Vitri�cation of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

Cristina Cuello Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia Cristina A. Martinez (  cristina.martinez-serrano@liu.se ) Linkopings universitet https://orcid.org/0000-0001-6811-0191 Josep M Cambra Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia Inmaculada Parrilla Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia Heriberto Rodriguez-Martinez Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynaecology, Faculty of Medicine and Health Sciences, Linköping University Maria A Gil Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia Emilio A martinez Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia

General transcriptome experiments of vitri ed-warmed have been done only in bovine.Vitri cation of in vitro-produced (IVP) bovine embryos caused overexpression of apoptosis-related genes [25] and the repression of genes involved in cell differentiation, cell adhesion and metabolism of lipids [28].It has been also demonstrated that vitri cation modi ed expression of genes related to stress response [29] in both IVP and in vivo-derived bovine blastocysts.Comparative studies on porcine are very scarce and limited to IVP embryos.Research based on qRT-PCR has demonstrated that vitri cation signi cantly upregulates genes involved in both mitochondria and death receptor-mediated apoptotic pathways in porcine parthenogenic blastocysts [22].It has also been reported that vitri cation causes the downregulation of POU5F1, which is related to embryo implantation [30], the upregulation of HSPA1A, which is involved in stress regulation [30], vitri cation also the modi es expression levels of IGF2 and IGF2R imprinted genes [23] in IVP-produced porcine blastocysts.Although these studies are useful, they are focused on few selected genes and therefore the information about the vitri cation impact on the embryo transcriptome is very limited.A better knowledge of the overall vitri cation effects on the embryonic transcriptome may help to understand sublethal cryoinjuries that could be associated with embryo developmental and pregnancy failure and it would yield new awareness about the response mechanisms of embryos undergoing vitri cation.Such studies should be done on in vivo-derived porcine embryos, which are the only embryos currently suitable for commercial embryo transfer in this species.
Microarrays or RNA-Seq analysis are the most e cient and comprehensive technologies to provide a whole-transcriptome coverage [31], enabling assessment of the expression of thousands of genes with a single experiment.Therefore, this study used a microarray approach with qRT-PCR con rmation to investigate the effect of vitri cation on the gene expression patterns of in vivo-derived porcine embryos at the morula and blastocyst stages, to determine if sub-lethal modi cations, escaping conventional morphology screening of embryo viability, are caused by the procedure.

Chemicals
Chemicals and media were acquired from Sigma-Aldrich Química S.A. (Madrid, Spain) unless otherwise indicated.

Animals
Embryos were obtained from hybrid donor sows (Landrace x Large-White) from the same genetic line (2 to 6 parities) located at a commercial farm (Agropor S.A., Murcia, Spain) and were maintained under eld conditions, and placed individually in crates in a mechanically ventilated con nement facility.The sows were fed a commercial ration twice daily according to their nutritional requirements, with constant access to water.

Detection of estrus and arti cial insemination
Weaning was used to synchronize the estrus of the sows.Sows were evaluated for estrus once a day (at 7:00 a.m.) beginning the day after weaning.Sows were exposed to a vasectomized mature boar allowing snout-to-snout contact and considered in estrus when they showed a standing estrus re ex when applying manual back pressure.Only sows with an interval between weaning and estrus of 4 to 5 days were used as embryo donors.Estrus sows were arti cially inseminated post-cervically at 6 and 24 h after the onset of estrus.Insemination doses (45 mL containing 1.5 × 10 9 spermatozoa) were prepared in a commercial arti cial insemination center with ejaculates extended in Beltsville Thawing Solution extender (BTS; [32].Sperm doses were stored at 17 °C for a maximum period of 24 h.

Embryo recovery and assessment
Morulae and blastocysts were surgically collected from the donor sows on Day 6 of the estrus cycle, with Day 0 being considered the onset of estrus.Sedation of embryo donors was performed with azaperone (Stresnil®, Landegger Strasse, Austria; 2 mg/kg body weight, i.m.) and general anesthesia was induced with sodium thiopental (B.Braun VetCare SA, Barcelona, Spain; 7 mg/kg body weight, i.v) and was maintained with 3-5% iso urane gas (IsoFlo®, Madrid, Spain).The genital tract was exposed by performing a mid-ventral laparotomy.Then, the corpora lutea present in each ovary were counted and embryos were collected as previously described [33] by ushing the tip of each uterine horn with 30 mL of Tyrode's lactate (TL)-HEPES-polyvinyl alcohol [34] with some modi cations (TL-HESPES-PVA; [15].After ushing, the embryos collected from the uterine horn were evaluated under a stereomicroscope at a 60 x magni cation, and the developmental stage and quality were assessed.One-cell structures and poorly developed embryos were classi ed as oocytes and degenerated embryos, respectively.Only morulae and unhatched full blastocysts with good or excellent morphology according to the criteria of the International Embryo Transfer Society [35] and an intact zona pellucida were selected for the experiments.Collected embryos were washed three times in TL-HEPES-PVA, placed Eppendorf tubes containing 1.5 mL of this medium and transported to the University of Murcia (Spain) in a transportable incubator set at 39 °C within 2 h after collection.

Vitri cation and warming
Vitri cation and warming were performed according to a previously described protocol [16]  air and 97% humidity atmosphere.After 24 h of in vitro culture, embryo morphology was assessed by stereomicroscopy to determine embryo viability and embryo developmental stage.The vitri ed-warmed morulae that had developed to the blastocyst stage and the vitri ed-warmed blastocysts that restructured their blastocoelic cavities after 24 h of in vitro culture and exhibited an excellent or good appearance were considered viable.Fresh control embryos that progressed after in vitro culture and showed good or excellent morphological features were classi ed as viable.The survival rate was calculated as the ratio of viable embryos to the total number of cultured embryos.

Sample preparation and microarray hybridization
Total RNA was extracted from embryo samples was performed with an RNeasy Micro kit (P/N 74004; Qiagen Iberica, Madrid, Spain) according to manufacturer instructions.The isolated RNA was checked with a Nanodrop 2000 (ThermoFisher Scienti c, Madrid, Spain) and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) to determine the total RNA amount and quality.The RNA integrity (RIN) values obtained ranged from 8 to 10.Then, ss-cDNA was synthesized from 650 pg of RNA from each sample using a GeneChip 3´ IVT Pico Reagent kit (P/N 902790; Affymetrix, ThermoFisher Scienti c, Madrid Spain), according to the protocol supplied by the manufacturer.The amount and quality of ds-cDNA was assessed by a Nanodrop 2000 (ThermoFisher Scienti c, Madrid, Spain) and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA); ds-DNA targets were cleaned, and after fragmentation and terminal labelling, 4.5 µg of fragmented and biotinylated ds-DNA were added to a hybridization mix from a GeneChip Hybridization, Wash and Stain kit (P/N 90720; Affymetrix) according to the recommendations of the manufacturer.The resulting preparations were hybridized to the GeneChip® Porcine Genome Array (P/N 900624; Affymetrix), which assesses 23,256 transcripts corresponding to 20,201 genes and provides widespread coverage of the Sus scrofa transcriptome.After scanning the array, microarray data were processed using the Affymetrix Expression Command Console (Affymetrix), and all samples met the quality criteria.

Microarray data analysis
The robust multiarray average (RMA) method was used to normalize the intensity data of each GeneChip® array [39], processing average intensity values according to the background adjustment.Raw values were then log 2 transformed, and quantile normalized in order to get a single intensity value for each probe set.Partek Genomics Suite and Partek Pathways software (Partek Incorporated, St. Louis, USA) were used for the statistical analysis and biological interpretation of data.Principal component analysis (PCA) was used to provide the general con guration of the evaluated dataset and to observe variations in the transcriptome between samples.Statistical analysis was based on tsingle-factor ANOVA with a restrictive threshold at an unadjusted P value lower than 0.05 for selecting differentially expressed genes (DEGs).The analysis of the overrepresented Gene Ontology (GO) terms and pathways were analyzed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.Pathway networks were constructed using ClueGo v2.0.3 application from the Cytoscape v3.0.0 [40].The ClueGo ontology source was KEGG pathway database.Pathways were functionally grouped based kappa score (≥ 0.4).
The following criteria were used for the ClueGo analysis: GO tree levels, 2-5 ( rst level = 0); minimum number of genes, 2; minimum percentage of genes, 2; GO term fusion; GO term grouping, initial group size of 2 and 50% for group merge.

Quantitative real-time PCR (qRT-PCR) analysis
For qRT-PCR we analyzed total RNA from the same samples used for the microarrays that was reversetranscribed to generate cDNA using a Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c); conditions were 25 °C for 10 min, 50 °C for 15 min and 85 °C for 5 min.Primers were designed using Primer Express™ software v3.0.1 (Applied Biosystems, Foster City, CA, USA) and were commercially synthesized (primer sequences are shown in Table 1).The qRT-PCRs were performed with iTaqTM Universal SYBR Green Supermix in 10-µL volumes with 500 nM of each set of primers.All reactions were carried out in a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems).The thermal cycling pro le was 50 °C for 2 min for uracil-DNA glycosylase activation, 95 °C for 10 min for initial denaturation followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Melt curve analysis was carried out to evaluate the speci city of each PCR by detection of one single peak on the dissociation curve pro le.A previous test with extra samples was conducted to calculate each primer pair e ciency according to the equation E = 10 (−1/slope) .qRT-PCRs were run in triplicate per gene and per sample, and relative mRNA levels were quanti ed according to the Pfa method [41].For data normalization, peptidylprolyl isomerase A (PPIA) was chosen as the housekeeping reference gene based on the results reported previously [42].Gene e ciencies were calculated according to the equation E = 10

Experimental design
To evaluate the effects of vitri cation on the transcriptome of porcine in vivo-produced morulae and blastocysts, 13 weaned sows were used as embryo donors in three replicates.A total of 50 morulae and 50 blastocysts were vitri ed and warmed, and then they were cultured in vitro for 24 h.Control embryos were fresh morulae (n = 40) and blastocysts (n = 40) cultured in vitro for 24 h.After in vitro culture, the viability of vitri ed-warmed and control embryos was assessed, and survival rates were calculated.Three pools of 10 viable embryos each were prepared from each experimental group (vitri ed and control) and each embryonic developmental stage (morulae and blastocysts).Embryos were placed in 5 µL Phosphate-buffered saline (PBS) in RNAase free Eppendorf tubes and then stored at -80ºC until transcriptome analysis.A total of 8 genes (5 upregulated genes and 3 downregulated genes according to the microarray results; Table 1) were selected to con rm microarray results by qRT-PCR.For this validation, three biological replicates and three technical replicates per sample were performed.

Embryo collection and embryo viability
The mean ovulation rate of donor sows was 20.8 ± 3.6 corpora lutea (range 14 to 25), and the recovery rate was 92.2%.Of the recovered structures, 96.0% were embryos, and the rest were unfertilized oocytes and/or degenerated embryos.The total number of embryos collected was 239, of which 42.3%, 54.8% and 2.9% were morulae, blastocysts and hatched blastocysts, respectively.A total of 90 morulae and 90 blastocysts were selected for use in this study, and the remaining embryos were used in other experiments.The survival rates of vitri ed-warmed morulae (92.0 ± 3.3%) and blastocysts (96.1 ± 3.4) were similar (n.s.) to each other and to those obtained in control embryos (100%).

Transcriptome pro les of vitri ed-warmed morulae and blastocysts
Vitri cation and warming affected the transcriptome pro le of porcine morulae and blastocysts.The PCA revealed that for 71.0% and 68.8% of variance was explained by the treatment (vitri cation or not) of morulae and blastocysts, respectively.First, lists of the differentially expressed transcripts were generated using an unadjusted p-value of 0.05 and various fold change cutoff values (Fig. 1).
Subsequent analyses were performed using the DEGs list produced from the selection criterion of fold change <-1.5 and > 1.5.Using these parameters, 233 DEGs were identi ed in vitri ed morulae compared to the control group.More speci cally, 38 genes were upregulated, whereas 195 were downregulated (Fig. 1).With regard to the blastocysts, the number of DEGs was 205.Interestingly, the proportion of upregulated genes (112) was higher than it was in the morulae (Fig. 1).The DEGs detected in vitri ed morulae and blastocysts are represented in the Volcano plots (Fig. 1).The unsupervised hierarchical clustering of transcriptome samples revealed that morulae and blastocysts vitri ed samples could be clearly distinguished from the control samples (Fig. 2).The transcriptome pro les obtained for vitri ed morulae and blastocysts were very different.Analysis of DEGs in vitri ed morulae and blastocysts showed that only 3 upregulated and 8 downregulated DEGs were shared between vitri ed morulae and blastocysts (Fig. 3).

Gene Ontology (GO) enrichment analysis of DEGs in vitri ed morulae and blastocysts
Gene ontology (GO) analysis identi ed the main biological processes targeted by the DEGs.A total of 105 and 671 enriched GO terms with an enrichment score ≥ 3 and an enrichment p-value < 0.05 were detected for vitri ed morulae and blastocysts, respectively.The most enriched GO terms for both developmental stages were related to biological process.Tables 2 and 3 summarize the top 10 most enriched GO terms corresponding to vitri ed morula and blastocysts, respectively.All DEGs in vitri ed morulae and blastocysts were classi ed within different functional categories based on molecular function (Fig. 4A and 4A'), biological process (Fig. 4B and 4B') and cellular component (Fig. 4C and 4C').Within each of the GO terms represented, vitri ed morulae displayed a higher proportion of downregulated DEGs than vitri ed blastocysts.

KEGG Pathway enrichment analysis of DEGs in vitri ed morulae and blastocysts
Pathway enrichment analysis of DEGs in vitri ed morulae detected ten enriched KEGG pathways for upregulated (Table 4) and seven for downregulated DEGs (Table 5).Enriched pathways for upregulated genes in morulae were mainly metabolism-related pathways.The most enriched pathways related to downregulated DEGs in morulae were glycolysis-gluconeogenesis and protein export.Partek detected a total of ten enriched pathways for upregulated DEGs in vitri ed blastocysts (Table 6); the most enriched pathways in this group were TGFβ, p53 and FoxO signaling pathways.Only four enriched pathways were obtained with downregulated DEGs (Table 7), including steroid biosynthesis, TGFβ and cGMP-PKG signaling pathways and gab junctions.Figure 5 represents the KEGG pathway networks analyzed with Cytoscape for vitri ed morulae (Fig. 5A) and blastocysts (Fig. 5B).

Validation of microarray results
Validation of microarray data was performed by qRT-PCR.The eight genes validated by qRT-PCR showed an expression trend that was similar to the results observed in the microarrays (Fig. 6).qRT-PCR analysis revealed that the mRNA levels for TP53INP, MGMT, DKK3 in blastocysts and WDR25 and PLEKHB1 in morulae were signi cantly (p < 0.05) upregulated.The expression of PAIP1 in blastocysts and DECR1 in morulae was signi cantly (p < 0.05) downregulated.The qRT-PCR analysis revealed that the expression of MYC was consistent with the results of the microarray, but the difference in expression levels between vitri ed and control blastocysts analyzed by qRT-PCR was not signi cant.

Discussion
To the best of our knowledge, this is the rst report about the impact of vitri cation/warming on the full transcriptome of in vivo-derived porcine morulae and blastocysts.This study contributes to the understanding of the consequences of vitri cation/warming procedures on embryo quality and developmental competence, not only in pigs, but also in other mammalian species.Considering our results, the impact of vitri cation in terms of the number of DEGs was similar for the morula (233 DEGs) and blastocysts (205 DEGs).Vitri cation/warming caused moderate transcriptome changes in both, morulae and blastocysts, in terms of fold-changes.We only found eight DEGs in morulae and 6 in blastocysts showing a fold change greater than three.Although the number of DEGs and fold change values were similar for both developmental stages, vitri ed morulae and blastocysts displayed very different transcriptome pro les, with only 12 (three upregulated and eight downregulated) DEGs in common.This is not surprising, considering that stage-speci c gene expression between morulae and blastocysts is characteristic of the preimplantation embryo development [43].
The GO term enrichment analysis of DEGs in vitri ed morulae revealed that, except for the biological process "reproduction", all the disturbed GO biological processes, GO cellular components and GO molecular functions were repressed.The most enriched GO terms in morulae were related to fatty acid and acyl-CoA metabolism.It is known that both fatty acids and acyl-CoA are metabolic switches linked to pluripotency that play an essential role in embryo development [44,45].In addition, acyl-CoA controls crucial cellular processes such as mitosis, autophagy and energy metabolism, and this control is either direct or is mediated by epigenetic regulation of gene expression [46,47].Fatty acids, on the other hand, are also key factors linked to metabolism, cell signaling, oxidative stress and gene expression in preimplantation embryos ([48, 49].The downregulation of genes in these two functional groups may have deleterious effects on vitri ed morulae development.It could be interesting to investigate whether supplementation of vitri cation-warming medium with acyl-CoA and/or fatty acids may offset these effects. For vitri ed blastocysts, all DEGs involved in the GO biological process of growth, cell population proliferation, cell aggregation and detoxi cation GO biological processes and those related to antioxidant activity and protein folding chaperone GO molecular functions were upregulated.These results re ect that the vitri cation/warming process induced a stress-related response in blastocysts.Among the upregulated DEGs, the following genes included in these categories, the TP53INP (tumor protein p53 inducible nuclear protein 1) and CDKN1A (cyclin-dependent kinase inhibitor 1A (p21, Cip 1) genes, are of special interest for their role in the regulation of cell death and survival under stress conditions [50,51].
The KEGG pathway enrichment analysis of up-and downregulated genes of vitri ed morulae and blastocysts showed that the impact of vitri cation was moderate in terms of the number of pathways altered and the percentage of transcripts within each pathway that showed disturbed expression (range 0.7-11.1%).Although, the disruption of gene expression can be considered moderate, we should pay particular attention to modi ed pathways that are crucial for embryo development.KEGG pathway analysis based on the upregulated DEGs in vitri ed morulae, revealed that nine pathways were enriched.Genes upregulated in these pathways (HEXA, SPAM1, ACAT2, HAAO, MAN1C1, PIP5K1A, PYGM and ELOVL1) are not directly linked to embryo development.However, the enrichment of the metabolic pathways, which has also been described in vitri ed in vivo-derived bovine blastocysts [50,51], could be sign of low embryo quality.The activation of metabolism-related pathways may cause detrimental effects on the development and implantation of vitri ed morulae according to the "quiet embryo" hypothesis, which proposes that preimplantation embryo survival is associated with a relatively "quiet" level of metabolism (Baumann et al., 2007;Leese, 2002;Leese et al., 2007).Among the seven enriched pathways for downregulated genes in vitri ed morulae, it is remarkably the Wnt signaling pathway that plays a critical role in embryo development [52][53][54].The Wnt signaling pathway regulates cell proliferation and differentiation in mammalian embryos [55,56], and modi ed genes in this pathway were GPC4, LRP6, MAPK8 and PPP3CB.The repression of these genes may impair the developmental potential of vitri ed morulae.
Enriched pathways in vitri ed blastocysts were different from those described for morulae because of the stage-speci c gene expression described above [57][58][59][60].Interestingly, when we examined enriched pathways for upregulated genes in vitri ed blastocysts, we observed important biological processes (cellular senescence and cell cycle) and signaling pathways (TGFβ, p53 and FoxO) that regulate the pluripotency of stem cells involved in embryo development and pregnancy.The enrichment of the TGFβ signaling pathway has also been reported in vitri ed porcine COCs [44,45], and upregulation of miRNAs related to this pathway was observed in vitri ed mouse blastocysts [61].The upregulated genes in the TGFβ signaling pathway were BMPR1B, ID4, SMAD3 and TGFβ1, and there were also DEGs involved in the cell cycle pathway (CDKN1A) and signaling pathways regulating the pluripotency of stem cells (BMPR1B, ID4 and SMAD3).These genes are key regulators of cell  [19,69].In conditions of reparable damage or transient stress, TP53INP induces cell cycle arrest by increasing the expression of CDKN1A/p21 [19,69], which was also upregulated in our vitri ed blastocysts.This response is related to repair, protection, and adaptation, and ultimately to cell survival [51].FoxO pathway is also activated under stress conditions [50,70]; FoxO molecules have been described in the inner cell mass of mouse blastocysts [71], and the upregulate target genes, thereby promoting cell cycle arrest genes in order to keep cells away from stress.If the cell cycle arrest is not su cient to recover cells, apoptosis is activated, thus producing cell death [71].In addition, the cellular senescence and cell cycle pathways, which play essential roles in the cell response and cell repair under stressful conditions [71], were also enriched in the vitri ed blastocysts.Our results show the triggering of essential repair mechanisms in the vitri ed blastocysts, which has not been observed in the vitri ed morulae.This fact could partly explain the vitri cation of blastocyst being better than of morulae, which has been observed in some studies [72].
Interestingly, when analyzing the downregulated DEGs, TGFβ-signaling pathway was enriched again; BAMBI and RBL1 are negative regulators of TGFβ and MYC encodes a nuclear phosphoprotein with a regulatory role [73].Among the four enriched pathways obtained from the downregulated DEGs in vitri ed blastocysts, the most remarkable is the steroid biosynthesis pathway.Historically, the production of steroids (estrogens) by porcine embryos has been considered the major signal for maternal recognition of pregnancy (Bazer et al., 1982).Recently, it has been demonstrated that although the production of embryonic estrogens is not essential for preimplantation development and early corpus luteum maintenance, it is indispensable for the maintenance of pregnancy beyond 30 days (Meyer et al., 2019).
Therefore, the disruption of the steroid biosynthesis pathway could be, in part, responsible for the increased number of pregnancy failures that we have observed after transfer of vitri ed blastocyst [17].
The enrichment of the Gap junction pathway that is involved in embryo-maternal interaction and implantation [77].The HTR2B, which is a gene that belongs to this pathway, is involved in morphogenesis and development [73]; therefore, its repression could be detrimental for the embryo.
Comparing our results with previous results on the gene expression of vitri ed porcine embryos performed by qRT-PCR, we observed very few matches due to the different origins of the embryos (parthenogenetic [78] or IVP [22]).Similar to Castillo-Martin et al. [23,30], we detected the upregulation of an HSP gene (HSPB1) in vitri ed blastocysts; HSPB1 encodes a small heat shock protein involved in the response to environmental stress [79].This result again shows the response of vitri ed blastocysts to vitri cation-induced stress.Noteworthy is to consider that these sub-lethal modi cations caused by the procedure of vitri cation/waring, escaped our conventional morphological screening of embryo viability post-warming, which calls for a further screening of the extent of the transcriptomic alterations and their impact on embryo viability, and of the development of more re ned methods to discover these modi cations at warming.

Conclusions
Taken together, our results demonstrate that vitri cation modi ed the transcriptome of in vivo-derived porcine morulae and blastocysts, resulting in different gene expression pro les based on the stage of development.The changes in gene expression that we observed could be considered moderate in terms of the number of DEGs and fold-change values.The vitri cation impact on morulae consisted mostly of gene expression repression, except for metabolism related pathways.In the case of blastocysts, we noted the activation of the cell cycle, cellular senescence, TFGβ, p53, FoxO and MAPK signaling pathways in response to vitri cation-induced stress.Disruption of pathways such as metabolic-related pathways in morulae, and steroid biosynthesis and gap junctions in blastocysts could be tightly related to the increased pregnancy loss observed after transfer of vitri ed embryos.Further research is needed to increase the knowledge about the biological implications of the GO terms and pathways modi ed by vitri cation procedures, as well as how to evolve screening methods for warmed embryos, beyond the apparently sub-optimal morphological evaluation currently used.

Figure 1
Figures

Table 1
(−1/slope).The qRT-PCR data were analyzed by Student's t-test using the IBM SPSS 24.0 Statistics package (IBM, Chicago, IL, USA).A p-value of < 0.05 was considered statistically signi cant.Sequences of the primers used for quantitative real-time PCR (qRT-PCR) analysis.

Table 2
Top ten most signi cant gene ontology (GO) terms for the differentially expressed genes in vitri ed morulae.
BP: biological process.MF: molecular function.* % of genes in group that are present in the differentially expressed genes list (vitri ed vs control morulae).

Table 3
Top ten most signi cant gene ontology (GO) terms for the differentially expressed genes in vitri ed blastocysts.
BP: biological process.MF: molecular function.* % of genes in group that are present in the differentially expressed genes list (vitri ed vs control blastocysts)

Table 4
Enrichment analysis of pathways for up-regulated differentially expressed genes in vitri ed morulae.

Table 5
Enrichment analysis of pathways for down-regulated differentially expressed genes in vitri ed morulae

Table 6
Enrichment analysis of pathways for up-regulated genes in vitri ed blastocysts

Table 7
Enrichment analysis of pathways for down-regulated genes in vitri ed blastocysts proliferation, stem-cell state, differentiation, and apoptosis at the earliest stages of embryo development [62] and the TGFβ1 and SMAD3 are also key factors during embryo implantation [63, 64].The activation of the TGFβ-signaling pathway seems to be a response to cell stress and injury, as has been reported in epithelial cells [65].The p53 signaling pathway is also an essential regulator of cellular stress, which may have opposite biological responses depending on many factors leading to cell death or cell survival [65].The expression of TP53INP can be induced by many different stress signals [66] that have been described as consequences of embryo vitri cation, such as oxidative stress [67, 68] or DNA damage