Effects of vitrification on blastomere viability and cytoskeletal integrity in mouse embryos.

Vitrification is widely used to cryopreserve supernumerary embryos following in vitro fertilization (IVF). The mouse model was used to investigate the effects of vitrification on blastomere viability, using viability markers, and on the cytoskeleton, by analysing spindle/chromosome configurations, using confocal scanning microscopy. Ninety cleavage and morula stage dimethyl sulphoxide (DMSO)/EG vitrified mouse embryos were either processed immediately following warming for viability assessment by labelling with the fluorescent markers carboxyfluorescein-diacetate succinimidylester (CFSE) and propidium iodide (PI) or were cultured to the blastocyst stage and immunostained with α-tubulin antibody to visualize microtubules and DAPI or PI to visualize DNA. Sixty-five fresh embryos were also used as the control. Vitrified embryos showed high survival rates following warming, but they had a higher incidence of damaged blastomeres compared with fresh embryos. Most mitotic spindles examined in all groups were normal, but multivariable analysis revealed that the proportion of abnormal spindles was significantly higher in vitrified/warmed embryos (P < 0.05). This study is the first to examine the immediate effects of vitrification on blastomere viability, using fluorescent markers and shows that although vitrification results in a higher incidence of damaged blastomeres, vitrified embryos may compensate for this limited number of damaged/abnormal cells, as development to the blastocyst stage was not compromised.

Semen analysis by electron and fluorescence microscopy in a case of partial hydatidiform mole reveals a high incidence of abnormal morphology, diploidy, and tetraploidy.

OBJECTIVE: To perform a highly detailed semen analysis in a man whose wife had a partial mole. DESIGN: Case report. SETTING: Gynecology departments of two university hospitals and a laboratory of histology/embryology. PATIENT(S): A 32-year-old man whose wife had a partial mole. INTERVENTION(S): Sperm characteristics were examined by light microscopy, morphology was analysed by electron microscopy (TEM), DNA fragmentation was evaluated by TUNEL using fluorescence microscopy, and chromosomal abnormalities were assessed by fluorescence in situ hybridization using probes for chromosomes 13, 15, 16, 18, 21, 22, X, and Y. MAIN OUTCOME MEASURE(S): Sperm count, motility, morphology, DNA fragmentation, and incidence of diploidy, tetraploidy, and aneuploidy. RESULT(S): Sperm concentration was 61 million/mL, with 31% progressive motility and 4% normal morphology. TEM revealed a high incidence of head, neck, and tail abnormalities as well as the presence of phagocytes. DNA fragmentation was within normal limits (11.6%). Aneuploidy levels were low for all chromosomes tested. However, there was a high level of diploidy, with XY, XX, and YY constitution. Tetraploid sperm (XXYY) were also noted. CONCLUSION(S): Semen analysis revealed a high incidence of abnormal morphology and increased diploidy. It may be important to perform FISH testing, to verify increased diploidy in sperm, in men whose wives have had partial moles. These couples could be informed of the option to have preimplantation genetic diagnosis as a means to distinguish between diploid and triploid embryos arising from fertilization of a haploid egg by diploid sperm.