ART Procedures and Human Embryo Spent Medium Collection

The embryo spent culture medium samples employed in this study were obtained from patients attending the Centro Scienze Natalità of the San Raffaele Hospital in Milano, Italy, with an indication to ART. All embryos were obtained after controlled ovarian stimulation that was performed using a gonadotropin-releasing hormone-agonist or antagonist protocol. Oocyte collection was performed 36 hours after triggering of ovulation. After 2–3 hours incubation in 5% HSA-supplemented Fertilization medium (Sage In-Vitro Fertilization, Inc. Trumbull, CT, USA) under oil, selected oocytes were allocated to conventional in vitro fertilization or ICSI. For ICSI, denudation of the cumulus oophorus was performed as previously described26, 43,44,45. Fertilized oocytes were sequentially cultured in group cultures of maximum 6 embryos in 50-µL microdrops of Quinn’s Advantage Cleavage medium (Sage In-VitroFertilization Inc., Cooper Surgical, Trumbull, CT, USA) supplemented with 10% SSS (Irvine, CA, USA) in a humidified atmosphere containing 5% O 2 and 6% CO 2 . On day 3 after fertilization, cleavage-stage embryos were transferred to fresh 50-µL drop of Quinn’s Advantage Blastocyst Medium (Sage In-VitroFertilization Inc., Cooper Surgical, Trumbull, CT, USA) supplemented with 10% SSS. The quality of the cleavage stage embryos and of the day 5 blastocysts was assessed and defined according to standard criteria26, 44, 45. The spent culture medium was collected from embryos at the cleavage stage or from expanded blastocysts without signs of degenerating cells during ART cycles and kept in −80 °C until use. The study (GFI2015) was approved by the Ethics Commitee of the Ospedale San Raffaele in Milan, Istituto di Ricovero e Cura a Carattere Scientifico. Informed consent was obtained from all participants, and the experiments were performed in accordance to the principles set out in the World Medical Association Declaration of Helsinki.

Isolation of EVs from spent media of human embryos at different development stages

Spent culture medium samples from embryos on day 3 or day 5 of development were subjected to differential centrifugation according to previous protocols46, 47. The same protocol of EVs isolation was used for all samples considered in this study. Briefly, in order to remove cells, dead cells, and cellular debris, conditioned media of embryos/blastocysts were centrifuged at 300 x g for 10 min, at 2,000 x g for 20 min and at 5,000 x g for 30 min; all centrifuges were performed at 4 °C. The supernatants were transferred in a 3,5-mL thick wall polycarbonate tubes (Beckman Coulter Inc., Brea, CA, USA, item no. 349622) and an equal amount of PBS was added. The sample was ultra-centrifuged at 110,000× g for 90 min at 4 °C in a TLA-100.3 rotor (Beckman Coulter Inc.) using Optima TLX centrifuge (Beckman Coulter Inc.) to pellet EVs. The supernatants were removed completely, thereby pellets were washed with 1 mL of PBS and centrifuged 90 min at 110,000 x g at 4 °C. The pellets containing embryo-derived EVs accumulated from day 1 to day 3 (D3-EVs) or from day 3 to day 5 (D5-EVs) of development were suspended in an adequate volume of PBS (50–100 µl) and the protein concentration was measured by Bradford assay and by NanoDrop8000 to assess EVs purification. We ensured that only samples with a protein content of at least 10 µg were utilized for the downstream experiments. The final EVs pellet was stored at −80 °C until use. Fresh media supplemented with 10% SSS or 5% HSA were subjected to the same procedures to ensure proper negative controls to each experiment.

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) was performed by an expert EV laboratory (Exosomics Siena S.p.A, Siena) in order to provide a validation of the results. NTA was performed using a NanoSight LM10-HS microscope (NanoSight Ltd., Amesbury, UK), as previously described48. For the analysis, 50 µl of spent media deriving from 6 Day 3 embryos, 50 µl of spent media of 6 Day 5 blastocysts, 50 µl of fresh media completed with 10% SSS and 50 µl of fresh media with 5% of HSA were diluted with concentrations adjusted, if necessary, in order to specifically fit the optimal working range (20–50 particles per frame) of the instrument. Three 60-s videos were recorded under the flow mode for each sample with camera level set at 16 and detection threshold set at 10. At least 200 completed tracks per video were collected. The camera level and overall settings were selected as optimal for the accurate detection of exosome-sized nanoparticles following the manufacturer recommendations and as confirmed during instrument calibration. Videos were analyzed with NTA software version 2.3 to determine the mean, mode and estimated concentration of measured particles with corresponding standard error. The NanoSight system was calibrated with polystyrene latex microbeads of 50, 100, and 200 nm (Thermo Scientific Waltham, MA, USA) prior to analysis and auto settings were used for blur, minimum track length and minimum expected particle size.

Transmission electron microscopy

Preparation for transmission electron microscopy (TEM) analysis was done using the method described by Théry et al.46 . EVs derived from single drops of spent media from six embryos at day 3 stage (D3-EVs), day 5 blastocyst stage (D5-EVs) and from 10% SSS or 5% HSA fresh media were mixed with an equal volume of 4% PFA and deposited by airfuge onto Formvar/Carbon coated EM grids (Ted Pella, Redding, CA, USA). Samples were contrasted and embedded by treatment with uranyl-oxalate solution (Electron Microscopy Science, Hatfield, PA, USA), pH 7, for 5 min, followed by methyl-cellulose-uranyl-acetate (Sigma, Saint Louis, MO, USA) on ice for 10 min. Negative stain immunogold labeling was carried out exclusively on fresh EVs. The live specimens in suspension were mounted onto formvar, carbon-coated grids, and allowed to adhere to the grids. The specimens were then exposed to the primary antibody followed by 10 nm gold-labelled secondary antibody (British Biocell, Cardiff, UK). The following primary antibodies were used: anti-CD9 (10 µg/ml; BD Pharmingen, #555370, San Jose, CA, USA) and anti-CD63 (10 µg/ml; BD Pharmingen, #556019, San Jose, CA, USA). After washing to eliminate any non-specific binding of either the primary or secondary antibody, the specimens were fixed using 2.5% glutaraldehyde and contrasted in 2% uranyl acetate and lead citrate. LEO 912AB Omega electron microscope (Carl Zeiss NTS, Oberkochen, Germany) was used for image analysis.

Western blot analysis

D3-EVs and D5-EVs were isolated from a pool of spent media from 50 embryos at different developmental stage and from an equal volume of 10% SSS- or 5% HSA- supplemented fresh media as negative controls and were used for immunoblotting analysis. Ten µg of isolated EVs (by Bradford assay and by NanoDrop8000 measurement) were lysed in reducing sample buffer [0.25 M Tris–HCl (pH 6.8), 40% glycerol, 8% SDS, 5% 2-mercaptoethanol and 0.04% bromophenol blue] and boiled for 5 minutes at 95 °C. For tetraspanins detection, non-reducing sample buffer (without 2-mercaptoethanol) was used. Proteins were resolved by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), electrophoretically transferred to polyvinylidene fluoride membranes, blocked in 5% non-fat powdered milk in TBS-T (0.5% Tween-20) and the membranes were incubated with the following antibodies: anti-ALIX (1:500, Santa Cruz, #sc-271975, Santa Cruz, CA, USA), anti-CD9 (1:1000, BD Pharmingen, #555370, San Jose, CA, USA), and anti-CD63 (1:1000; BD Pharmingen, #556019, San Jose, CA, USA). Protein bands were detected using X-ray film and enhanced chemiluminescence reagent (ECL, Amersham, Buckinghamshire, UK).

RNA extraction, Reverse transcription and Whole Transcriptome Amplification

Since the RNA content was expected to be limited, D3-EVs and D5-EVs isolated from a pool of spent media from 50 embryos at different developmental stage and an equal volume of 10% SSS- or 5% HSA- supplemented media as negative controls, were subjected to RNA extraction, reverse transcription and whole transcriptome amplification using REPLI-g Cell whole genome amplification (WGA) & whole transcriptome amplification (WTA) Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, lysis buffer was added to 13 µl of EVs resuspended in PBS and heated at 95 °C for 5 min to lyse and release the EV contents. Lysed EVs were used for WTA of total RNA. Genomic DNA was removed, cDNA was synthesized and subjected to ligation and amplification steps. The RT-reaction was included in the kit which used T-Script reverse transcriptase combined with random and oligo-dT primers. REPLI-g SensiPhi DNA Polymerase with high proofreading was used for isothermal amplification of cDNA. The DNA derived from amplification of cDNA was stored at −80 °C or immediately used. To avoid contamination among samples, we adopted precautions normally used during routine viral diagnostic PCR analysis at SSI, where extraction, amplification and analyses were physically separated and negative samples were included in all steps.

Touch-down PCR of pluripotency-related genes

EV-derived amplified cDNA from spent medium of embryos at different development stages was used for the detection of pluripotency gene transcripts. In order to quantify and perform the PCR amplification using the same amount of cDNAs from each sample, purification of amplified DNA by LiCl/EtOH precipitation was performed, according to supplementary protocol instructions of REPLI-g kit. In order to ensure optimal normalization of results on total amplified cDNA quantity, 5 ng of the amplified cDNA of EVs isolated from embryo spent medium were subjected to PCR using AmpliTaq Gold® DNA Polymerase, LD (low DNA) (Applied Biosystems, Foster City, CA, USA). The PCR amplification was carried out for one cycle of denaturation at 95 °C for 10 min and subsequent 40 cycles. To increase specificity, sensitivity and yield of amplification, a touch-down PCR was performed starting from an annealing temperature of 72 °C to 57 °C over the course of 15 cycles, followed by 25 standard cycles with denaturation at 95 °C, annealing and extension at 60 °C for 30 sec. Primer sequences are listed in Supplementary Table S1. Ten microliters of PCR products were loaded on a 2% agarose gel and stained with EtBr. The positive control was cDNA of human neuronal iPS kindly provided from Dott. Giacomo Frati and Dott. ssa Angela Gritti (San Raffaele Telethon Institute for Gene Therapy)49 and the negative controls were amplified cDNAs from EVs from fresh media supplemented with 10% of SSS or with 5% of HSA.

HLA-G detection: Bio-Plex system

Covalent coupling of antibodies to microsphere and HLA-G Bio-Plex assay were performed as described previously50. Briefly, covalent coupling of the anti-HLAG antibodies, MEM-G9 moAb (Exbio, Prague, Czech Republic) to the carboxylated polystyrene microspheres (Bio-Rad, Hercules, CA, USA) has been performed using the Bio-Plex amine coupling kit (Bio-Rad). Five μl of HLA-G standards assayed in duplicate or sonicated D5-EVs and D3-EVs from a pool of spent media from 50 embryos at different developmental stage have been incubated with 50 μl of anti-HLA-G conjugated beads (5000 beads/well) in 96-well filter plates for 60 min at room temperature with shaking. An equal volume of 10% SSS or 5% HSA supplemented media as negative controls was used. For the analysis of sHLA-G not associated with EVs, the same procedure was applied to 50 µl of 110,000 x g supernatant (EV-depleted spent culture media) of each sample. Plates have been washed by vacuum filtration with 100 μl of Bio-Plex wash buffer, 25 μl of biotinylated antibody W6/32 (10 µg/ml) (Dako, Glostrup, Denmark) has been added, and plates were incubated for 30 min at room temperature with shaking. After filter washes, 50 µl of streptavidin-phycoerythrin has been added, and the plates have been incubated for 15 min at room temperature with shaking. Finally, plates have been washed by vacuum filtration, beads have been suspended in 125 µl of Bio-Plex assay buffer, and samples have been analyzed on the Instrument Bio-Plex system in combination with the Bio-Plex Manager software. The standard curves for HLA-G have been used from 100 to 0.5 pg/ml and the minimum detectable dose was 1.0 pg/ml. The specificity of this assay has been validated with an isotype control (Mouse IgG1 Isotype control, code 1B-457-C100 biotin, Exbio, Praha, Czech Republic) used in place of W6/32 biotin moAb. The background observed was lower than the selected detection limit (data not shown). The intra-assay coefficient of variation (CV) was 1.4% and the inter-assay CV was 2.0%. The limit of detection is 0.5 pg/ml.

HLA-G detection: Flow cytometry assay

The flow cytometry technique was also used to analyze the presence of HLA-G protein51 in EVs. Dynabeads M-280 Tosylactivated (Dynal Biotech, Oslo, Norway) were prepared according to the manufacturer’s recommendations. Briefly, the dynabeads were incubated for 24 hours at 37 °C with MEM-G9 MoAb (Exbio, Prague, Czech Republic), with a ratio of 107 dynabeads to 3 μg MEM-G9 MoAb. After several washes in recommended buffers, the Dynabead-MEM-G9 conjugates were stored in PBS, pH7.4 with 0.1% BSA and 0.02% sodium azide at 4 °C. Transfected HeLa-G5 cell culture supernatants were used as positive control, and HeLa wild-type cells culture supernatants as negative control. After sonication, the EV content was incubated with 106 dynabeads-MEM-G9 conjugated for 1 hour at 37 °C, then washed twice with PBS and labeled with anti-beta2microglobulin-FITC MoAb (Biodesign International, Saco, MN, USA) for 15 min at room temperature. The flow cytometric assay was performed on FACS CantoII (Becton Dickinson, San Jose, CA, USA) using standard settings and data analysis performed with Cell Quest software (Becton Dickinson).

Establishing primary endometrial cell cultures from human endometrial samples

Human endometrial samples were obtained from women undergoing laparoscopy for ovarian benign pathology using a surgical curette. Women with previous autoimmune or neoplastic disorders were excluded from the study. Moreover, they were not taking oral contraceptives, progestins, GnRH analogues/antagonists or other hormonal medications. All subjects were <40 years of age and had regular menstrual cycles. All samples of uterine endometrium were obtained at the time of laparoscopy after informed consent was given. Endometrial tissues were used to establish primary endometrial cultures52. Cytofluorimetric analysis showed that leukocyte contamination (CD45-positive cells) of our cultures was less than 2%. Briefly, using a scalpel, the tissue was transected into small pieces (1–2 mm3) and washed in fresh medium to remove mucus or debris. Tissue was then incubated 2 hours at 37 °C in a shaking water bath in 0.2% of collagenase A (Hoffmann-La Roche, Basel, Switzerland) diluted in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). The digested suspension was filtered through a 45 μm pore sterile filter to remove undigested tissue. The endometrial cell suspension, containing mainly stromal and epithelial glandular endometrial cells, was centrifuged at 300 xg for 5 min and fresh media was added to the pelleted cells. Some experiments were also performed dividing the two components of endometrium, the epithelial and the stromal cells, according to a previous protocol52 including differential sedimentation and selective attachment to the dishes. The cells were finally plated for EV uptake assay. Endometrial cells were cultured for a maximum of 7–8 days.

Labelling of EVs with Fluorescent Dye and EV Uptake Assay

EVs-D3 and EVs-D5 were labelled with the Vybrant™ DiO cell-labeling solution (#V22886, Molecular Probes, Eugene, OR, USA) following the procedures described by Nazarenko and colleagues53 with some modifications. Briefly, 100 μg of EVs sedimented by ultracentrifugation were resuspended in 200 μl of cold PBS. One µl of dye was diluted to 200 µl EV suspension and incubated 30 min at 37 °C by rotation. EVs labelled were purified by ultracentrifugation at 110,000 × g for 1 hour at 4 °C. Vybrant DiO-labelled EVs (5 µg, 10 µg/ml, EV proteins by Bradford assay and by NanoDrop8000 measurement) were added to 8 × 104 ECs plated on glass coverslips onto 24-wells. Co-cultures were maintained at 37 °C for different time periods (30 min, 1, 2 and 4 hours). EV-containing medium was then removed and the cells were washed gently with 2X PBS to eliminate unbound EVs. The ECs were then fixed in 4% paraformaldehyde for 20 min at room temperature and washed three times with PBS. Then, coverslips with treated cells were mounted on a microscope slide with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) carefully avoiding building of air bubbles. As negative controls, PBS mixed to the dye, labelled HSA-EVs or SSS-EVs were processed similarly to other samples and added to culture wells. Non-treated endometrial cells (NT-ECs) were also used as a further negative control in order to exclude potential autofluorescence. Internalization of EVs was observed by immunofluorescence microscopy and virtual images were generated using the Axio Vision Imaging Software (Axiovision Rel 4.8®) on an Axio Imager M2 microscope (Carl Zeiss, Oberkochen, Germany). For standardization and evaluation of image quality, at least 10 images were randomly acquired by using a Zeiss color AxioCam MR5. All fluorescence images were captured using a fixed exposure time of 250 ms. The cells that internalized Vybrant DiO-labelled EVs were quantified analyzing the intensity of signal which was scored as EV− cells (no signal), EV+ cells (moderate signal: fluorescent dots) and EV++ cells (strong signal: cytoplasmic diffusion of fluorescence).