1 in fibroblasts-derived exosomes promotes epithelial-mesenchymal transition of ovarian cancer cells. Oncotarget 2017, 8, 96035–96047. [Google Scholar] [CrossRef] [PubMed]
  60. Huang, S.; Yan, F.; Qiu, Y.; Liu, T.; Zhang, W.; Yang, Y.; Zhong, R.; Yang, Y.; Peng, X. Exosomes in inflammation and cancer: From bench to bedside applications. Mol. Biomed. 2025, 6, 41. [Google Scholar] [CrossRef] [PubMed]
  61. Yoo, K.H.; Thapa, N.; Chwae, Y.J.; Yoon, S.H.; Kim, B.J.; Lee, J.O.; Jang, Y.; Kim, J. Transforming growth factor β family and stem cell derived exosome therapeutic treatment in osteoarthritis (Review). Int. J. Mol. Med. 2022, 49, 62. [Google Scholar] [CrossRef] [PubMed]
Applsci 15 08866 g001
Figure 1. Study design: (A) Up: Primary EVT cells were cultured as a monolayer in complete DMEM/F12 medium supplemented with 10% serum until confluency (n = 4). Forty-eight hours before EVT-CM collection, cells were washed with PBS buffer and the culture medium was replaced with serum-free DMEM/F12. The EVT-CM was collected 48 h later. Middle: Sw71-GFP EVT-like trophoblasts were cultured for differentiation of Sw71 spheroids. Down: Teff-CM was collected from TCR stimulated, differentiated, polyclonal activated, effector T lymphocytes with predominating Th1 cytokine producing CD4 T helpers. (B) sEVs were precipitated from serum-free EVT-CM (n = 4) and characterized by protein concentration (BCA, n = 4), transmission electron microscopy (TEM, n = 2), and immunogold electron microscopy (IEM, n = 2) analyses. (C) Sw71 spheroids were treated with sEVs during compromised conditions (Teff-CM-induced excessive inflammation) versus differentiation or migration under optimal conditions.
Figure 1. Study design: (A) Up: Primary EVT cells were cultured as a monolayer in complete DMEM/F12 medium supplemented with 10% serum until confluency (n = 4). Forty-eight hours before EVT-CM collection, cells were washed with PBS buffer and the culture medium was replaced with serum-free DMEM/F12. The EVT-CM was collected 48 h later. Middle: Sw71-GFP EVT-like trophoblasts were cultured for differentiation of Sw71 spheroids. Down: Teff-CM was collected from TCR stimulated, differentiated, polyclonal activated, effector T lymphocytes with predominating Th1 cytokine producing CD4 T helpers. (B) sEVs were precipitated from serum-free EVT-CM (n = 4) and characterized by protein concentration (BCA, n = 4), transmission electron microscopy (TEM, n = 2), and immunogold electron microscopy (IEM, n = 2) analyses. (C) Sw71 spheroids were treated with sEVs during compromised conditions (Teff-CM-induced excessive inflammation) versus differentiation or migration under optimal conditions.
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Figure 2. Differentiation of Sw71 spheroids. Up: representative, Sw71-GFP BLS in the FITC channel, showing changes in the shape and dimensions of the 3D structure during the 48 h of differentiation; middle: morphological characterization—measurements of the same Sw71 BLS during differentiation on day 0, day 1, and day 2; down: schematic view of the Sw71-GFP cells’ compactization into an Sw71 spheroid within 48 h. Magnification: 4× on day 0 and 10× on day 1 and day 2.
Figure 2. Differentiation of Sw71 spheroids. Up: representative, Sw71-GFP BLS in the FITC channel, showing changes in the shape and dimensions of the 3D structure during the 48 h of differentiation; middle: morphological characterization—measurements of the same Sw71 BLS during differentiation on day 0, day 1, and day 2; down: schematic view of the Sw71-GFP cells’ compactization into an Sw71 spheroid within 48 h. Magnification: 4× on day 0 and 10× on day 1 and day 2.
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Figure 3. Visualization and characterization of the isolated EVT-sEVs. (A) TEM of homogeneous, nano-sized EVT-sEVs (arrows, representative for n = 2) and CD63 expression confirmed by IEM (arrows), magnification: 15,000× (left), 100,000× (right); (B) size distribution of the measured EVT-sEVs (n = 114) according their mean diameter; (C) protein content of the precipitated sEV samples in µg/mL and as mean ± SD (n = 4).
Figure 3. Visualization and characterization of the isolated EVT-sEVs. (A) TEM of homogeneous, nano-sized EVT-sEVs (arrows, representative for n = 2) and CD63 expression confirmed by IEM (arrows), magnification: 15,000× (left), 100,000× (right); (B) size distribution of the measured EVT-sEVs (n = 114) according their mean diameter; (C) protein content of the precipitated sEV samples in µg/mL and as mean ± SD (n = 4).
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Figure 4. Effect of EVT-sEVs on normal and Th1-compromised differentiation of Sw71 BLS. (A) Differentiation of a Sw71 spheroid compromised by Th1-driven excessive inflammation or in optimal conditions. Up: Percentage of intact Sw71 BLS in Th1-induced excessive inflammation as compared to optimal conditions and effect of EVT-sEV treatment. Middle: Differentiated Sw71 spheroid (Day 2) primed with Teff-CM (1, representative for n = 10) or in optimal conditions (2, representative for n = 15). Down: 1. Differentiated Sw71 spheroid primed with Teff-CM and treated with EVT-sEVs for 48 h (representative for n = 10), 2. EVT-sEV treatment of Sw71 spheroid in optimal conditions (representative for n = 15); (B) the EVT-sEV treatment of Sw71 BLS during differentiation in optimal conditions had no effect on their morphology (mean diameter, area, and volume); (C) the EVT-sEV-treated Sw71 BLSs during differentiation in optimal conditions were equally functional to non-treated spheroids. Note that the pattern of the bars in the graphs corresponds to the pattern of the images and indicates the groups of Sw71 spheroids in Experiment 1. Statistical significance is defined as p < 0.05 (*).
Figure 4. Effect of EVT-sEVs on normal and Th1-compromised differentiation of Sw71 BLS. (A) Differentiation of a Sw71 spheroid compromised by Th1-driven excessive inflammation or in optimal conditions. Up: Percentage of intact Sw71 BLS in Th1-induced excessive inflammation as compared to optimal conditions and effect of EVT-sEV treatment. Middle: Differentiated Sw71 spheroid (Day 2) primed with Teff-CM (1, representative for n = 10) or in optimal conditions (2, representative for n = 15). Down: 1. Differentiated Sw71 spheroid primed with Teff-CM and treated with EVT-sEVs for 48 h (representative for n = 10), 2. EVT-sEV treatment of Sw71 spheroid in optimal conditions (representative for n = 15); (B) the EVT-sEV treatment of Sw71 BLS during differentiation in optimal conditions had no effect on their morphology (mean diameter, area, and volume); (C) the EVT-sEV-treated Sw71 BLSs during differentiation in optimal conditions were equally functional to non-treated spheroids. Note that the pattern of the bars in the graphs corresponds to the pattern of the images and indicates the groups of Sw71 spheroids in Experiment 1. Statistical significance is defined as p < 0.05 (*).
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Figure 5. Migration of Sw71 spheroids in the Th1-inflamed and optimal microenvironment in the presence/absence of EVT-sEVs. (A) Migration capacity presented as a percentage of migrating from all tested spheroids. The migration rate of Sw71 spheroids in optimal conditions (gray) was significantly higher than that in the Th1-inflamed microenvironment (black). The addition of EVT-sEVs to Teff-CM primed Sw71 spheroids increased their migration rate to 100% (black, squared), while it had no significant impact in optimal conditions (gray, squared). (B) Migration efficacy presented as size of the migration zone. The migration zone of Sw71 spheroids was severely compromised by the Th1-induced excessive inflammation (black) as compared to the spheroids in optimal conditions (gray). Although the EVT-sEV treatment partially restored the migration zone of Teff-CM primed spheroids (black, squared), it was significantly smaller than that of Sw71 spheroids migrating in optimal conditions (gray, squared) treated with EVT-sEVs. (C) Migration of single Sw71 cells in Teff-CM primed spheroids (up) that increased in number in EVT-sEV-treated Sw71 BLSs (down). (D) No significant change was observed in the migration zone of Sw71 BLSs in optimal conditions regardless of the EVT-sEV treatment. Note that the pattern of the bars in the graphs corresponds to the pattern of the images and indicates the groups of Sw71 spheroids in Experiment 2. Statistical significance is defined as p < 0.05 (*) and p < 0.001 (***).
Figure 5. Migration of Sw71 spheroids in the Th1-inflamed and optimal microenvironment in the presence/absence of EVT-sEVs. (A) Migration capacity presented as a percentage of migrating from all tested spheroids. The migration rate of Sw71 spheroids in optimal conditions (gray) was significantly higher than that in the Th1-inflamed microenvironment (black). The addition of EVT-sEVs to Teff-CM primed Sw71 spheroids increased their migration rate to 100% (black, squared), while it had no significant impact in optimal conditions (gray, squared). (B) Migration efficacy presented as size of the migration zone. The migration zone of Sw71 spheroids was severely compromised by the Th1-induced excessive inflammation (black) as compared to the spheroids in optimal conditions (gray). Although the EVT-sEV treatment partially restored the migration zone of Teff-CM primed spheroids (black, squared), it was significantly smaller than that of Sw71 spheroids migrating in optimal conditions (gray, squared) treated with EVT-sEVs. (C) Migration of single Sw71 cells in Teff-CM primed spheroids (up) that increased in number in EVT-sEV-treated Sw71 BLSs (down). (D) No significant change was observed in the migration zone of Sw71 BLSs in optimal conditions regardless of the EVT-sEV treatment. Note that the pattern of the bars in the graphs corresponds to the pattern of the images and indicates the groups of Sw71 spheroids in Experiment 2. Statistical significance is defined as p < 0.05 (*) and p < 0.001 (***).
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MDPI and ACS Style

Alexandrova, M.; Ivanova, M.; Vangelov, I.; Hristova, I.; Dimova, T. Effect of EVT-Derived Small Extracellular Vesicles on Normal and Impaired Human Implantation. Appl. Sci. 2025, 15, 8866. https://doi.org/10.3390/app15168866

AMA Style

Alexandrova M, Ivanova M, Vangelov I, Hristova I, Dimova T. Effect of EVT-Derived Small Extracellular Vesicles on Normal and Impaired Human Implantation. Applied Sciences. 2025; 15(16):8866. https://doi.org/10.3390/app15168866

Chicago/Turabian Style

Alexandrova, Marina, Mariela Ivanova, Ivaylo Vangelov, Iana Hristova, and Tanya Dimova. 2025. "Effect of EVT-Derived Small Extracellular Vesicles on Normal and Impaired Human Implantation" Applied Sciences 15, no. 16: 8866. https://doi.org/10.3390/app15168866

APA Style

Alexandrova, M., Ivanova, M., Vangelov, I., Hristova, I., & Dimova, T. (2025). Effect of EVT-Derived Small Extracellular Vesicles on Normal and Impaired Human Implantation. Applied Sciences, 15(16), 8866. https://doi.org/10.3390/app15168866

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