Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation

In the present study we investigated the relevance of spermimmobilization prior to intracytoplasmic sperm injection (ICSI)in the fertilization process. Using supravital staining of thespermatozoa with eosin and studying sperm decondensation with2 mM dithiothreitol (DTT) in conditions imitating sperm handlingduring ICSI, we demonstrated that immobilization of the spermatozoonby squeezing its tail between the glass pipette and the bottomof the dish damages the sperm plasma membrane. Polyvinylpyrrolidone(PVP), which is usually present in the drop with the spermatozoonto facilitate its handling, was found to impede the access ofboth eosin and DTT to the sperm nucleus. We conclude that (i)sperm immobilization prior to ICSI damages the sperm plasmamembrane, that (ii) this damage is sufficient for thiol-reducingagents to gain access to the sperm nucleus, and finally that(iii) PVP possibly interferes with sperm nucleus decondensation.     

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin

Purpose

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 Μg/ml puromycin for 6–8 hr and further cultured for 12–15 hr.

Results

Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%).

Conclusions

Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.     

Visualization and cytogenetic analysis of second polar body chromosomes following its fusion with a one-cell mouse embryo

Purpose This study was designed to visualize the second polar body (2PB) chromosomes using its electrofusion with a one-cell-stage mouse embryo to approach preconception diagnosis of chromosomal disorders. Results Eighty to 90% hybridization efficiency has been achieved by electrofusion of 2PB with mouse zygotes. 2PB chromosomes were visualized in 40–50% of hybrids. Sixty-five percent of 2PB chromosomes were visualized when fused with the cytoplast obtained microsurgically by removing pronuclei from a one-cell embryo. As much as 33–43% of these resulting metaphases appeared to contain chromosomal aberrations. The follow-up of the development of the reconstructed one cell-stage hybrids in vitro revealed a significant decrease in their viability. The hybrid embryos resulting from 2PB electrofusion with enucleated zygotes did not develop beyond the two-cell stage. Conclusion Electrofusion is an efficient approach for hybridization of 2PB with a one-cell mouse embryo and may be useful for visualization and cytogenetic analysis of 2PB chromosomes. The visualization rate of 2PB chromosomes is higher if 2PB is fused with enucleated zygotes. However, the method induces over 30% of chromosomal aberrations and may lead to a significant decrease in the viability of the resulting one-cell embryos.     

Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates

OBJECTIVE: To elucidate the relative predictive value of implantation markers at different stages of preimplantation development. DESIGN: Correlation of pronuclear morphology with embryo morphology and implantation rates in retrospective and prospective analysis of in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) treatment cycles. SETTING: Private infertility center. PATIENT(S): A total of 441 couples undergoing infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Size of pronuclei and distance between them, the number and polarization of nucleolus precursor bodies (NPB) at the one-cell stage, embryo cleavage and fragmentation rates on days 2 and 3, and pregnancy and implantation rates. RESULT(S): Polarization of the NPB in both pronuclei had a statistically significant correlation with normal membrane breakage during ICSI (40%, compared with 33% easy, and 31% difficult membrane breakage) and also with faster cleavage and lower fragmentation rates of embryos. Sixty-one percent of implanting embryos had polarization of the NPB in both pronuclei compared with 37% for all embryos. Larger distance between pronuclei and their unequal size had a statistically significant correlation with slower cleavage and inferior embryo quality. Embryo selection based on only pronuclear morphology or on only day-3 embryo morphology yielded implantation rates of 15.1% and 12.1%, respectively. Embryo selection based on sequential evaluation of both pronuclear morphology and embryo morphology on day 3 resulted in a 21.1% implantation rate. CONCLUSION(S): Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.     

Andrology: Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

When intracytoplasmic sperm injection (ICSI) is performed, itis important to know the capacity of sperm cells to activatethe oocytes, although knowledge of their ability to fuse withthe oocytes is not vital. Hamster oocytes are not suitable forthis purpose because they are easily activated by the injectionprocedure itself. We therefore investigated whether mouse oocytescould be used to assess the activation properties of human spermatozoa.Mouse oocytes were randomized for injection with initially motilespermatozoa, medium, heat-treated or salt-extracted spermmatozoa,and the survival and activation rates were examined. About halfof the mouse oocytes survived the intracytoplasmic injectionof a human sperm cell. Unlike hamster oocytes, the rate of activationprovoked by the injection procedure itself was acceptably low(20%), resembling in this respect the behaviour of human oocytes.Following the injection of initially motile human spermatozoa,all mouse oocytes were activated. The injection of heat-treatedor salt-extracted human spermatozoa resulted in activation ratesof 14 and 15% respectively, comparable with the results followingsham ICSI. These data support the hypothesis of a sperm-associatedoocyte activation factor. In most activated oocytes, the humansperm nucleus decondensed to form a male pronucleus. Cytogeneticanalysis at the first metaphase revealed that human sperm chromosomeswere able to undergo replication in a heterologous environment.From our data we concluded that human spermatozoa can be injectedsuccessfully into mouse oocytes, resulting in a reasonable survivalrate, and that mouse oocytes provide a useful model for boththe assessment of the sperm-associated oocyte activation factorand the cytogenetic analysis of human spermatozoa.     

Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection

The fertilization rates and further development of 528 humanmetaphase IT oocytes directly injected by a single spermatozoonwere analysed with respect to their morphological features atthe light microscopy level at the time of retrieval. The deviationsof oocyte morphology which were most frequently observed, afterremoval of cumulus cells, were dark incorporations, dark zonapellucida, large peri-vitelline space, spots, vacuoles, refractilebodies and irregular shape. These deviations correlated neitherwith the fertilization rate nor with the embryo quality score,as compared to ‘ideal’ oocytes. Since the majorityof oocytes displayed deviations from the ‘ideal’morphotype but were still fertilized and developed in cultureat a normal rate, they were probably as normal as ‘ideal’oocytes. Since some of these morphotypes, such as refractilebodies, have been shown to be associated with failure of fertilization,it seems that intracytoplasmic sperm injection may be an appropriatemethod of treatment for couples in whom repeated failure ofin-vitro fertilization is associated with the retrieval of dysmorphicoocytes in the presence of normal semen characteristics.     

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm- oocyte interaction

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.      

Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay.

OBJECTIVE: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Plasmid relaxation. RESULT(S): Ham's F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. CONCLUSION(S): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.