The metaphase II (MII) spindle of the human oocyte may be damaged by cryopreservation. High performance confocal microscopy was used to assess meiotic spindle and chromosome organization in oocytes after vitrification by the cryoleaf system. Three hours after retrieval, donor mature oocytes were fixed or vitrified. Vitrification was performed by equilibration in 7.5% ethylene glycol (EG) and 7.5% dimethylsulphoxide (DMSO), transfer to 15% EG, 15% DMSO and 0.5 mol/l sucrose, and loading onto cryoleaf strips. Tubulin staining was found in all survived vitrified-warmed oocytes, the majority (62.8%) of which displayed a bipolar spindle. A normal bipolar spindle configuration and equatorial chromosome alignment was observed only in a part of vitrified-warmed oocytes (32.6%). This frequency was significantly lower in comparison to fresh oocytes (59.1%). In another fraction of vitrified-warmed oocytes (30.2%), spindle bipolarity was associated to one or more non-aligned scattered chromosomes that often appeared tenuously associated with the lateral microtubules of the spindle. Furthermore, in cryopreserved oocytes with a bipolar spindle, a significantly increased pole-to-pole distance (14.9 +/- 2.3 microm) was found in comparison to the fresh control (12.4 +/- 2.6 microm) (P = 0.001). Therefore, under the conditions tested, vitrified-warmed oocytes maintain a MII spindle with a bipolar organization. However, chromosome alignment appears to be partly compromised.
"Ideally, new ART procedures should be introduced into clinical practice only following well-planned safety studies. The information available on the neonatal outcome and the long-term follow-up of children conceived after oocyte cryopreservation is limited (Chian et al., 2008, 2014; Noyes et al., 2009; Wennerholm et al., 2009; Cobo et al., 2014) and only small studies have specifically addressed the safety of oocyte vitrification (Coticchio et al., 2009; Bonetti et al., 2011; Chen et al., 2012; Forman et al., 2012; Khalili et al., 2012; Monzo et al., 2012; Dominguez et al., 2013; Gugliemo et al., 2014; Konc et al., 2014; Nohales Có rcoles et al., 2014; Palmerini et al., 2014). "
[Show abstract][Hide abstract] ABSTRACT: Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analyzed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after ICSI were compared in three different assays. First, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at day 1. Secondly, embryo development up to day 3 was analyzed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analyzed by immunostaining on day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older subfertile / infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.
Molecular Human Reproduction 03/2015; 21(6). DOI:10.1093/molehr/gav013 · 3.75 Impact Factor
"Cryopreservation of ovarian tissues is practised by two methods: controlled slow freezing and vitrification (Cao et al. 2009). Several studies have provided evidence of the detrimental effects of freeze–thawing on oocytes such as chromosomal misalignment, modification in reactive oxygen species (ROS) status and DNA fragmentation (Huang et al. 2008; Katz–Jaffe et al. 2008; Coticchio et al. 2009; Martínez- Burgos et al. 2011). These changes may impair chromatin conformation in oocytes, ultimately affecting the genetic and functional competence of oocytes and embryos (Aye et al. 2010; Smitz et al. 2010). "
[Show abstract][Hide abstract] ABSTRACT: It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze–thawing. Using a mouse model, here we have investigated the functional competence and phosphoryla-tion of H2AX (g-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with g-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P , 0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P , 0.05) and control (P , 0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P , 0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome. Additional keywords: g-H2AX, in vitro maturation, vitrification.
Reproduction Fertility and Development 07/2014; DOI:10.1071/RD14048 · 2.40 Impact Factor
"Several studies have provided evidence on the detrimental effects of freeze-thawing in oocytes such as chromosomal misalignment, modification in reactive oxygen species (ROS) status, and DNA fragmentations [Coticchio et al. 2009; Huang et al. 2008a; Katz-Jaffe et al. 2008; Martínez-Burgos et al. 2011]. These changes may impair gene expression in oocytes or follicular cells, ultimately affecting genetic and functional competence of oocytes and embryos [Aye et al. 2010; Smitz et al. 2010]. "
[Show abstract][Hide abstract] ABSTRACT: Abstract Ovarian tissue cryopreservation is the primary treatment modality currently available to women at risk of losing their ovarian function due to cytotoxic therapy. However, the impact of these techniques on the oocyte DNA integrity is not elucidated. Here we have investigated the effect of vitrification and conventional slow freezing of eight week old Swiss albino mouse ovarian tissues on the oocyte and granulosa cell DNA integrity using the comet assay. The intracellular levels of reactive oxygen species in oocytes was measured by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. The cryopreservation of ovarian tissue by the slow freezing technique resulted in a significantly higher level of DNA fragmentation in oocytes in comparison to vitrification (p < 0.05) whereas DNA fragmentation in granulosa cells was significantly higher than the control (p < 0.01). Further, reactive oxygen species were significantly elevated in oocytes derived from slow freezing when compared to vitrification (p < 0.05). Therefore, we conclude that the ovarian tissue slow freeze-thawing makes the oocyte and granulosa cells more vulnerable to DNA damage whereas vitrification appears to be a safer method than slow freezing for ovarian tissue cryopreservation.
Systems Biology in Reproductive Medicine 06/2014; 60(6):1-6. DOI:10.3109/19396368.2014.923542 · 1.60 Impact Factor
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