Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration

BACKGROUND: Recently, interest in oocyte cryopreservation has steadily increased. Newly developed protocols have dramatically improved survival rates, removing perhaps the major hurdle that has prevented this approach from becoming a fully established form of treatment. However, the clinical efficiency of these protocols has not been exhaustively explored and therefore remains controversial. METHODS: Morphologically normal oocytes displaying the first polar body were frozen-thawed with a slow cooling protocol that utilized 1.5 mol/l propane-1,2-diol (PrOH) and 0.3 mol/l sucrose. RESULTS: A total of 927 oocytes from 146 patients were frozen-thawed, achieving a 74.1% survival rate. Over 76% of microinjected oocytes displayed two pronuclei 16 h post-insemination, while the proportion of embryos at 44-46 h post-insemination was 90.2%. At this time point, the majority (68.3%) of embryos were at the two-cell stage, showing in most cases (78.7%) minimal or moderate fragmentation. Eighteen clinical pregnancies, three of which were twin, were observed, giving rise to rates of 12.3 and 9.7%, calculated per patient and per embryo transfer, respectively.The implantation rate was 5.2%. To date, four children have been born and three pregnancies resulted in spontaneous abortions, while the remaining pregnancies are ongoing. CONCLUSIONS: Our data indicate that although the combination of slow cooling and high sucrose concentration ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency

Embryos with morphokinetic abnormalities may develop into euploid blastocysts

Irregular cleavage divisions are expected to produce chromosomally deviant embryos. We investigated whether embryos from irregular cleavages could develop into euploid blastocysts, and, if so, whether any evidence existed of a self-correction mechanism of the embryo. We also investigated the role of different dynamic aspects of morula compaction in this process. A total of 791 embryos from 141 patients undergoing pre-implantation genetic screening were retrospectively analysed using a time-lapse imaging system, and multiple cell divisions were evaluated. A total of 276 embryos developed into blastocysts suitable for biopsy and chromosome screening through array-comparative genomic hybridization. As well as testing trophectoderm biopsy specimens for aneuploidy, excluded cells of 18 blastocysts, which developed from partially compacted morulas, were also analysed. Unique data on the developmental fate of embryos with cleavage abnormalities are presented, and a potential mechanism of 'aneuploidy rescue' is postulated through which mosaic embryos may form partially compacted morulas to exclude aneuploid cells. In addition, this process seems to be less efficient in older women. The data obtained also provide further evidence that excluded cells should not be used to infer the cytogenetic status of the embryo

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop

This study describes and compares the possible effects of vitrification oil the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, all intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern wits observable in only 30% of the oocytes subjected to vitrification, regardless of the Support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained it higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification