Cumulus cells apoptosis as an indicator to predict the quality of oocytes and the outcome of IVF-ET.

PURPOSE: Our purpose was to establish an evaluation system for oocyte quality based on the incidence of cumulus cells apoptosis and to examine the effect of coculture, using autologous cumulus cells, on the outcome of IVF-ET according to proliferative activities of helper cells and the incidence of cumulus cells apoptosis. METHODS: Cumulus cell masses were collected from 91 mature oocytes among 330 oocytes retrieved from a total of 34 IVF-ET cycles with tubal infertility and unexplained infertility. The incidence of apoptosis in cumulus cells was assessed by apoptosis detection kit fluorescein. On ovum pick up, 2nd day embryos were cocultured with autologous cumulus cells. Prior to coculture, in vitro proliferative activity of cumulus cells was evaluated. RESULTS: Cumulus cells from patient groups over 40 years old had a significantly increased apoptosis incidence, a lower fertilization rate, and the decreased number of oocytes retrieved compared to the other age groups (P < .05). The incidence of cumulus cells apoptosis was significantly lower when the number of oocytes retrieved was 5 or less (P < .05). Cumulus cells from fertilized oocytes (0.43 +/- 0.07%) and those from patients who became pregnant (0.44 +/- 0.11%) following IVF-ET showed a significantly lower incidence of apoptosis compared to those of unfertilized oocytes (1.80 +/- 0.35%; P < .001) and the nonpregnant group (0.81 +/- 0.10%; P < .05). Embryo quality also had a negative correlation with the incidence of cumulus cells apoptosis. Coculture of fertilized oocytes with cumulus cells with high proliferative activity resulted in improved rates of implantation and pregnancy compared to that with poor active cumulus cells. No significant difference was found between the in vitro proliferative activity of cumulus cells and the incidence of cumulus cells apoptosis (P < .063). CONCLUSIONS: The age of women might influence the incidence of apoptosis in cumulus cells, and the increased incidence of apoptosis is associated with the number of oocytes retrieved, the fertilization rate, and the pregnancy outcome following IVF-ET. These results suggest that the incidence of cumulus cells apoptosis can be used in predicting oocyte quality, outcome of IVF-ET, and age-related decline in fertility.

The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components.

OBJECTIVE: To elucidate the mechanism for the mode of action of coculture by the use of a coculture system for mouse one-cell embryos with human oviductal epithelial cells. DESIGN: Prospective, controlled in vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): Female ICR strain mice aged between 6 and 8 weeks. INTERVENTION(S): Flushed one-cell embryos were cultured in human tubal fluid medium alone (control), in coculture system with human oviductal cells, in five kinds of conditioned media, and in a contactless coculture system using a cell-culture insert. MAIN OUTCOME MEASURE(S): The percentage of the embryos developed to hatching blastocyst stage and the level of superoxide anion in the supernatant from each culture condition. RESULT(S): The rates of embryo development to the hatching blastocyst stage were significantly higher in the coculture group (43%) than in the control group (none) (P <.05). The embryo development rate in the control group was similar to that of the embryos in the five kinds of conditioned media. The effects of coculture on embryo development disappeared in the contactless coculture group. The level of superoxide anion was significantly reduced in the coculture group compared to the control group. CONCLUSION(S): The present coculture system overcomes the two-cell block in vitro and improves the embryo development. The beneficial effect may be a result of direct cell-to-cell contact between the embryo and helper cells and the removal of deleterious components from medium, rather than a result of embryotrophic factors.

Relationships between concentrations of tumor necrosis factor-alpha and nitric oxide in follicular fluid and oocyte quality.

PURPOSE: Our objective was to explain a relationship between concentrations of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in follicular fluid, oocyte quality, and outcomes of in vitro fertilization--embryo transfer (IVF-ET). METHODS: The concentrations of TNF-alpha and NO were measured in 115 follicular fluid samples collected from 43 patients undergoing IVF-ET program, due to tubal obstruction, some with endometriosis (8 patients) or hydrosalpinx (5 patients). A correlation of these factors concentrations and the oocyte quality, the oocyte maturity, and infertility-associated diseases was analyzed. RESULTS: No correlation was found between concentrations of NO and TNF-alpha in follicular fluid. NO concentrations in follicular fluids were significantly higher in patients with endometriosis (P < 0.001) or hydrosalpinx (P < 0.01) compared to the patients with just tubal obstruction. Follicular NO concentration differences according to oocyte maturity and oocyte quality were not found. In contrast, TNF-alpha concentrations in follicular fluids were significantly higher in poor quality oocytes (P < 0.05) but were not associated with infertility-associated diseases, such as hydrosalphinx or endometriosis,and the oocyte maturity. No significant differences in follicular levels of NO and TNF-alpha as well as IVF-ET parameters of pregnant and nonpregnant groups were revealed. CONCLUSIONS: There is no significant correlation between the concentrations of NO and TNF-alpha in follicular fluid. NO levels in follicular fluid are altered in infertility-associated diseases. However, TNF-alpha levels but not NO levels influence oocyte quality. These results suggest that the production of NO and TNF-alpha in follicular fluid may be regulated via different pathways and can be tempered with infertility-associated diseases, thereby influencing oocyte quality locally.

The effect of nitric oxide on sperm cell function and embryo development.

PROBLEM: Nitric oxide (NO) has been known to have multifunctional roles both in the male and female reproductive systems. We investigated the effects of sodium nitroprusside (SNP)-dependent NO release on sperm cell function and embryo development to elucidate the mechanisms of action of NO. METHOD OF STUDY: Semen samples from 20 healthy men were processed by the swim-up method. Sperm motility, hyperactivation, and acrosome reaction were examined following incubation with various concentrations of SNP. The concentration of 10 nM to 1 mM was used for sperm motility and hyperactivation measurement and 1 microM to 1 mM for examining the effect on acrosome reaction. Embryo development to blastocyst stage was assessed using 100 nM to 1 mM of SNP added before transferring the mouse embryos into the culture medium. Finally, to understand the mechanism of action of NO, changes in embryo development were examined after zygotes were treated with various concentrations ranging up to 1 mM of 8-bromo-cGMP, an analog of cGMP. RESULTS: Both sperm motility and hyperactivation were significantly reduced at 100 microM and 1 mM concentrations of SNP after 6 hr of incubation. After 24 hr of incubation, they were greatly decreased with all, except the 10 nM concentration of SNP. The percentage of acrosomal-reacted spermatozoa was increased with the increasing concentration of SNP following incubation with 10 microM and 1 mM of SNP. Embryo development was arrested since the two-cell embryonic stage with all except the 100 nM concentration of SNP, and inhibited by 200 microM of SNP regardless of SNP treatment stage. However, embryo development was not influenced by 8-bromo-cGMP. CONCLUSIONS: We concluded that SNP-inhibited sperm cell function and embryo development in a dose- and time-dependent manner, and the inhibitory effect on embryo development, may not be a stage-specific treatment mediated via a cGMP-independent pathway. This result suggests that NO may be enough to affect the fecundity potential in vivo.