Effect of slow freeze versus vitrification on the oocyte: an animal model.

OBJECTIVE: To determine whether there is a deleterious effect on dynamic events in the nucleus and cytoplasm of oocytes by using different cryopreservation protocols in an animal model. DESIGN: Prospective study. SETTING: University hospitals. PATIENT(S): Not applicable. INTERVENTION(S): Immunostaining and confocal laser scanning microscope techniques were used. MAIN OUTCOME MEASURE(S): The spindle and chromosomal configurations, as well as dynamic changes of the cortical granules (CGs) and mitochondria in different cryogroups. RESULT(S): After thawing/warming of bovine oocytes, CGs became more dispersed in the cytoplasm, particularly in the DMSO group. A significant reduction in normal spindle and chromosomal configurations was observed in all three cryogroups, particularly in the propylene glycol (PROH) group, when compared with the fresh group. Global DNA methylation levels were significantly reduced in the slow and DMSO groups, as compared with the fresh group; however, methylation levels were significantly increased in the PROH group. The proportion of severely apoptotic oocytes was dramatically increased in all three cryogroups, compared with the fresh group. CONCLUSION(S): Overall, results demonstrate that using DMSO as the cryoprotectant is better for preserving the cellular and nuclear integrity of the oocyte. The PROH method makes the oocyte more vulnerable to increased DNA methylation, which may be associated with imprinting gene alteration. This study adds to the increasing body of evidence that cryopreservation protocols vary in their impact upon the oocyte.

Embryo manipulation and imprinting.

As the use of assisted reproductive technologies (ART) continues to rise worldwide, it remains of the upmost importance to maintain the safety of those techniques used in ART. Many of these practices are unique to this discipline; as such, it becomes difficult to assess the true risks that the potential offspring may be subjected to under this type of treatment. Removal of oocytes from a woman's body during an in vitro fertilization (IVF) cycle offers an increased opportunity for routine cellular processes to go awry. Specifically, epigenetic modifications and imprinting diseases are rare among the general population; however, although their incidence among IVF-conceived children is also rare, their frequency in this population remains elevated compared with universal rates. Recent investigations have directly attributed their occurrences to the use of ART and IVF to achieve a successful pregnancy. This review discusses the major cellular manipulations of a typical IVF cycle to assess the potential risks versus the reported risks. These manipulations include preimplantation genetic diagnosis and screening, intracytoplasmic sperm injection, ooplasmic transfer, embryo culture, in vitro maturation, and cryopreservation. Oocyte and embryo handling is a delicate part of the IVF process that continues to improve. The safety of those potential improvements is also discussed.

Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis.

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus-oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B+M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75-75 00 mIUmL(-1)) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIUmL(-1)) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P>0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 75 00 mIUmL(-1) Gn; however, when the concentration was increased to 75 000 mIUmL(-1), the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).

Effect of gonadotropins on dynamic events and global deoxyribonucleic acid methylation during in vitro maturation of oocytes: an animal model.

Although the redistributions of mitochondria and cortical granules and global DNA methylation status were not altered in a dose-response manner, high dosages of gonadotropin induced spindle and chromosomal abnormalities. The present study highlights the importance of judicious use of gonadotropins and can be applied to clinical stimulation protocols to reduce the potential risks.

The effect of semen processing on sperm DNA integrity: comparison of two techniques using the novel Toluidine Blue Assay.

OBJECTIVE: The first goal of this study was to determine the effect that semen processing has on sperm DNA integrity. The second goal was to assess which processing technique (modified swim-up versus density gradient centrifugation) results in a superior sample. DNA integrity was measured using a novel Toluidine Blue Assay. STUDY DESIGN: Side-by-side comparison. MATERIALS AND METHODS: Raw semen samples were collected from thirty-two male individuals and scored for routine semen analysis. Prior to discarding the specimens identical aliquots were divided and processed by density gradient centrifugation and a modified swim-up technique. The Toluidine Blue Assay was used to analyze raw and processed samples. RESULTS: Both density gradient centrifugation and the modified swim-up improved DNA quality compared to the unprocessed sample. However, the modified swim-up technique proved superior. CONCLUSIONS: The swim-up technique generates a sperm sample with better DNA integrity. Should DNA integrity correlate with better pregnancy rates in IUI and IVF, respectively, the swim-up may be the sperm processing technique of choice for these procedures.

Current assessment of sperm DNA integrity.

Conventional semen analysis is rapidly losing its place as the gold standard of diagnosis and the cornerstone of treating the infertile male in modern times. Recent technology allows scientists to analyze sperm fertilizing potential and subsequent embryonic growth by studying factors that have previously escaped traditional parameters. It has become increasingly evident that nuclear DNA arrangement is essential to the fertilizing potential of sperm. A vast array of tests are now available to examine the genetic makeup of individual spermatozoa, ranging the entire gamut from simple bench top assays performed routinely to complex flow cytometric assays requiring highly-skilled technologists. Future research to compare these new tests to those more commonly in use, correlating them with reproductive outcome promises to fill the current void in the field of male infertility, paring innovative diagnostic (and prognostic) technological standards to the already existing sophisticated assortment of successful treatment modalities.