The aim of this study was to describe a system for embryo morphology scoring at the morula stage and to determine the efficiency of this model in selecting viable embryos for transfer. In total, 519 embryos from 122 patients undergoing intracytoplasmic sperm injection (ICSI) were scored retrospectively on day 4 according to the grading system proposed in this article. Two separate quality scores were assigned to each embryo in relation to the grade of compaction and fragmentation and their developmental fate was then observed on days 5 and 6. Secondly, the prediction value of this scoring system was compared with the prediction value of the traditional scoring system adopted on day 3. Morulas classified as grade A showed a significant higher blastocyst formation rate (87.2%) compared with grades B, C and D (63.8, 41.3 and 15.0%, respectively), (P < 0.001). Furthermore, the ability to form top quality blastocysts was significantly higher for grade A morulas with respect to grades B, and C and D (37.8% vs. 22.4% vs. 11.1%), (P < 0.001). Finally, the morula scoring system showed more prediction power with respect to the embryo scoring a value of 1 [Akaike information criterion (AIC) index 16.4 vs. 635.3 and Bayesian information criterion (BIC) index −68.8 vs. −30.0 for morulas and embryos respectively]. In conclusion, results demonstrated that the presented scoring system allows for the evaluation of eligible embryos for transfer as a significant correlation between the grade of morula, blastulation rate and blastocyst quality was observed. Furthermore, the morula scoring system was shown to be the best predictive model when compared with the traditional scoring system performed on day 3.

The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.