Vitrification slightly affected embryo morphology by increasing the percentage of embryos showing low grade (less than 20%) blastomere cytofragmentation. Embryo fragmentation is clinically considered as an indicator of reduced embryo viability and developmental ability, reduced number of high quality embryos available for establishing pregnancies, thus as a relevant problem for assisted reproduction outcome. In fresh embryos, blastomere fragmentation has been reported to be associated to apoptosis [24, 35, 36] or to apoptosis and necrosis . A recent study  revealed novel early transcription mechanisms by which maternal genotype affects cytofragmentation by alterating regular cytoskeletal functions. In the present study, in which embryos were examined no more than two hours after warming (including staining procedures), these mechanisms could be only hypothesized. Thus, it can be concluded that vitrification increases only mild cytofragmentation, thus allowing the preservation of embryo blastomeres integrity, and not reducing the number of embryos available for transfer.
Vitrification increased low level chromatin damage P < 0.05). Moreover, only intermediate stages of development were affected. Although not many studies have been published so far, our observations are in line with results of previous studies demonstrating, with different methods, lower damaging effects of vitrification compared with slow freezing on embryo chromatin integrity and function. Vutyavanich et al., 2009  reported significantly higher average number of nuclei in blastocysts derived from embryos vitrified at the 2-cell stage, and cultured in vitro, compared with those obtained after slow freezing. Other studies examined the effects of cryopreservation methods on DNA integrity and stability, as assessed by TUNEL test. Tsang and Chow, 2010  reported significant reduction of DNA integrity after both procedures. AbdelHafez et al., 2011 , reported higher DNA damage extent in vitrified blastocyst versus in early cleavage stage vitrified embryos, may be attributable to differences in blastocoel shrinkage after exposure to vitrification solutions. To our knowledge, this is the first study including all developmental stages and comparing stage-specific effects of vitrification on chromatin integrity of mouse embryos.
Bioenergy/oxidative stress analysis can be performed with several molecular and biochemical methods . Global assessment strategies, such as OMICS technologies, such as Transcriptomics, Proteomics, Metabolomics, are becoming increasingly valuable in this area of investigation . For the specific purpose of assisted reproduction, particularly for research on oocytes and embryos, confocal imaging allows global qualitative and quantitative evaluation of bioenergy/redox parameters in individual samples, also enabling the localization and quantification of functional aberration. As for parameters of cytoplasmic maturity, reduced percentages of embryos showing mt P/P distribution pattern were observed only at early stages of development (4/16-cell stage, P < 0.001) compared with fresh control embryos. Moreover, at any stage of development the rate of vitrified embryos showing P/P pattern never dropped below 50% and in total samples, more than 70% of embryos retained this mt pattern. The qualitative analysis of fluorescence, indicative of mt activity, conducted on fresh 4/16-cell stage embryos demonstrated that: 1) mt localization was perinuclear and pericortical, 2) there were blastomeres with intense mt activity while others with almost no activity, and 3) spots of intense mt activity were evident at the level of blastomere cell junctions. This observation is in agreement with those reported by Van Blerkom, 2009 , who showed that mt activity can influence or can be influenced by intercellular contacts. Our observations are in agreement with a previous study by Zhao et al., 2009  who reported that in fresh 2PN mouse embryos stained with JC-1, red colored mitochondria (high Δψ) were distributed primarily around pronuclei and along the cell membrane whereas in vitrified-warmed 2 PN embryos, red mt were greatly diminished with green mt (low Δψ) evenly distributed throughout the cytoplasm. At the same time, these authors found that the proportion of fresh 2PN embryos with normal aggregation of high Δψ mt (84%) was significantly higher than that of vitrified 2 PN embryos (27%). Observed altered mt distribution could be due to modifications of cytoskeletal elements which have been reported to be involved in cellular movement on a rapid timescale of these organelles , other cytoplasmic components, such as endoplasmic reticulum , or to modifications of specific proteins involved in mt anchoring to cytoskeletal microfilaments or microtubules . Previous studies in the mouse reported no significant differences after vitrification in microfilament distribution in zygotes, 2 cell embryos, morulae and blastocysts  and in microtubule formation in 2PN embryos .
In embryos at the morula and blastocyst stage, the qualitative analysis showed that mt compartimentalization, which at these stages is indicative of developmental stage-dependent acquisition of blastomeres cytoplasmic maturity, was not affected by vitrification. This observation could be related to major cryotolerance but also to a greater difficulty in visualizing mt distribution modification in embryos at these stages due to reduced blastomere cytoplasmic size. Qualitative analysis also showed that mt activity of trophoectodermal cells in blastocyst stage embryos is more intense than that observed in the cells of the inner cell mass and that blastomeres showing strong mt activity were located nearby the blastocoelic cavity. This observation is in agreement with those reported by Van Blerkom, 2011,  who showed that the maintenance of the blastocoele and its rapid recovery after collapse and hatching phase, are morphodynamics activities that require huge production of ATP from the cells of the trophoectoderma. Instead, cells in the inner cell mass, which are not involved in these activities, appear to be metabolically quiescent in these developmental phases. Quantitative analysis of ATP production in mouse blastocysts showed that approximately 80% of ATP produced by the embryo is from the trophoectoderma and that the number of mt observed by confocal laser microscopy are located well below in the inner cell mass .
Quantification analysis in the present study was performed in embryos at the morula and blastocyst stage. A statistically significant reduction of MitoTracker fluorescence intensity was found in vitrified blastocysts compared with their fresh counterparts, indicating significant reduction of mt activity at this stage of development. In embryos at the morula stage, vitrification had no effects on mt activity. More interestingly, both in embryos at the morula and blastocyst stage, significant increase of DCF fluorescence intensity, indicative of an excess production of ROS, was found after vitrification. This finding could be interpreted considering that the conditions used for the vitrification method could have resulted in the onset of oxidative stress condition. This observation could be consistent with the reported up-regulation of genes involved in the mechanisms of oxidative stress (Hsp70, MnSOD, CuSOD) in mouse vitrified embryos . Increased oxidative stress in vitrified/warmed embryos in the present study, kept in vitro no more than two hours after warming, could be an initial effect, as suggested by Tsang and Chow, (2010; ) who reported that stress-related gene expression dropped down to normal levels within 7 hours after warming. Colocalization of intracellular free radicals (ROS) and actively respiring mt has been reported as indicative of higher ATP turnover resulting from a more intense mt activity and thus indicative of healthy cell conditions in ovine in vivo matured ovulated metaphase II oocytes  and in hepatocytes. By this analysis, it came out that mt/ROS colocalization was not affected by vitrification in blastocyst stage embryos and it was increased in embryos at the morula stage, may be due to increased ROS generation. To our knowledge, this is the first study reporting mt/ROS colocalization, objectively expressed as Pearson’s correlation coefficient, for the comparison between fresh and vitrified mouse embryos.
Taken together our data allow to confirm that morula and blastocyst are good stages from the standpoint of embryo viability after vitrification. In vitrified morulae, only low level chromatin damage was found and bioenergy/redox parameters were positively affected. Infact, mt pattern was not affected and increased oxidative activity and consequent increased mt/ROS colocalization were found. In vitrified blastocysts, neither nuclear chromatin nor mt pattern were affected; a significant reduction of mt activity was found but ADU absolute values remained at consistent levels (395.1 ± 89.3 versus 522.4 ± 176.8 for vitrified vs fresh, respectively), indicating that vitrified embryos retained/kept a good/substantial part of mt activity. As well, in vitrified blastocysts increased respiratory/oxidative activity was found, as observed by increased ROS generation.