Oocyte vitrification modifies nucleolar remodeling and zygote kinetics-a sibling study.

PURPOSE: Oocyte vitrification does not affect embryo quality after oocyte warming, making this method effective in the preservation of female fertility. Morphokinetic parameters can be used to predict the competence of an embryo produced from fresh oocytes. Our aim was to study the effect of oocyte vitrification on zygote-embryo kinetics (pl). METHODS: The embryo-kinetics of fresh and sibling vitrified/warmed oocytes were compared to determine the consequences of oocyte preservation on the timing of embryo development. A 44-hours time-lapse analysis, from the time of ICSI (t0), of 179 fertilized fresh oocytes was compared to 168 fertilized sibling vitrified/warmed oocytes. RESULTS: Oocyte vitrification accelerated pronuclear disappearance, one-cell stage timing and modified nucleoli activity by increasing their number and decreasing their diameter at the zygote stage. In contrast, embryo kinetics during cleavage were similar to those observed for fresh sibling oocytes based on the parameters examined in this study. CONCLUSIONS: At the zygote stage, oocyte vitrification induces changes in pronuclei stability, probably due to pronuclei envelop instability as well as modifications in nucleoli functionality. Therefore, the predictive morphokinetic parameters on embryo competence found from fresh oocytes must be revised when applied on embryos from vitrified/warmed oocytes.

Meiotic spindle presence and oocyte morphology do not predict clinical ICSI outcomes: a study of 967 transferred embryos.

With a view to correlating oocyte morphology and meiotic spindle presence to clinical intracytoplasmic sperm injection (ICSI) outcomes, 967 oocytes that led to 967 transferred embryos in 404 embryo transfers were studied. No relationship was found between oocyte morphology (ooplasm texture, perivitelline space largeness, perivitelline space granulation absence/presence and the first polar body shape) or meiotic spindle presence or absence and clinical pregnancy per transfer and implantation rates after ICSI. It was concluded that oocyte morphology and meiotic spindle presence or absence can only predict fertilization, cleavage rates and embryo quality, as previously described in the literature, but do not help in daily ICSI practice in the choice of the metaphase II oocyte that will lead to the embryo that starts clinical pregnancy.

Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes.

In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P < 0.001) and embryo quality (grade I embryos over total embryos: 36.7 and 22.2% with fresh oocytes in IVF and ICSI; 12.1% with frozen-thawed oocytes in ICSI; respectively P < 0.001 and P < 0.05) were statistically lower after oocyte cryopreservation. The significant decrease in meiotic spindle retrieval rate before freezing (62.4%) and after thawing procedures (43.4%; P < 0.001) suggests that cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.

BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.