MicroRNAs in spent blastocyst culture medium are derived ......and SBM samples revealed that 96.6%...

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ORIGINAL ARTICLE: REPRODUCTIVE SCIENCE

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MicroRNAs in spent blastocystculture medium are derivedfrom trophectoderm cells andcan be explored for humanembryo reproductivecompetence assessment

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Antonio Capalbo, Ph.D.,a,b Maria Ubaldi Filippo, M.D., M.Sc.,a,b Danilo Cimadomo, M.Sc.,a,b Laila Noli, M.Sc.,c

Yakoub Khalaf, M.D., M.Sc.,c Alessio Farcomeni, Ph.D.,d Dusko Ilic, M.D., Ph.D.,c and Laura Rienzi, M.Sc.a,b

a GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; b GENETYX, Molecular Genetics Laboratory,Vicenza, Italy; c Division of Women's Health and Assisted Conception Unit, King's College of London, Guy's Hospital,London, United Kingdom; and d Department of Public Health and Infectious Diseases, Sapienza University of Rome,Rome, Italy

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Objective: To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM)and used as embryonic biomarkers.Design: Prospective cohort study.Setting: Private and academic in vitro fertilization centers.Patient(s): Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts.Intervention(s): Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated onhuman embryonic stem cells (hESCs) and on SBM with and without biological variability.Main OutcomesMeasure(s): Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles betweenimplanted and unimplanted euploid blastocysts.Result(s): Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence ofmiRNAs. The SBM were prospectively collected from euploid implanted (n¼ 25) and unimplanted blastocysts (n¼ 28) for comparison.We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TEand SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3–99.6) of the miRNAs detected in the SBM were expressed from TE cells,suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, sug-gesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid im-planted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in theformer and were predicted in silico to be involved in 23 implantation-related pathways.Conclusion(s): MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this

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approach can be further explored for noninvasive embryo selection. (Fertil Steril� 2015;-:-–-. �2015 by American Society for Reproductive Medicine.)Key Words: Biomarkers of implantation, blastocyst-endometrial dialogue, blastocystevaluation, embryo quality, microRNA

Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/capalboa-mirnas-blastocyst-culture-medium/

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Received July 30, 2015; revised and accepted September 10, 2015.A.C. has received grants from Merck-Serono. M.U.F. has nothing to disclose. D.C. has nothing to disclose. L.N. has nothing to disclose. Y.K. has nothing to

disclose. A.F. has nothing to disclose. D.I. has nothing to disclose. L.R. has nothing to disclose.Supported by the Merck Serono Grant for Fertility Innovation (2013) and a Saudi Arabia government studentship (to L.N.).Reprint requests: Antonio Capalbo, Ph.D., GENERA, Centre for ReproductiveMedicine, Clinica Valle Giulia, via De Notaris 2/B, Rome, Italy (E-mail: capalbo@

generaroma.it).

Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc.http://dx.doi.org/10.1016/j.fertnstert.2015.09.014

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M icroRNAs (miRNAs) are evolutionarily conserved,single-stranded, noncoding RNA molecules of�22 nucleotides in length. Over 1,000 miRNAshave been identified in the human genome (miRBase,www.mirbase.org), and they are considered to be major tran-scriptional/posttranscriptional regulators of gene expression.A miRNA can modulate the levels of many target genes atonce through partial sequence complementation, usually atthe 30 untranslated regions (UTRs) of mRNAs, and subsequentinterference with mRNA stability and/or protein translation(1, 2). MicroRNAs are secreted in membrane-bound exosomesand microvesicles, are bound to stabilizing proteins, and canbe detected in virtually all body fluids, including blood, urine,saliva, tears, breast milk, semen, amniotic fluid, cerebrospinalfluid, peritoneal fluid, and pleural fluid as well as in culturemedia collected from different cell lines (3–5). It is believedthat miRNAs that are secreted from donor cells can beinternalized into recipient cells, where they can exert theirgenetic regulatory function. Chen et al. (6) tested bloodserum samples and found that miRNAs were highly stableand able to withstand extreme environmental conditionssuch as freezing and thawing; thus, they may be effectivebiomarkers. Consequently, there is great interest inidentifying miRNAs within biofluids that can be used asnoninvasive biomarkers for the early detection of diseases.For example, miR-141, miR-499, and miR-122 are associatedwith prostate cancer, myocardial infarction, and drug-induced liver injury, respectively (3,6–9).

We hypothesized that human embryos could also secretespecific miRNAs in the extracellular environment as part ofthe blastocyst–endometrium interaction, which is necessaryfor implantation success. This interaction could determinethe establishment of phase synchrony, ultimately aiming tocreate a hospitable environment for competent embryos atthe implantation site, which is a prerequisite for successfulimplantation. Several studies have already illustrated dy-namic changes in miRNA expression in gametes and duringearly embryonic development of mammalian species (10–14). Other studies have recently reported that the humanendometrial epithelium releases exosomes into the uterinecavity. If these exosomes transfer their contents to eitherthe blastocyst or the adjacent endometrium, they couldinfluence implantation by affecting endometrial geneexpression (15, 16). Furthermore, new communicationsystems mediating the cross-talk between the blastocystand the endometrium based on secreted miRNAs have beenrecently discovered in animal models. In particular, LIF-dependent STAT activation and its impact on modulation oftrophoblast functions during embryo implantation wasdescribed in several animal models (17–20) and recentlydirect evidence of the role of miR-181 secreted by endometrialcells in modulating embryo implantation through the regula-tion of LIF has been reported in mice (21).

However, to date no comprehensive data have been re-ported regarding blastocyst-derived miRNAs in the extracel-lular environment. In this context, growing embryos in vitroduring IVF cycles offer a unique possibility to determine thepresence of miRNAs in easily collectable embryonic culturemedia and identify noninvasive biomarkers associated with


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embryo reproductive competence. We comprehensively char-acterized the profile of miRNAs secreted by human preim-plantation embryos in spent culture media and investigatedwhether secreted miRNAs can be used as predictive markersof clinical outcomes in in vitro fertilization (IVF) cycles.

MATERIALS AND METHODSStudy Design and Outcome Measures

In our prospective cohort study, the primary hypothesis wasthat human embryos secrete miRNAs in culture media,which can be used as biomarkers of embryo quality duringIVF cycles. First, a protocol for miRNA purification andanalysis based on quantitative polymerase chain reaction(qPCR) was set and validated on human embryonic stemcells (hESCs) of different concentrations and on spent blas-tocyst culture media (SBM) with and without biological vari-ability. A miRNA profile of trophectoderm (TE) cells andtheir relative culture media was then generated andcompared among these specimens. Next, SBM samplescollected from embryos at different stages of preimplanta-tion development (cleavage, morula, and blastocyst stages)were profiled for miRNAs to investigate when the secretionprocess begins in the preimplantation window. Finally, miR-NAs profiles of SBMs prospectively collected from implantedversus unimplanted euploid blastocysts were compared toinvestigate the potential for the use of these biomarkersfor embryo selection.

Embryos

The work described here was performed under a license fromthe United Kingdom Human Fertilization and EmbryologyAuthority (research license numbers: R0075 and R0133) andalso received local ethics approval (U.K. National Health Ser-vice Research Ethics Committee Reference: 06/Q0702/90).Informed consent was obtained from all participants, andthe experiments conformed to the principles set out in theWorld Medical Association Declaration of Helsinki and theNational Institutes of Health Belmont Report. No financialdonations were offered. Cryopreserved embryos that wereno longer needed for therapeutic use by patients were ob-tained from Guy's Assisted Conception Unit and externalunits through the Human Embryonic Stem Cell Coordinator's(hESCCO) network (22). To obtain a pure TE fraction, the innercell mass (ICM) was biopsied from expanded blastocysts12 hours after warming as described elsewhere (23). WholeTE samples and 25 mL of SBM from each embryo, collectedimmediately before TE isolation, were snap-frozen and laterprocessed individually for miRNA analysis.

Human Embryonic Stem Cells

The hESC line KCL034 was cultured as previously describedelsewhere (24–26). Smaller (approximately 10–50 cells) andlarger (approximately 500–1,000 cells) clumps of hESCswere microdissected free of feeders, transferred directly tolysis buffer, and snap-frozen in liquid nitrogen.

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Spent Culture Medium from Single-embryoTransfer Cycles

The spent culture medium was collected from embryos at thecleavage and morula stages or from expanded blastocysts ofgood quality and without signs of degenerating cells duringin vitro fertilization (IVF) cycles. The ethics committee ofClinica Valle Giulia approved the study.

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IVF Procedures and Spent Medium Collection

All embryos were obtained after controlled ovarian stimula-tion that was performed using a gonadotropin-releasinghormone-agonist long protocol and IVF. Fertilized oocyteswere sequentially cultured in separate 25-mL microdrops(Sage) up to the blastocyst stage (days 5–6) in a humidified at-mosphere containing 5%O2 and 6% CO2. On day 3 after fertil-ization, cleavage-stage embryos were transferred to a freshindividual 35-mL drop of Quinn's Advantage Blastocyst Me-dium (Cooper Surgical) with 5% Quinn's Advantage HumanSerum Albumin (Cooper Surgical). The quality of the blasto-cysts was assessed immediately before cryopreservation andwas defined according to the standard criteria (27).

The blastocyst vitrification and warming procedures wereperformed using a Vitrification and Warming Kit (KitazatoBioPharma) (28, 29). The entire vitrification and warmingprocedures were performed at room temperature aspreviously described elsewhere (30). Immediately afterwarming, the blastocysts were allocated to individuallynumbered 35-mL microdrops of blastocyst mediumsupplemented with 5% human serum albumin undermineral oil (Sage). The TE biopsy, 24-chromosome aneuploidyscreening, and vitrification were performed as previouslydescribed elsewhere (31). Euploid blastocysts were selectedfor transfer based on the morphologic score and were warmedand cultured at 37�C (6% CO2 and 5% O2) until transfer. Wetransferred 25 mL of SBM to individually labeled PCR tubescontaining 120 mL of lysis solution. The PCR tubes werethen briefly centrifuged and stored at �20�C before miRNAisolation. Only cycles with single embryo transfers wereincluded. The endometrial preparation and transfer proce-dures were performed as previously described elsewhere(32). Ongoing implantation was defined by the presence ofa fetus with heart activity beyond 20 weeks of gestation (33).

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MicroRNA Isolation, Retrotranscription, andPreamplification

The miRNA isolation and purification procedures were per-formed using the TaqMan miRNA Anti-miRNA Bead Cap-ture (ABC) Purification Kit (Applied Biosystems), whichwas designed specifically for the rapid purification of miR-NAs from small inputs of all human sample types includingcell cultures. Human Panel A v3.0 beads are a type of superparamagnetic Dynabeads that are covalently bound to aunique set of 381 anti-miRNA oligonucleotides. The miRNAisolation relies on hybridization of endogenous miRNAs tothe corresponding anti-miRNA oligonucleotides attachedto the beads. The entire recoverable volume of sample wasbrought to a total volume of 150 mL with ABC buffer (and

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to a total volume of 200 mL with lysis buffer in the caseof hESCs).

The purification protocol was followed according to themanufacturer's instructions. The bead hybridization and themiRNA elution steps were conducted on a Thermomixer (Ep-pendorf). All the washing steps were performed using theDynaMag-2 magnet (Applied Biosystems) to remove DNA,proteins, contaminants, and residual binding solution whilekeeping the miRNAs bound to the beads. The miRNAs wereeluted in a final volume of 10 mL to concentrate them in thesmallest possible volume (20 mL in the case of hESC). ThecDNA was then generated using the specific Megaplex RTPrimer Pools A v3.0 (Applied Biosystems) on a 7900 HTReal-Time PCR System (Applied Biosystems) according tothe manufacturer's protocol.

The preamplification reaction was modified because twocDNA synthesis reactions were performed in parallel persample to double the final sample volume. Specifically,two independent reactions were performed in two differenttubes to create products that were joined at the end of theprocess; each tube contained 3.5 mL of retrotranscriptionproduct, 12.5 mL of TaqMan PreAmp Master Mix (2X),2.5 mL of Megaplex PreAmp Primers (10X), and 6.5 mL ofnuclease-free water. The thermal protocol used was thesame as that reported by the manufacturer and consistedof eight cycles.

MicroRNA Analysis Using a TaqMan Low-DensityArray

MicroRNA expression was evaluated using TaqMan Low-Density Array (TLDA) miRNA Cards (Panel A) (Applied Bio-systems) with an Applied Biosystems Viia7 Real-Time PCRSystem. Card A contains 381 TaqMan miRNA assays,enabling the simultaneous quantitation of 377 human miR-NAs plus three endogenous controls and a negative control.Fifty microliters of the preamplification product was addedto 400 mL of nuclease-free water and mixed with 450 mL ofTaqMan Universal Master Mix II, No UNG. The mixture wasthen dispensed into 384 wells by centrifugation on a HeraeusMegafuge 40 (Thermo Scientific) with the proper TLDA cardadapters. The reactions were incubated in a 384-well plateat 95�C for 10 minutes followed by 40 cycles of 95�C for15 seconds and 60�C for 1 minute on a ViiA 7 Real-TimePCR System (Applied Biosystems).

After a preliminary manual inspection of all amplifica-tion plots, the raw data were analyzed using SDS software(Applied Biosystems), and the cycle threshold (Ct) valueswere used as a readout. All Ct values below 35 were consid-ered valid in order to capture all potential meaningful signalsfrom such low-input samples where no comprehensive dataabout miRNAs spectra have been reported. False amplifica-tion curves were manually excluded from the downstreamanalysis. The data were processed using Real-Time StatMiner5.0 software (Integromics).

Two blank media control drops (hereafter referred to asnegative controls) were incubated in the same dishes as thosewith media drops containing embryos. They were processedfor miRNA analysis as previously described.

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Cross Validation of miRNAs Detected in TE andSBM Samples Using Single Assays

To cross-validate the results obtained through the protocolpreviously described, a completely different method formiRNA quantification was used, which instead involvedcolumn-based miRNA purification and analysis with noneed of preamplification and using a SYBR green-basedqPCR quantification. In particular, microRNAs isolationfrom TE and SBMs was conducted with miRCURY RNA Isola-tion Kit for Biofluids (EXIQON) according to manufacturer'sinstructions. The sample initial input was 25–30 mL, 1 mgcarrier-RNA MS2 (Roche) was added to each sample beforeprotein precipitation step, and elution was performed in20 mL. Five different biologic replicates for both SBM andTE were analyzed.

Retrotranscription was conducted according to miR-CURY LNA Universal RT individual microRNA assays proto-col, modifying only the sample input volume. In particular,2 mL of 5X reaction buffer, 1 mL of enzyme mix, and 0.5 mL ofUniSP6 spike-in control (a synthetic template to monitorprotocol efficiency) were added to 6.5 mL of sample. Thethermal protocol respected the manufacturer's indications.Retrotranscription product was diluted 1:8 with nuclease-free water.

The qPCR reaction was performed according to the stan-dard protocol on a Viia7 instrument (Applied Biosytems). Inparticular, the following assays selected as TE specific (miR-512-5p, miR-522-3p), two miRNAs significantly different be-tween implanted and unimplanted blastocysts (miR-20a-5p,miR-30c-5p), five randomly selected from those present inSBM samples (miR-373-3p, miR-302b-3p, miR-146a, miR-512-3p, miR24-3p), and two cellular housekeeping (RNU44and RNU48) were tested. We added 5 mL of ExiLENT SYBRGreenMaster Mix and 1 mL of specific assay to 4 mL of sample,thus obtaining a final dilution of the elute input volume of1:31. A no-template control of real-time (RT) and qPCRwere also included in the analysis, and also a blank culturemedia sample never exposed to embryo culture.

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Statistical Analysis

After inspection of putative normalizers, a global expression-based normalization strategy was used, where the meanexpression Ct values of all detectors with valid Ct were usedas the normalization factor to normalize the data for a givensample. Whole-genome RT-qPCR based miRNA profiling incombination with a global mean normalization strategy hasproven to be the most sensitive and accurate approach forhigh throughput miRNA profiling (34).

The mean and the range of the fold change for eachmiRNA were calculated as 2�DDCt, using the estimatedDDCt value� standard error (SE) (35). Hierarchical clusteringanalysis based on normalized Ct values was performedthrough single linkage and Pearson's correlation as a similar-ity method to test the relationship between hESCs of differentcell contents. The scale bar values represent the DDCt values.The heat map was produced by median centering expressionvalues for each miRNA and then performing hierarchicalclustering.

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To investigate the ability of the miRNA protocol to cap-ture biological variation in SBM samples, the mean Ct valuecorrelation coefficients between mixed and unrelated SBMsamples were assessed and compared. The resulting P valueswere adjusted for multiplicity as described herein. The scalebar values represent the DDCt values. The heat map was pro-duced bymedian centering expression values for each miRNAand then performing hierarchical clustering. The differentialexpression analysis of SBM versus TE samples and of SBMsamples from implanted versus unimplanted blastocysts wasbased on the nonparametric Wilcoxon test.

Each family of tests was made by several hypothesestested simultaneously. Many false positives could arise byrandom chance if a single-inference significance level of5% was used. We therefore collected all raw test statistics, as-sessed their dependence, and verified that [1] it was weakenough and [2] a permutation could be found leading to adecreasing pattern (36, 37). As both checks were passed, wedeemed the Benjamini and Hochberg (38) method to bevalid; we thus proceeded by collecting all raw P values,sorting them, and comparing the i-th ranked P value withthe threshold 0.05i/m, where m is the total number of Pvalues. This would guarantee that the expected proportionof false positives over the number of rejections would bebelow 5%. A volcano plot was generated using statisticalsignificance versus fold-change on the y-axis and x-axis,respectively.

Target Prediction and Pathway Analyses

The analyses were performed with DIANAmiRPath v.2.0 soft-ware (http://diana.cslab.ece.ntua.gr) (39). The software ex-ploits the miRNAs and pathway information provided bymiRBase (http://www.mirbase.org) and the Kyoto Encyclo-pedia of Genes and Genomes (KEGG) v58.1 (http://www.ge-nome.jp/kegg). Both in silico-predicted and experimentallyvalidated gene targets were selected in the present analysis.The software performs the prediction in silico using the algo-rithm DIANA-microTCDS, which detects and scores bothmiRNA binding sites in the 30-UTR and coding sequences ofthe predicted targets. The default 0.8 threshold score wasadopted.

The experimentally validated miRNA-gene interactionswere instead provided by the DIANA TarBase v6.0 database(http://diana.cslab.ece.ntua.gr/tarbase). The ‘‘union of genes’’clustering option was set, which selects unions of genes tar-geted by at least one of all the uploaded miRNAs, before per-forming the statistical calculation. The software then adoptsthe Fisher's combined probability method to calculate amerged P value for each single pathway. The false discoveryrate was determined according to the Benjamini and Hoch-berg method (38) before displaying the results.

RESULTSMicroRNAs in Spent Blastocyst Medium AreExpressed Also in Trophectoderm Cells

To determine whether sample low-input volume would influ-ence the sensitivity and accuracy of the assay, we compared

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FIGURE 1 FIGURE 1 Continued

Comparison of the miRNA profiles between the trophectoderm (TE)and spent blastocyst culture media (SBM) samples. (A) Schemeillustrating the sample collection. Samples subjected to miRNAprofiling are highlighted with a red frame. ICM ¼ inner cell mass.(B) The majority of miRNAs detected in more than three TE and/orSBM samples were shared among all samples examined. (C) TheVenn diagram shows that after a quality control (QC) filter, 57 of59 miRNAs detected in SBM samples were also present in TE. (D)Correlation heat map among the miRNA expression profiles. (E)Volcano plot showing differential expression between the TE andSBM samples. Blue dots indicate assays detectable in both groups;yellow and red dots indicate assays detectable only in the SBMsample or TE sample, respectively. The Log10 of the relativequantitation is plotted on the x-axis, and the statistical significance(expressed as the negative Log10 of the adjusted P value) is plottedon the y-axis. The green line represents the cutoff to define themiRNAs that were more abundant in the SBM samples fromimplanted blastocysts, the red line represents the cutoff to definethe miRNAs that were more abundant in the SBMs fromunimplanted blastocysts, and the horizontal black line representsthe cutoff for the statistical significance after a Benjamini-Hochbergfalse-detection rate correction. (F) Five miRNAs were statisticallysignificantly more abundant in the TE samples compared with theSBM samples (miR-16, miR-17, miR-20b, miR-518b, and miR-9),whereas two miRNAs were statistically significantly more abundantin the SBM samples with respect to the TE samples (miR-223 andmiR-328; P

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Ten miRNAs were common in all SBM samples, 41 miRNAswere common in four out of five samples, and 59 were com-mon in three out of five samples (see Fig. 1B and C).

The TaqMan Low Density Array (TLDA) cards includedthree assays (RNU-44, RNU-48 and U6-snRNA) for miRNAsthat are commonly used as putative normalizers. The expres-sion of all of these miRNAs was detected with high consis-tency in all the TE biologic replicates but only in four out offive SBM samples (Supplemental Fig. 3, available online).The nonhuman negative control miRNA, ath-miR-159a, wasnegative in all samples tested. In light of this evidence, aglobal normalization strategy was adopted in the subsequentanalysis based on the geometric mean of all the Ct values ob-tained for all assays for each sample. The blank samplesshowed a consistent amplification for few miRNAs (miR-150, miR-891a, miR-155, miR-184, miR-212, miR-320,miR-486-3p, miR-126, miR-202, miR-370, miR-433, miR-518d, miR-548c, miR-520d) that were interpreted as false-positive signals and excluded from the subsequent analysisof test samples.

To optimize the selection of stably expressed miRNAs,only the miRNAs that were detected in at least three outof five biological replicates were included in the subsequentanalysis. The majority of the detected miRNAs were sharedby R3 samples (85.0%–94.1% for TE and 68.3%–95.8%for SBM; see Fig. 1B). After this filtering step, 128 differentmiRNAs were flagged as valid for the TE samples, and 59were identified for the media samples, representing approx-imately 34% and 16% of the miRNAs on the arrays, respec-tively (see Fig. 1C). The mean Ct values of miRNAs includedwere 28.1 � 3.5 and 29.9 � 2.7 for TE and SBM samples,respectively.

As expected, the mean Ct values for TE was statisticallysignificantly higher compared with the SBM samples(P< .01). Even though 7.9% and 15% of the miRNAs detectedshowed a Ct value above 33 for TE and SBM samples, respec-tively, we decided to include these targets for future analysisto describe the whole population of miRNAs that can be de-tected in this unique data set. A comparative analysis of theTE and SBM samples revealed that 96.6% (57 of 59; 95% CI,88.3–99.6) of the miRNAs detected in the SBM were indeedexpressed from TE cells, suggesting that the miRNAs detectedin the media can be released from TE cells (see Fig. 1C). A bio-informatics analysis of the predicted and experimentally vali-dated target genes for each miRNA detected in the SBMsamples (Supplemental Table 1, available online) pointed toseveral pathways and biologic processes known to beinvolved in embryo implantation, including apoptosis, cellproliferation, and cell communication and differentiation(Supplemental Table 2, available online).

Hierarchical clustering analysis showed a sharp separationbetween TE and SBM samples based on miRNAs profiles (seeFig. 1D). A differential expression analysis showed that twomiRNAs were statistically significantly more abundant in theSBM with respect to TE (miR-223, and miR-328, with foldchanges of 44.7, and 58.0, respectively; P< .01), and five miR-NAs were statistically significantly more abundant in the TEcompared with the SBM samples (miR-16, miR-17, miR-20b,miR-518b, and miR-9, with fold-changes of 5.75, 105.7, 7.7,

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19.4, and 26.7, respectively; P< .02) (see Fig. 1E and F). More-over, two miRNAs were detected only in the SBM samples andnot in the TEs. The absence of these miRNAs in TE cells can beassumed as a process of very high secretion from the TE, ahigher stability into the extracellular environment, or anICM origin that was not investigated here. Furthermore, 71miRNAs were unique to the TE samples (see Fig. 1C and E).Although there may be several possible explanations for thisdifference between the cellular and extracellular miRNA pop-ulations, including cell lysis and preferential stability inside oroutside of the cell, these results are consistent with the hypoth-esis that there is a specific packaging and export mechanismfor extracellular miRNAs (Wang et al., 2010). Q

MicroRNAs in Conditioned Culture Media can BeConsistently Detected Only after Blastulation

The miRNAs in the SBM may be secreted not only by TE butalso by the embryo at earlier stages of development withina limited period of time (e.g., at the cleavage or morula stages).We therefore collected spent media samples from the sameembryos (n ¼ 3) at the cleavage, morula, and expanded blas-tocyst stages within the same IVF cycle (Fig. 2A). Analysis ofmiRNAs in the media collected from embryos at the cleavageand morula stages revealed a profile similar to that which wasobserved in the negative controls. In particular, the miRNAsdetected in the spent culture media collected from embryosat the cleavage (11 miRNAs) and morula stages (11 miRNAs)were all previously detected in the negative controls (culturemedium only), suggesting that this group of miRNAs is relatedto same intrinsic and systematic false-positive amplifications(see Fig. 2B and C). In the SBM samples, absolute numbers of51, 88, and 65 different miRNAs were detected, with 43 ex-pressed in all of the biologic replicates (see Fig. 2B). Excludingfalse positives, blastocyst SBMs showed 34 different miRNAs.This analysis suggests that the miRNAs can be consistentlydetected in culture media only after blastulation.

MicroRNAs that Are Differentially Expressed inImplanted versus Unimplanted Blastocysts HaveBeen Associated with Implantation RelevantProcesses

Next, we investigated in a proof of principle analysis the po-tential of miRNAs detected in the SBM samples to serve asbiomarkers for predicting the reproductive competence ofblastocysts. The SBM samples from single vitrified-warmedembryo transfer of euploid blastocysts were compared amongembryos that resulted in an ongoing implantation and em-bryos that failed to implant (Fig. 3A). To rule out the signifi-cant confounding factor of aneuploidies on implantation,only blastocysts shown to be chromosomally normal afterTE biopsy and comprehensive chromosomal screening ana-lyses were considered.

The SBM samples were prospectively collected from allembryos reaching the blastocyst stage from 44 consecutivepatients attending a blastocyst-stage preimplantation geneticscreening (PGS) cycle, with embryo cryopreservation and em-bryo transfer performed on the subsequent natural cycle.

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MicroRNA (miRNA) detection in spent culture media collected at different stages of preimplantation embryo development. (A) Spent medium wascollected at three different stages of preimplantation development (cleavage, morula, and blastocyst stages) from three embryos (E1, E2, and E3)cultured from the cleavage to the blastocyst stage. Marked increases in the number of miRNAs detected were observed simultaneously at theblastocyst stage in all three embryos. (B) Most of the miRNAs detected at each stage were shared among all the samples. (C) Only nine miRNAswere detected in all samples at all stages (miR-126, miR-184, miR202, miR-28-3p, miR-320, miR-212, miR-370, miR-433, and miR-486-3p).These same miRNAs were also detected in the negative controls.Capalbo. MicroRNAs in blastocyst culture media. Fertil Steril 2015.


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Eight patients transferred a euploid blastocyst twice. Fifty-three euploid blastocysts were transferred, 25 of which re-sulted in an ongoing implantation. The laboratory andclinical outcomes for these IVF-PGS cycles are reported inSupplemental Table 3 (available online).

After normalization and filtering steps (>60% detectionrate across each biologic group), the total number of miRNAsdetected in the SBM of implanted blastocysts was somewhat

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higher (n ¼ 38) compared with that of unimplanted blasto-cysts (n ¼ 33). The mean Ct values of miRNAs detected inSBM of implanted and unimplanted blastocysts were 28.5 �4.3 and 28.5 � 4.5 (not statistically significant). The meandelta Ct values were also not statistically significantlydifferent between the two groups; in particular, the valueswere 2.8� 5.6 for implanted blastocysts and 3.4� 4.9 for un-implanted blastocysts.

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FIGURE 3 FIGURE 3 Continued

Differential expression of two miRNAs in the SBM of implantedembryos. (A) Scheme of the sample collection from embryos thatwere transferred into patients. Embryos were cultured to theblastocyst stage in vitro. Spent blastocyst culture media (SBM)samples were collected immediately before trophectoderm (TE)biopsy. Blastocysts were vitrified immediately after the biopsy.Quantitative polymerase chain reaction–based comprehensivechromosomal screening (CCS) was performed on the TE biopsies.Only euploid blastocysts were thawed for single-embryo transferand SBM profiling. COC ¼ cumulus oocyte complexes. (B) Volcanoplot showing a comparison of the miRNAs in SBMs from implanted(n ¼ 25) and unimplanted blastocysts (n ¼ 28). Blue dots indicateassays detectable in SBMs from both groups; yellow dots indicateassays detectable preferentially in SBMs from implanted blastocysts;red dot indicates the assay preferentially detected in SBM ofunimplanted blastocysts. The Log10 of the relative quantitation isplotted on the x-axis, and the statistical significance (expressed asthe negative Log10 of the adjusted P value) is plotted on the y-axis.The green line represents the cutoff to define the miRNAs thatwere more abundant in the SBM from implanted blastocysts, thered line represents the cutoff to define the miRNAs that were moreabundant in the SBM from unimplanted blastocysts, and thehorizontal black line represents the cutoff for statistical significanceafter a Benjamin-Hochberg false-detection rate correction. TwomiRNAs were identified as statistically significantly more abundantin the SBM from implanted blastocysts: miR-20a and miR-30c. (C)Box plot built on delta cycle threshold (Ct) values of miRNAs thatwere differentially expressed between SBM samples from implantedand unimplanted blastocysts. (D) Frequency of detection of miRNAsthat were differentially expressed in the SBM of implanted andunimplanted blastocysts. (E) Receiver operating characteristicanalysis showing the clinical predictivity of miR-20a and miR-30c onimplantation potential of euploid blastocysts. (F) Pathway analysisfor miR-30c and miR-20a. These miRNAs are predicted to regulategenes involved in 25 KEGG pathways, 23 of which may have a rolein blastocyst–endometrial communication: cell-to-cellcommunication/cell signaling, cell-to-cell adhesion, and cell growth.Capalbo. MicroRNAs in blastocyst culture media. Fertil Steril 2015.

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A statistical analysis comparing the miRNA profiles be-tween chromosomally normal implanted (n ¼ 25) and unim-planted blastocysts (n ¼ 28) revealed two differentiallyexpressed miRNAs (miR-20a and miR-30c; P< .05) thatshowed increased concentrations in SBM from implantedblastocysts (see Fig. 3B). The mean Ct values for these miR-NAs were 22.3 � 3.0 versus 24.0 � 2.1 (miR-20a) and 23.4� 3.4 versus 26.1 � 2.7 (miR-30c) for implanted and unim-planted blastocysts, respectively. The normalized Ct valuesfor these two miRNAs were statistically significantly lowerfor implanted blastocysts (see Fig. 3C). Furthermore, fivemiRNAs (miR-220, miR-146b-3p, miR-512-3p, miR-34c,miR-375) were preferentially detected in SBM samples ofimplanted blastocysts compared with blastocysts that failedto implant (see Fig. 3B). A combined secretion of these twomiRNAs (miR-20a and miR-30c) was detected in the vastmajority of implanted embryos (96%), and they were some-what less prevalent in embryos that did not implant (seeFig. 3D). The receiver operating characteristic analysis per-formed on implantation with these two miRNAs showedthat each single miRNA was predictive of implantation,with an area under the curve of 0.773 (95% CI, 0.637–0.908) and 0.786 (0.663–0.909) for miR20 and miR30c,respectively (see Fig. 3E).

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1004100510061007100810091010101110121013101410151016101710181019

Based on in silico-predicted and experimentally vali-dated miRNA targets, miR-20a and miR-30c are involvedin 25 different pathways and biologic processes (seeFig. 3F; Supplemental Table 4, available online). The path-ways represented are mainly involved in cell-to-cellcommunication and signaling (6 of 25, 24%), cell-to-celladhesion (1 of 25, 4%), and cell growth/cancer (16 of 25,64%). It is interesting that, based on bioinformatics analysis,miR-20a has been experimentally validated to modulate fivegenes (PTEN, NRAS, MAPK1, MYC, and CCND1) and is pre-dicted to regulate two additional transcripts (SOS1, TCF7L1)involved in endometrial cell proliferation and growth.Furthermore, miR-30c is predicted to regulate five distincttranscripts (APC, KRAS, PIK3CD, SOS1, and FOXO3) thatare also involved in endometrial cell proliferation andgrowth.

1020102110221023102410251026102710281029103010311032103310341035103610371038103910401041104210431044104510461047104810491050105110521053105410551056105710581059106010611062

Single Assay-based Cross-validation of RelevantmiRNAs from TE and SBM Samples

To cross-validate the results obtained from the comparison ofSBM samples from implanted and unimplanted blastocysts, acompletely different method of miRNA analysis was used,which instead involved column-based miRNA purificationand analysis with no need of preamplification and using aSYBR green-based qPCR quantification (SupplementalTable 5, available online). We assessed the expression oftwo distinct miRNAs that were exclusively detected in TEsamples and never in SBM samples (miR-512-5p; miR-522-3p), confirming a stable expression in five different TE sam-ples and the absence of detection in five unrelated SBMsamples.

Next, we confirmed a consistent expression of the twomiRNAs showed to be more abundant in SBM of implantedblastocysts (miR-20a and miR30c). Then another five miRNAswere randomly selected from the ones expressed in the SBMof euploid blastocysts and confirmed by single assay analysison five unrelated SBM and five TE samples. Only one miRNA(miR-24-3p) was not confirmed in the majority of SBMreplicates.

Finally, the two putative cellular normalizers, RNU44 andRNU 48, were also confirmed by single assays showing lowstability and detection rate in SBM samples, as expected.One exogenous miRNA (UniSp6) was also run in paralleland showed a very stable and consistent detection acrossthe different replicates, thus highlighting low technical vari-ability across the samples analyzed. Blank samples, namelywater and culture media never exposed to blastocyst culture,were run in parallel and showed no amplification for any ofthe tested miRNAs.

DISCUSSIONHere, for the first time, we have comprehensively character-ized the population of miRNAs secreted from human blasto-cysts that are present in the spent culture media, where theymay act to directly transfer information from the blastocystto the surrounding endometrial cells, thus altering the poten-tial for implantation success. These biological findings sug-gest the possibility of using miRNAs from spent culture

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media, which is easy to collect, as noninvasive biomarkersof embryo quality during IVF cycles.

First, one of the main challenges of the study was todevelop and validate a reliable protocol for miRNAs quantifi-cation from low-input samples, such as 25 mL of culture me-dia conditioned after 12 hours of blastocyst culture afterwarming. As preliminary validation step of the protocol, largeand small clusters of hESCs were compared, showing the pro-tocol to be highly robust even when scaled down on low-input samples. Furthermore, mixed SBM samples showed astatistically significantly higher correlation compared withunrelated samples, suggesting that the protocol was able tocapture biologic variation between SBM samples.

Next, we characterized the miRNA signature of SBM sam-ples showing that 96.6% of the miRNAs detected are ex-pressed also from TE cells with few miRNAs being moreabundant in the SBM or TE fraction. These results are consis-tent with a specific packaging and exporting hypothesis forextracellular miRNAs and in agreement with previous studiesperformed on cell lines where exosomes derived frommyoblast or PC-prostate cancer cells contained differentiallysorted miRNA (40, 41).

Intriguingly, the SBM samples were collected after12 hours of blastocyst culture after warming, a time framethat was sufficient to allow a consistent quantification ofmany miRNAs. Several studies already claimed a consistentisolation of substantial amounts of miRNAs in themedium af-ter culture of different cell types at variable timings. In agree-ment with our findings, a previous study by Wang et al. (4)that investigated the kinetic of miRNAs exportation fromcultured cells after different biologic stimuli found thatmost of the miRNAs tested appeared to be actively exportedin a short pulse lasting about an hour after serum deprivation(4).

Fourteen of the 59 miRNAs detected in the SBM sampleswere predicted to be involved in endometrial cell growth andproliferation, suggesting the existence of a previously unrec-ognized potential communication system between the em-bryo and the implantation site on the uterine wall.However, important pieces of the puzzle are still missing.Although there is evidence that vesicles containing miRNAsmay be taken up by the cells (42), direct proof that endome-trial cells are internalizing miRNA-containing complexessecreted by blastocysts is not yet available. Secreted miRNAsmay target and convey information to neighboring TE cellsand not to the uterus. Regardless, the elucidation of this novelcommunication system between an embryo and its implanta-tion site will certainly be of the utmost importance in under-standing the many biologic processes that governimplantation.

To our knowledge, only one study has attempted to pro-file miRNAs from blastocyst culture media so far (43). Howev-er, due to many specific differences in the protocol formiRNAs extraction and quantification, only 10 miRNAswere detected in culture media samples in that study, witheight of them shown to be false-positive signals.

Finally, to investigate the potential role of miRNAs anal-ysis from SBM as embryonic biomarker, single-embryo trans-fer of euploid blastocysts was compared between implanted

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and unimplanted embryos. This analysis revealed two differ-entially expressed miRNAs showing higher concentration inSBM samples of implanted blastocysts. Bioinformatics anal-ysis of these miRNAs showed several biologic pathways andcellular function regulated from them, including cell prolifer-ation, cell communication, and cell signalling. Importantly,these two miRNAs have been already reported as circulatingin human body fluids (44–50). Even if further data areneeded to confirm these preliminary findings, this is thefirst report highlighting the potential role of miRNAsquantification from the easily collectable SBM as a newbiomarker to be further explored for embryo selection.

In the context of human embryo selection for IVF, theideal embryonic biomarker would allow noninvasive assess-ment of the embryo, would be stable over time, specific to em-bryos, and easily measurable to allow rapid and accurateassessment of embryo quality, all features fitting this newlyproposed approach. Indeed, a prominent feature of circulatingmiRNAs is their remarkable stability (51): they are protectedby forming complexes with proteins such as Argonaute (52)and GW182 (53) or are encapsulated in exosomes that provideadditional protection from degrading enzymes (54). More-over, miRNAs are a specific product of the embryo that canbe related to the quality of many differentiation processesthat beset blastocyst differentiation and are not supposed tobe already present in culture media. Furthermore, changingconcentration of extracellular miRNAs has been already asso-ciated with a variety of pathologies (6, 8,55–57). Finally, real-time qPCR–based miRNA analysis can be performed in-housein a matter of hours with high accuracy, rendering fresh blas-tocyst transfers feasible with same-day media analysis.

It should be acknowledged as a limit of the study that dueto statistical distribution there is always a high level of Ctvariation when target quantities approach single copy (usu-ally for Ct values >33 after the preamplification step), as inthe case of conditioned culture media samples. In our analysis15% of the miRNAs described in SBMs showed a Ct value>33. Therefore, miRNAs that yield Ct values in this rangewill unavoidably give rise to poorer precision and conse-quently less power to detect low-fold changes. However, wedecided to include these miRNAs to capture all the biologic in-formation from SBM samples, and differentially expressedmiRNAs between implanted and unimplanted blastocystsshowed a mean Ct value 1.000 discovered in humancells. Accordingly, untested miRNAs might still exist inSBM samples and may play a role in the blastocyst-endometrial dialogue. Furthermore, direct proof that endome-trial cells are internalizing miRNA-containing complexessecreted by blastocysts is not yet available and is underinvestigation.

In conclusion, these biological and preliminary clinicalfindings open the possibility to further explore the analysisof miRNAs from the easily collectable spent culture mediaas a noninvasive biomarker of embryo quality during IVFcycles. In this study we provided a proof of principle thatprofiling miRNAs from SBM may enhance blastocyst assess-ment during IVF cycles or might be used together with TE-

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based aneuploidy screening to further improve selectionamong euploid blastocysts. This preliminary clinical investi-gation used a high-throughput approach to test for 381different humanmiRNA sequences. A prospective multicentrestudy involving both private and academic IVF centers iscurrently ongoing where a custom panel of 46 selected miR-NAs will be used to corroborate the present findings on ahigher and defined sample size that will guarantee a two-sided 95% confidence interval of total length at most 6% todefine the clinical power of embryo selection based on miR-NAs profiling from SBM samples.

The use of this custom platform is also the first step to-ward the reduction of complexity and the development of adiagnostic assay based on the analysis of informativemiRNAsonly from SBM to select reproductively competent embryos atreasonable cost and with a low turnaround time of analysis.All these aspects will better fit with the clinical requirementsof embryo selection IVF, providing a novel and practical toolfor the noninvasive assessment of embryo quality.

Acknowledgments: We are especially indebted to the pa-tients who donated embryos.

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SUPPLEMENTAL FIGURE 1

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Correlation matrix of microRNA (miRNA) profiles from large (n ¼ 3)and small (n ¼ 3) clusters of human embryonic stem cells (hESCs).Red represents the highest correlation, and green represents thelowest correlation. The numbers in squares express the exactPearson's correlation values.Capalbo. MicroRNAs in blastocyst culture media. Fertil Steril 2015.

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To evaluate whether the protocol for microRNA (miRNA) quantification can capture biologic variations, five spent blastocyst culture media (SBM)samples were collected from different embryos, pooled together in a single tube, and divided again in five equal amounts to remove biologicvariation between samples (A). Mean correlation coefficient of the cycle threshold (Ct) values for miRNAs detected in mixed samples (0.93 �0.2) was statistically significantly higher compared with unrelated SBM samples (0.77 � 0.06; P


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Cycle threshold (Ct) values of microRNAs (miRNAs) commonly used asputative normalizers in the trophectoderm (TE) and related spentblastocyst culture media (SBM) samples. The expression of all thesemiRNAs was detected with high consistency in all the TE biologicreplicates but not among the SBM samples. For SBM sample 3, noresults could be detected. RNU44 was not detected in any of themedia samples analyzed.Capalbo. MicroRNAs in blastocyst culture media. Fertil Steril 2015.


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MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo repr ...Materials and methodsStudy Design and Outcome MeasuresEmbryosHuman Embryonic Stem CellsSpent Culture Medium from Single-embryo Transfer CyclesIVF Procedures and Spent Medium CollectionMicroRNA Isolation, Retrotranscription, and PreamplificationMicroRNA Analysis Using a TaqMan Low-Density ArrayCross Validation of miRNAs Detected in TE and SBM Samples Using Single AssaysStatistical AnalysisTarget Prediction and Pathway Analyses

ResultsMicroRNAs in Spent Blastocyst Medium Are Expressed Also in Trophectoderm CellsMicroRNAs in Conditioned Culture Media can Be Consistently Detected Only after BlastulationMicroRNAs that Are Differentially Expressed in Implanted versus Unimplanted Blastocysts Have Been Associated with Implantat ...Single Assay-based Cross-validation of Relevant miRNAs from TE and SBM Samples

DiscussionAcknowledgmentsReferences

MicroRNAs in spent blastocyst culture medium are derived ......and SBM samples revealed that 96.6%...

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