Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth

Extracellular vesicles (EVs) are important mediators of embryo attachment and outgrowth critical for successful implantation. While EVs have garnered immense interest in their therapeutic potential in assisted reproductive technology by improving implantation success, their large‐scale generation remains a major challenge. Here, we report a rapid and scalable production of nanovesicles (NVs) directly from human trophectoderm cells (hTSCs) via serial mechanical extrusion of cells; these NVs can be generated in approximately 6 h with a 20‐fold higher yield than EVs isolated from culture medium of the same number of cells. NVs display similar biophysical traits (morphologically intact, spherical, 90–130 nm) to EVs, and are laden with hallmark players of implantation that include cell‐matrix adhesion and extracellular matrix organisation proteins (ITGA2/V, ITGB1, MFGE8) and antioxidative regulators (PRDX1, SOD2). Functionally, NVs are readily taken up by low‐receptive endometrial HEC1A cells and reprogram their proteome towards a receptive phenotype that support hTSC spheroid attachment. Moreover, a single dose treatment with NVs significantly enhanced adhesion and spreading of mouse embryo trophoblast on fibronectin matrix. Thus, we demonstrate the functional potential of NVs in enhancing embryo implantation and highlight their rapid and scalable generation, amenable to clinical utility.

Similarly, human trophectodermal EVs modulate endometrial polarity (implications in embryo homing to implantation site) and upregulate endometrial expression of receptivity (SOD2, GPX3, DPP4) and implantation players (LGALS3, ANXA2, CD44) [23].Further, embryo EVs increase embryo developmental competency (blastomere count, apoptotic index, outgrowth) and implantation rates in utero [42].These functions are purported to be influenced by the small RNA [41,44] and protein [23,46] content of EVs, which are internalised by target cells to alter their molecular landscape [23,45,46].EVs are emerging as an attractive therapeutic modality [48] to enhance implantation success in assisted reproductive technology (ART) [49].However, technical challenges associated with scalable generation of natural EVs (including those formed through either endosomal sorting or derived from the plasma membrane) have impeded their clinical utility [49].This has led to the development of innovative strategies to generate EV-like NVs directly from cells that display similar functional repertoire as natural EVs (reviewed [50]).Indeed, we [51,52] and others have previously reported serial extrusion [53][54][55] of parental cells as a rapid means to generate NVs from different cell source, with these NVs retaining donor cell functional properties for tissue/cell repair [54,55].In this process, cells are fragmented through mechanical extrusion through membrane pore filters, generating an enrichment of smaller sized cell membrane fragments that self-assemble into NVs that retain intracellular molecules and surface components from their parental cells [51,54].In this study, we employed a similar extrusion-based methodology to generate NVs from human trophectodermal cells (hTSC) derived from earlystage human embryos [56].Like natural EVs, hTSC-generated NVs carry known pro-implantation factors, reprogram the endometrium to enhance hTSC spheroid attachment, and promote mouse embryo adhesion and outgrowth on fibronectin matrix.
Cells were grown on flasks coated with 0.5% gelatin prior to experimental seeding and passaged using Trypsin-EDTA (Gibco).For EV isolation and NV generation, T3-TSC cells were passaged and cultured in EV-depleted FCS (obtained by FCS ultracentrifugation at 100,000 × g for 18 h).
T3-TSC spheroids were generated as described [27,57,58] with slight modifications [59].T3-TSC cells were seeded at a density of 1500 cells per well in an ultra-low adhesion round-bottom 96-well plate in 100 µl of trophectoderm medium.This promoted aggregation of cells into a single spheroid per well.
The endometrial cells were routinely maintained in DMEM/F12 supplemented with 1% P/S, and 5% v/v FCS and incubated at 37

Isolation of T3-TSC EVs
Cells were cultured in T75 flasks in EV-depleted FCS (obtained post-100,000 × g for 18 h) for 48 h.Conditioned media was collected and centrifuged at 500 × g, and 2000 × g to remove cellular debris.The supernatant was then ultracentrifuged at 100,000 × g for 1 h to pellet crude EVs.Crude EVs were washed with 500 µl of PBS and pelleted at 100,000 × g for 1 h at 4 • C. Pellets were resuspended in 100 µl PBS and subjected to total protein quantification using Micro BCA Protein Assay (Thermo Fisher Scientific) and stored in aliquots at 1 µg/ul at −80 • C until further use.Presence of classic EV markers (CD81, TSG101) was performed using western blot analysis.
Briefly, samples were reduced with 10 mM DTT at RT for 1 h (350 rpm), alkylated with 20 mM iodoacetamide (IAA) (Sigma-Aldrich) for 20 min at RT (light protected), and immediately quenched with 10 mM DTT.A Sera-Mag SpeedBead carboxylate-modified magnetic particle mixture (hydrophobic and hydrophobic 1:1 mix, 65152105050250, 45152105050250, Cytiva) was added to each protein extract, washed in 50% ethanol, and incubated for 10 min (1000 rpm) at RT. Beads were sedimented on a magnetic rack, supernatants removed, and beads washed three times with 200 µL 80% ethanol.Beads were resuspended in 100 µL 50 mM TEAB pH 8.0 and digested overnight with trypsin (1:50 trypsin: protein ratio; Promega, V5111) at 37 • C, 1000 rpm.The peptide and bead mixture was centrifuged at 20,000 × g for 1 min at RT.Samples were placed on a magnetic rack and supernatant was collected and acidified to a final concentration of 1.5% formic acid (FA), frozen at −80 • C for 20 min, and dried by vacuum centrifugation for ∼1 h.Peptides were resuspended in 0.07% trifluoroacetic acid (TFA), quantified by Fluorometric Peptide Assay (Thermo Fisher Scientific, 23290) as per manufacturer's instructions, and samples normalised to 1 µg/µl with 0.07% TFA.

Liquid chromatography-tandem mass spectrometry
Peptides were analysed on a Dionex UltiMate NCS-3500RS nanoUH-PLC coupled to a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer equipped with a nanospray ion source in positive, datadependent acquisition mode as described [66].Peptides were loaded (Acclaim PepMap100 C18 5 µm beads with 100 Å pore-size, Thermo Fisher Scientific) and separated (1.9-µm particle size C18, 120Å, 0.075 × 250 mm, Nikkyo Technos Co. Ltd) with a gradient of 2%-80% acetonitrile containing 0.1% formic acid over 110 min at 300 nL min-1 at 55 • C (in-house enclosed column heater).An MS1 scan was acquired from 350-1650 m/z (60,000 resolution, 3 × 10 6 automatic gain control (AGC), 128 msec injection time) followed by MS/MS data-dependent acquisition (top 25) with collision-induced dissociation and detection in the ion trap (30,000 resolution, 1 ×10 5 AGC, 60 ms injection time, 28% normalized collision energy, 1.3 m/z quadrupole isolation width).Unassigned precursor ions charge states and slightly charged species were rejected and peptide match disabled.Selected sequenced ions were dynamically excluded for 30 s.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [67] partner repository with the dataset identifier PXD038856.

Data processing and bioinformatics
Peptide identification and quantification were performed as described previously [27,66] using MaxQuant (v1.6.14) with its built-in search engine Andromeda [68].Tandem mass spectra were searched against Homo sapiens (human) reference proteome (74,811 entries, downloaded 12-2019) supplemented with common contaminants.Search parameters included carbamidomethylated cysteine as fixed modification and oxidation of methionine and N-terminal protein acetylation as variable modifications.Data was processed using trypsin/P as the proteolytic enzyme with up to 2 missed cleavage sites allowed.The search tolerance and fragment ion mass tolerance were set to 7 ppm and 0.5 Da, respectively, at less than 1% false discovery rate on peptide spectrum match (PSM) level employing a target-decoy approach at peptide and protein levels.Label free quantification (LFQ) algorithm in MaxQuant was used to obtain normalised quantification intensity values, log2 transformed, and single batch analysis using Perseus as described [59].For GO annotation protein accession IDs were submitted to g:Profiler [69].Hierarchical clustering was performed in Perseus using Euclidian distance and average linkage clustering.Proteins upregulated in HEC1A cells treated with NVs or EVs include those that were identified in at least 80% of replicates, either uniquely or which fold change (log2) ≥0.1 compared to HEC1A cells treated with PBS (control).Functional enrichment analyses (Gene Ontology (GO), KEGGs) were performed using g:Profiler [69].Pathway Enrich-mentMap analysis was performed using Cytoscape (v3.7.1) [70].

Statistical analysis
Data were analysed using GraphPad Prism v8.4.3, with all data pretested for normality.If the data was non-parametric, a Kruskal-Wallis with a Tukey's post-hoc test or Mann-Whitney U analysis was performed.If parametric, one-way ANOVA with a Tukey's post-hoc test or unpaired t-test was performed.All data presented as mean plus/minus standard deviation (mean ± SD).In all analyses, *p < 0.05 is considered statistically significant.
We next subjected hTSCs to serial extrusion using microfilters of decreasing pore size (10, 5, 1 µm, 13 times per membrane) (performed n = 3-5 biological replicate as indicated).The extruded material was subjected to density gradient separation to isolate NVs as previously reported [51] (Figure 1A).For comparative analysis, EVs were isolated using sequential centrifugation, as previously described [72].Using cryo electron microscopy, we show that NVs (buoyant density of 1.10-1.20 g/cm 3 ), appear as membrane-limited, morphologically intact, spherical structures, similar to EVs (Figure 1B), consistent with our previous reports [51,52].Nanoparticle tracking analysis revealed NV particle size distribution of 90-130 nm, and EV particle size distribution of 100-170 nm (Figure 1C).Despite similar biophysical properties, EVs are enriched in EV marker proteins TSG101 and CD81 compared to NVs, indicating biochemical differences in their composition due to different modes of generation (Figure 1D).Compared to EVs isolated from conditioned media that takes ∼14 days, NVs were generated in 6 h with a 20-fold higher yield (140 µg for NVs and 7 µg for EVs) from 6.25 × 10 6 T3-TSCs (Figure S1).
TA B L E 1 Players of embryo implantation and established markers of endometrial receptivity significantly enriched in NVs and EVs compared to donor cells.

ADAMTS1
A disintegrin and metalloproteinase with thrombospondin motifs 1 Established as a critical player of embryo implantation and female fertility, involved in epithelial cell remodelling during embryo implantation in the uterus and expression is induced in endometrial stromal cells surrounding the implanting blastocyst.
[190] AMIGO2 Amphoterin-induced protein 2 Cell adhesion molecule and scaffold protein that regulates AKT signalling via PDK1 membrane localisation.Also plays roles in angiogenesis and cell survival. [191]

ANXA2
Annexin A2 Critical at the site of embryo implantation and facilitates attachment of the embryo to endometrial cells by regulating cytoskeletal proteins RHOA, ROCK, and F-actin.
[ 73,74,192,193] ANXA4 Annexin A4 Expressed in the endometrium during the mid-secretory phase (window of implantation) following stimulation by progesterone.[194] CLU Clusterin Expression in the endometrium is indicative of tissue remodelling and reorganisation [195] COTL1 Coactosin-like protein Endometrial receptivity marker included in ERA, differentially expressed in the receptive endometrium.Also significantly downregulated in the receptive endometrium of obese patients (who are prone to infertility).
[ 91,196] DPP4 Dipeptidyl peptidase 4 Established critical player of embryo implantation and endometrium receptivity and regulates ongoing pregnancy by mediating adhesion with trophinin.Associated with endometrial receptivity.
[ 77,114,193] EZR Ezrin Key regulator of cytoskeleton that facilitates uterine receptivity and embryo-endometrium attachment.[197] F3 Tissue factor Forms a proteolytically active complex with coagulation factor VIIa to activate cell signalling.This complex cleaves EphA2, downregulating EphA2 expression in the embryo (reducing its motility), and in the endometrium during the secretory phase (prevents repulsion of the implanting embryo).[198][199][200] FAP Prolyl endopeptidase FAP Facilitates active tissue remodelling in the endometrium during implantation that supports embryo implantation and growth, potentially by mediating levels of chemokines, proinflammatory factors, and angiogenic factors in the microenvironment. [201]

GJA1 Gap junction alpha-1 protein
Established role in uterine decidualisation/remodelling required for successful embryo implantation.Highly expressed in decidual cells and can upregulate expression of other decidualisation genes.Loss of GJA1 results in severe fertility defects due to impaired endometrial stromal responsiveness to the implanting embryo.[202,203] HLA-A HLA class I histocompatibility antigen, A alpha chain Regulator of immune system by presenting peptides from within the cell, although role in implantation and pregnancy remains unclear. [204]

ICAM1
Intercellular adhesion molecule 1 ICAM1 expression peaks at the apical membrane of uterine epithelial cells at the time of implantation.Its expression in the endometrium also increases in response to trophoblast signals, which also increases attachment rate.[205,206] IGFBP7 Insulin-like growth factor-binding protein 7 Critical for pregnancy establishment as its inhibition induced pregnancy failure.Increase in its expression may be attributed to signals from the blastocyst.It localises to endometrial glands and stroma and regulates uterine receptivity through Th1/Th2 lymphocyte balance and decidualization.
[ Expression increased in endometrial epithelial cells when in contact with trophoblast cells. [231]

NVs can be taken up by endometrial cells
An important feature of engineered NV lies in their ability to interact with and be taken up by recipient cells of interest [51].Hence, we incubated NVs labelled with fluorescent lipophilic DiI dye with HEC1A cells for 2 h and analysed NV uptake using confocal fluorescence microscopy (Figure 3A).NVs labelled with DiI were subjected to density-centrifugation to remove unbound dye.Confocal fluorescence microscopy revealed that NVs, similar to EVs, were readily taken up by HEC1A cells.Both NVs and EVs appeared as punctuate structures in recipient cells, typical of EV uptake [23,59].More-over, confocal microscopy along the z-axis revealed that NVs were internalised by HEC1A cells (Figure 3B).

Functional dissection of NVs in attachment and outgrowth
We have previously reported that trophectoderm/embryo [23] and endometrium [26,27,59] secreted EVs can promote embryo attachment and outgrowth.Here, to assess NV function on embryo attachment to the endometrium, we performed a co-culture attachment assay that we previously developed to demonstrate EVs capacity to promote trophectoderm spheroids attachment to endometrial cells [29,57].We treated HEC1A monolayer with NVs or EVs for 24 h and assessed the rate of hTSC spheroid attachment to these primed HEC1A cells (Figure 4A).We show that NV and EV treatments significantly increased hTSC spheroid attachment rate by ∼ 20% compared to PBS control (Figure 4B).
Next, to assess NV function on embryo attachment and outgrowth, we performed a classical fibronectin-binding assay [103,104] with mouse embryos which is an indicator of implantation potential in vivo.
We seeded day-3 partially hatched mouse embryos onto fibronectincoated wells and treated them with a single dose of NVs or EVs (50 µg/mL), or PBS (volume matched) (Figure 4C), monitoring attachment and outgrowth across 72 h.We show that NVs, like EVs, significantly (p < 0.005) enhanced mouse embryo outgrowth on fibronectin matrix at 72 h compared to PBS vehicle alone (Figure 4D, E).

NVs significantly upregulate expression of proteins and processes in HEC1A cells implicated in embryo implantation
To gain molecular insights into how NVs enhance receptivity of low receptive HEC1A cells, we performed proteomic analyses on NV/EVprimed HEC1A cells (Figure 5A).PCA revealed distinct proteome profiles of NVs and EVs-treated HEC1A cells compared to PBS (control)treated HEC1A cells (Figure 5B).A total of 3109, 2993, and 3051 proteins were detected in NV-treated, EV-treated, and PBS-treated HEC1A cells, respectively (n = 4 replicates) (Figure 5C, Table S3).
We highlight that NVs could be a promising avenue in oxidative stress protection [29,146,147].Oxidative stress due to an imbalance between reactive oxygen species (ROS) and antioxidants has been established as an important factor that can negatively impact outcomes of ARTs.In ART, up to 75% of embryos are exposed to oxidative stress partially or throughout their in vitro development [148].Gardner and colleagues, by supplementing antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine, a-lipoic acid) into human embryo culture media, significantly increased implantation and pregnancy rates in 35-40year-old patients [149].Further, exposure to supplements in the embryo culture medium (e.g., L-ascorbic acid and α-tocopherol) has been shown to enhance the development of porcine denuded oocytes and to protect against DNA fragmentation of cumulus cells [150].In this study we highlight that several proteins (SOD1/2, CAT, PRDX1/2) in NVs were similarly identified in EVs derived from uterine fluid of fertile women in the receptive phase, which elicit antioxidative effects in vitro, significantly decreasing levels of ROS [29].NVs thus present as a potential regulator of redox levels, promoting embryo attachment to and remodelling of the endometrium to maintain embryo viability and development.
EVs released from preimplantation embryos promote embryo hatching and increase implantation rates [43,165] SOD1) and proteins involved in embryo development [41] (ARFGEF2, LDLR, PYCR1, TPP1) may thus elicit similar effects.The embryo-tofibronectin binding assay we used in this study assesses integrinmediated adhesion between the blastocyst surface and fibronectin [103], and is a close proxy to rate of mouse embryo implantation in vivo [104].This assay thus determines embryo developmental and implantation competency with higher accuracy than morphological grading.Indeed, our NVs demonstrated embryotrophic effects by significantly enhancing the attachment and outgrowth rate of mouse embryos and is thus potentially a safe and feasible supplement for ET media.Altogether, our hTSC-derived NVs present as a promising implantation-enhancing modality capable of simultaneously promoting embryo development and improving endometrial receptivity.
EV-based strategies have been explored to improve fertility treatment candidates such as hCG [153][154][155][156], which have immunomodulatory and endometrial receptivity-enhancing effects.Indeed, uterine fluid EVs (UF-EVs), when actively loaded with hCG via sonication, prolonged hCG release and increased in vitro attachment rates at higher levels compared to native UF-EVs co-supplemented with hCG.This design retains the beneficial effects on native UF-EVs on embryo health [29,177] and implantation [33,178] while enhancing their functionality via loaded molecule/s.Likewise, selective loading of cargo into NVs may be explored further to address additional determinants of implantation success, including immunomodulation [21,[179][180][181] and redox balance [29,91].For example, the extrusion strategy used for NV generation can be modified for effective loading of factors including hCG (NCT01786252 [182], NCT01030393 [183]) and antioxidative enzymes [184] without genetically modifying parental hTSCs [185].
Alternatively, multiple characteristics or functions may be achieved simultaneously by generating fused NVs from multiple cell types [186].
For example, NVs with abundant expression of cytokine receptors and SARS-CoV-2 receptor ACE2 were engineered by co-extruding monocytes and ACE2-overexpressed embryonic kidney cells, efficiently adsorbing viruses, and inflammatory cytokines to intervene viral infection and cytokine storm [186].Likewise, cells with immunomodulatory effects favourable to implantation, such as peripheral blood mononuclear cells (PBMCs) (currently undergoing clinical trials for treatment of recurrent implantation failure (NCT03267797, NCT05421364)), are excellent candidates for co-extrusion with hTSCs to engineer NVs with multipotent functions.

SUMMARY
Extrusion of cells is an efficient method of acquiring a large yield of NVs, overcoming limitations with scalability associated with EV-based therapies, while enabling a plethora of EV engineering strategies and designs to suit various purposes [48,51].We show that NVs from hTSCs, enriched in implantation and endometrial receptivity players,

HEC1A endometrial epithelial cells
were used to model a low-receptive endometrium[60][61][62][63].Endometrial cells were seeded at confluency onto round-bottom 96-well plates before overnight starvation with basal media (DMEM/F12 supplemented with 1% v/v P/S).T3-TSC spheroids (1500 cells per spheroid, 1 spheroid per well) were transferred to endometrial cells treated with NVs or EVs at 50 µg/mL (protein) final concentration, or PBS (volume matched), and incubated for 1 h, after which the media was aspirated and washed gently once with phosphate-buffered saline (PBS).Spheroid adhesion was calculated by the number of attached spheroids to the HEC1A cells, divided by the total number of spheroids seeded per treatment (n = 12, N = 5), and expressed as a percentage.Significance Statement Dynamic reprogramming of the molecular composition of trophectoderm and endometrial cells is critical to embryo implantation and pregnancy establishment.Extracellular vesicles (EVs) are essential cell-secreted signalling modalities facilitating embryo-maternal crosstalk to regulate embryo implantation to endometrium.The trophectoderm produces embryotrophic and pro-implantation factors which are packaged into EVs; we reasoned that direct extrusion of human trophectoderm cells will generate EV-like nanovesicles (termed NVs) that contain these molecules.These trophectodermal NVs retain features of their cell source, including the enrichment of pro-implantation and embryotropic factors and regulators of endometrial receptivity.Functionally NVs enhance hallmarks of implantation biology endometrial/trophectoderm cell attachment and embryonic outgrowth.Thus, we demonstrate the functional potential of NVs in enhancing embryo implantation and highlight their rapid and scalable generation, amenable to clinical utility.

F I G U R E 2
Quantitative mass spectrometry-based proteomics of TSC-derived NVs, EVs, and WCL.(A) Venn diagram of proteins identified in NV, EV, or WCL.(B) Principal component analysis of proteins identified in NVs, EVs, and WCL.(C) Venn diagram comparing NVs and EVs, highlighting 1032 commonly identified proteins.(D) EnrichmentMap of Gene Ontology (biological process) of the 1030 proteins commonly identified in NV and EV.(E) Venn diagram comparing commonly identified proteins in NVs and EVs compared to ERA and other implantation players [91, 92, 193].(F) Two-way scatter plot highlighting the most significantly upregulated and downregulated proteins in NVs and EVs compared to their parental cells.To investigate how NVs and EVs are distinct from WCL, we compared NV and EV proteomes to WCL (log 2 fold change (FC) > 0.3, p < 0.05) and highlight 128 significantly upregulated proteins (Table

F I G U R E 3
Uptake of NVs and EVs by HEC1A endometrial cells.(A) Confocal fluorescent microscopy images demonstrating uptake of DiI lipophilic fluorescent dye labelled NVs or EVs (red) by HEC1A endometrial cells after 2 h incubation (n = 3).(B) Fluorescent Z-stack image displaying intracellular distribution of DiI-labelled NVs (red).Nuclei were stained with Hoechst.Scale bar 10 µm.

F I G U R E 4
TSC-derived NVs and EVs enhance trophectodermal spheroid attachment to low receptive endometrial cells and mouse embryo outgrowth.(A) Experimental workflow for co-culture attachment assay.Created with Biorender.(B) Box plot indicating percentage of TSC-spheroid attachment to HEC1A endometrial cells following PBS control, NV, or EV treatment (n = 5).(C) Experimental workflow for mouse embryo fibronectin attachment and outgrowth assay.(D) Box plot indicating quantified area of mouse embryo outgrowth 72 h following PBS control, NV, or EV treatment.(E) Bright-field microscopic images of mouse embryos 72 h following PBS control, NV, or EV treatment (n = 4).Nuclei were stained with Hoechst.Scale bar 500 µm.F I G U R E 5 Proteome profiling of HEC1A cells treated with NVs, EVs, or PBS.(A) Workflow for mass spectrometry-based proteomic profiling, comprising sera-bead sample preparation with nano-liquid chromatography tandem mass spectrometry and data processing/informatics.Created with Biorender.(B) Principal component analysis of HEC1A cellular proteome treated with NVs, EVs, or PBS control (volume matched).(C) Venn diagram of proteins identified in HEC1A cells after treatment with NV, EV, or PBS.(D) Two-way scatter plot highlighted the most significantly upregulated and downregulated proteins in NV-treated and EV-treated HEC1A cells compared to PBS control.(E) EnrichmentMap of Gene Ontology (biological process) and Reactome processes overrepresented in significantly upregulated proteins in both NV-treated and EV-treated HEC1A cells compared to PBS control.
have multipotent functional effects on trophectodermal-endometrium attachment and mouse embryo outgrowth, promoting two critical hallmarks of implantation success.Upon treatment onto low-receptive endometrial cells, hTSC-NVs upregulated expression of implantation players and surface adhesion molecules that facilitate embryoendometrial attachment critical for implantation success.Further evaluation of their therapeutic potential in vivo, mechanism of action, and safety in mammalian models are required.Future directions from this work include engineering NVs to enhance or modify its therapeutic benefit; as the extrusion process allows incorporation of specific small molecules, proteins, or RNAs into NVs [187-189], or even co-extrusion of different cells to combine their characteristics for desired functions.NVs thus represent a feasible and robust method of large-scale generation of therapeutic vesicles for improving embryo implantation.AUTHOR CONTRIBUTIONS Qi Hui Poh, Alin Rai, and David W. Greening conceived and designed experiments.Qi Hui Poh carried out majority of experiments.Alin Rai provided critical experimental insight and with Qi Hui Poh/Alin Rai with informatics analyses.Mulyoto Pangestu assisted with experimental work (mouse embryo training and handling).Lois A. Salamonsen provided editorial assistance.Qi Hui Poh, Alin Rai, and David W. Greening wrote, reviewed, and edited the manuscript.All authors approved the final manuscript.
Increases in expression in the window of implantation at the site of embryo implantation, potentially regulated by embryonic signals, possibly facilitates implantation by binding to osteopontin, functional blockage of this integrin led to decreased implantation sites.
Adhesion proteins and established markers of endometrial receptivity significantly enriched in HEC1A endometrial cells treated with NVs and EVs versus PBS.at the site of embryo implantation and serves as a marker of endometrial receptivity.It may facilitate embryo invasion into the endometrium by disrupting uterine epithelial cell adhesion to the basal lamina.Significantly downregulated in patients with RIF.May facilitate embryo implantation via the focal adhesion pathway, allowing intercellular connection between the embryo and endometrium.Mediates firm attachment of the embryo to endometrium.