Resveratrol improves in vitro maturation of oocytes in aged mice and humans.

OBJECTIVE: To evaluate the effects of resveratrol on oocyte maturation in aged mice and humans. DESIGN: Experimental laboratory study. SETTING: University-based reproductive medicine center. PATIENT(S): A total of 64 women 38-45 years of age undergoing intracytoplasmic sperm injection (ICSI) and 48-52-week-old female C57BL/6J mice. INTERVENTION(S): In vitro culture in the presence of three different concentrations of resveratrol (0.1, 1.0, and 10 µm) or dimethylsulfoxide. MAIN OUTCOME MEASURE(S): Parameters of oocyte nuclear maturation, fertilization, immunofluorescence intensity of mitochondria, and normal morphology of spindle and chromosome of oocytes undergoing in vitro maturation (IVM) in aged mice and humans; blastocyst formation and levels of SRIT1, CAT, SOD1, and GPX4 gene expressions in aged mice. RESULT(S): Resveratrol at 1.0 µm significantly increased first polar body emission rate in oocytes derived from aged mice and humans, and an increased percentage of fertilization and blastocyst formation was observed in aged mice. In addition, immunofluorescence intensity of mitochondria and normal morphology of spindle and chromosome of oocytes undergoing IVM were notably improved compared with control samples in aged mice and human. Furthermore, the use of resveratrol exhibited enhanced expression patterns of SRIT1, CAT, SOD1, and GPX4 in aged mice. CONCLUSION(S): Resveratrol induced oocyte maturation and blastocyst formation in aged mice, and improved oocyte maturation and quality was examined in aged humans. In conclusion, 1.0 µm resveratrol was the appropriate concentration in IVM medium.

Comparison of the Developmental Potential and Clinical Results of In Vivo Matured Oocytes Cryopreserved with Different Vitrification Media.

BACKGROUND: Oocyte vitrification is widely used throughout the world, but its clinical efficacy is inconsistent and depends on the vitrification media. This study compared the developmental potential and clinical results of in vivo matured oocytes cryopreserved with different vitrification media. METHODS: This retrospective study involved vitrified-warmed oocytes at one in vitro fertilization laboratory. Vitrification media kits comprised the MC kit (ethylene glycol [EG] plus 1,2-propanediol [PROH]), the KT kit (EG plus dimethyl sulphoxide [DMSO]), and the Modified kit (EG plus DMSO and PROH kit). Rates of oocyte survival and subsequent developmental potential were recorded and analyzed. The t-test and the Chi-square test were used to evaluate each method's efficacy. RESULTS: Oocyte survival rate was significantly higher for the Modified kit (92.0%) than for the MC kit (88.2%) (P < 0.05) and the KT kit (77.3%) (P < 0.001). The rate of high-quality embryo development in the Modified kit group (35.8%) was significantly higher than in the MC kit group (29.0%) and the KT kit group (28.3%) (P < 0.001). No significant differences were observed in the clinical pregnancy and implantation rates among the MC, KT, and Modified kit groups (37.2% vs. 30.2% vs. 39.6%; 21.9% vs. 18.8% vs. 27.4%, respectively) (P > 0.05). The high-quality embryo rate per warmed oocyte was significantly higher (23.4%) in the Modified kit group than in the other groups (P < 0.001). The embryo utilization and live birth rates per warmed oocyte were the highest in the Modified kit group, but not significantly (P > 0.05). CONCLUSIONS: Modified vitrification media are efficient for oocyte vitrification and, with further verification, may be able to replace commercially available media in future clinical applications.

[Application of calcium ionophore A23187 in ICSI for globozoospermia: A report of 2 cases and review of the literature].

OBJECTIVE: To investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men. METHODS: We collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease. RESULTS: Quality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group. CONCLUSION: Calcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.

Pregnancy with oocytes characterized by narrow perivitelline space and heterogeneous zona pellucida: is intracytoplasmic sperm injection necessary?

PURPOSE: This retrospective study analyzed fertilization protocols and pregnancy outcomes for oocytes with with narrow perivitelline space and heterogeneous zona pellucid (NPVS/HZP). METHODS: In 63 in-vitro fertilization cycles filled with NPVS/HZP oocytes (abnormal oocytes group) and 521 cycles with normal oocytes (normal oocytes group), major clinical and laboratory parameters were recorded and compared in different fertilization cycles (conventional IVF cycles, rescue ICSI cycles, and traditional ICSI cycles). RESULTS: NPVS/HZP oocytes meant lower MIIoocytes rates in both IVF and ICSI cycles compared with normal oocytes (p < 0.05). The 2PN rates for abnormal oocytes were significantly lower than those for normal oocytes in both conventional IVF cycles (58.8% VS 71.3%, P < 0.05) and rescue ICSI cycles (58.0% VS 78.0%, P = 0.0000). The high-quality embryo rates in normal oocytes groups were significantly higher than those in abnormal oocytes groups in different fertilization cycles (52.2% VS 35.0%, P < 0.01; 42.9% VS 23.9%, P < 0.001; 50.6% VS 31.0%, P = 0.0000, respectively). No clinical pregnancy was obtained from abnormal oocytes in 11 conventional IVF cycles. The clinical pregnancy rates in rescue ICSI and traditional ICSI cycles were comparatively lower in abnormal oocytes groups, but there was no significant difference as compared with normal oocytes groups (35.0% VS 48.1% and 26.7% VS 50.7%, P > 0.05, respectively). CONCLUSIONS: Retrieval of oocytes characterized by NPVS/PZP from cycle to cycle was one of the reasons for obscure infertility. ICSI may be the right way to avoid fertilization failure and get pregnancy in women with NPVS/HZP oocytes.

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles.

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3-5 h (Group B) or 24-28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P<0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P<0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

Evaluation of the immature human oocytes from unstimulated cycles in polycystic ovary syndrome patients using a novel scoring system.

OBJECTIVE: To establish a novel scoring system for human immature oocytes and estimate its prognostic effects on the in vitro maturation (IVM) and developmental potential of human immature oocytes. DESIGN: Prospective study. SETTING: Hospital-based IVF center. PATIENT(S): Patients with polycystic ovary syndrome (PCOS) undergoing unstimulated cycles of oocyte retrieval. INTERVENTION(S): Immature (germinal vesicle and metaphase I) oocytes were collected from PCOS patients and were evaluated based on morphologic characters. After IVM, the maturation rate, fertilization rate, and cleavage rate were evaluated. The quality of the embryos and the apoptosis of the granulosa cells (GCs) were analyzed as well. MAIN OUTCOME MEASURE(S): Morphologic characters of immature oocytes and maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and the apoptosis of the GCs after IVM. RESULT(S): The maturation rate, fertilization rate, cleavage rate, and high-quality embryo formation rate of both MI and GV oocytes reduced with the decrease of the score. There were no differences in maturation, fertilization, and cleavage rates between the MI and GV phase. The apoptosis rate of GCs of grade 1' and 0' oocytes was obvious higher than those of grade 3-4'. CONCLUSION(S): Evaluating cumulus layer around the oocyte was a simple but useful method, which played an important role in terms of yielding good prognostic parameters in an embryology laboratory. The scoring system of immature oocytes can correctly reflect the development potential of human oocytes.

[Comparison of vitrification and slow-freezing of human day 3 cleavage stage embryos: post-vitrification development and pregnancy outcomes].

OBJECTIVE: To compare the effects of vitrification with slow-freezing on the developmental ability of day 3 cleavage stage embryos. METHODS: Patients who had no less than 4 high quality embryos were included in this study. These embryos were cryopreserved using the methods of vitrification or slow-freezing. In the cryopreserved embryo transfer cycles, the embryos which were cryopreserved using one of the methods were chosen randomly. The developmental ability of embryos was compared between these two groups. RESULTS: A total of 80 patients were included in this study with 160 embryos. In the group of slow-freezing, 73 (91%) embryos were survived and achieved 15 (38%) clinical pregnancies. Among these, 3 were twins and the implantation rate was 25% (18/73). In the group of vitrification, 71 (89%) embryos were survived and achieved 19 (48%) clinical pregnancies. Among these, 9 were twins and the implantation rate was 39% (28/71), which was significantly higher than the slow-freezing group (P < 0.05). Otherwise, the clinical pregnant rate and multiple pregnant rate was higher in the group of vitrification than the slow-freezing group, but had no significance. CONCLUSION: Vitrification is more benefit for the developmental ability of the thawed embryos and more suitable for the cryopreservation of day 3 cleavage stage embryos.

[Spindle and chromosome configuration of human in vitro matured oocytes after slow freezing-fast thawing].

OBJECTIVE: To evaluate the development of immature oocytes after freezing-thawing by conventional cryopreservation method for mature oocytes. METHODS: Immature oocytes were collected from stimulated ovaries of intracytoplasmic sperm injection (ICSI) cycles. Immature oocytes were in vitro matured directly or after slow freezing-fast thawing and immunostained for tubulin and chromatin and at last visualized by confocal microscopy. RESULTS: No statistical difference was found in maturity rate between freezing groups and the controls. There was a statistically significant increase in abnormalities of chromosome (23.7% vs. 50%) and spindle (28.9% vs. 53.9%) in the GV freezing group compared with the GV control (P < 0.05). MI groups gave the same results. But we did not find any statistical difference between GV frozen group and MI frozen group. CONCLUSIONS: Cryopreservation decreases the ability of immature oocytes to form normal meiosis spindle and induce abnormal division of chromosomes. This suggests that the routine slow freezing-fast thawing procedure for mature oocyte couldn't preserve the ability well to form normal meiosis spindle during in vitro maturation.

[Comparison of the outcome of intracytoplasmic single sperm injection between two motile sperm separation methods].

OBJECTIVE: To evaluate the efficiency of density gradient and improved swim-up methods for motile sperm isolation from fresh semen samples in intracytoplasmic sperm injection and embryo transfer (ICSI-ET) program, thus guiding the clinical application. METHODS: The fertilization rate, cleavage rate, high-quality embryo rate, clinical pregnancy rate, sperm abnormal rate and recovery rate of 42 cycles were studied prospectively. The cycles were divided into two groups with respect to the motile sperm isolation methods. RESULTS: No obvious difference was found in the fertilization rate, cleavage rate, high-quality embryo rate and clinical pregnancy rate between the two methods. The abnormal sperm rate induced by the improved swim-up method was significantly higher than that of the density gradient method (P < 0.01). For severe oligozoospermia, the recovery rate of motile sperm with density gradient was higher than that of improved swim-up (P < 0.01). CONCLUSION: In the ICSI-ET, there is no obvious difference in clinical pregnancy outcome between the two methods. The method of improved swim-up can be used for all patients but those with severe oligozoospermia.

[Oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection].

OBJECTIVE: To investigate the effect of assisted oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). METHODS: All 113 discarded oocytes that showed no evidence of fertilization at 16 - 18 hours after in vitro maturation (IVM)-ICSI cycles and conventional ICSI were assigned to four groups according to the time after ICSI: IVM-ICSI 22-hour group (n = 33), IVM-ICSI 44-hour group (n = 18), ICSI 44-hour group (n = 37) and ICSI 68-hour group (n = 25). All unfertilized oocytes were exposed to calcium ionophore A23187 (5 micromol/L) for 5 minutes and subsequently incubated with puromycin (10 microg/ml) for 4 hours. After incubation, the oocytes were cultured in vitro for 3 - 5 days. The activation rate, proportion of oocytes that showed pronucleus formation and cleavage rate were calculated after activation. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH) on the embryos that displayed two pronuclei and a second polar body. RESULTS: The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 - 68 hours after ICSI. Best results were achieved in IVM-ICSI 22-hour group, which elicited 88% (29/33) of activation rate, 62% (18/29) of cleavage rate and 28% (5/18) of 4-cell embryos. One embryo in this group developed to the morular stage. The activation rate and developmental potential of the activated embryos in IVM-ICSI 44-hour group, ICSI 44-hour group and ICSI 68-hour group decreased. FISH analysis showed 4 embryos with XX and 9 embryos with XY in 16 embryos. CONCLUSIONS: The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 - 68 hours after ICSI. The cultured time of unfertilized oocytes after ICSI affects activation efficiency and developmental potential of the activated embryos. The activated zygotes that display two pronuclei and a second polar body can develop normally.

[Sex chromosome analysis and IGF-II expression on activated human unfertilized oocytes after ICSI with calcium ionophore A23187 and puromycin].

OBJECTIVE: To investigate the sex chromosomes and the expression of insulin-like growth factor-II (IGF-II) on activated human unfertilized oocytes after intracytoplasmic sperm injection(ICSI) with calcium ionophore A23187 and puromycin. METHODS: All 95 discarded oocyes that showed no evidence of fertilization at 16-18 h after in vitro maturation and intracytoplasmic sperm injection cycles (IVM-ICSI)/conventional ICSI were exposed to calcium ionophore A23187 (5 micromol/L) for 5 min and then were incubated with puromycin (10 microg/mL) for 4 h. After activation, the oocytes were cultured in vitro for 3-5 days. The sex chromosome analysis was performed by dual color fluorescence in situ hybridization. The expression of IGF-II on the activated embryos, normal embryos, and parthenotes was examined. RESULTS: The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 h after ICSI. The activated rate, cleavage rate, and quality of activated embryos of the IVM-ICSI group were similar to those of ICSI group, respectively. Sex chromosome analysis indicated that 8 male and 5 female embryos had been derived from two pronucleus and a second polar body. The expression of IGF-II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes. CONCLUSION: The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 h after ICSI. Moreover, the unfertilized oocytes activated by calcium ionophore A23187 and puromycin had normal sex chromosomes and expression of IGF-II like the normal embryos. These suggest that oocyte activation may be considered as a remedial measure in the presence of total or nearly total fertilization failure in ICSI.

[In vitro maturation and fertilization of oocytes from unstimulated cycles in women with infertility due to polycystic ovary syndrome].

OBJECTIVE: To investigate the effect of in vitro maturation (IVM) and fertilization of oocytes from unstimulated cycles in women with infertility due to polycystic ovary syndrome (PCOS). METHODS: Seventy women with PCOS, who came to our clinic during March to were involved in this trial. June 2003. Every cycle was given human chorionic gonadotropin 10,000 IU 36 h before oocyte retrieval. Immature oocytes were matured in vitro and fertilized, and the resulting embryos were replaced back to the uterus. RESULTS: A total of 94 IVM cycles were performed and 1283 oocytes were obtained. The overall maturation, fertilization and cleavage rates were 65.3%, 66.0% and 48.0% respectively. Maturation, fertilization, cleavage and high quality embryo rates had no difference between metaphase I stage oocytes (69.7%, 71.7%, 52.2% and 26.1% respectively) and germinal vesical (GV) stage oocytes (67.7%, 66.4%, 47.6% and 24.1% respectively), but the rates in those oocytes which could not be classified (44.8%, 53.8%, 46.2% and 16.9% respectively) were much lower than the former two groups. After embryo transfer, 18 pregnancies were reported (24%). CONCLUSION: IVM/IVF-ET in unstimulated cycles is a feasible treatment for women with PCOS.

[The cryopreservation of human oocytes at different maturity stages].

OBJECTIVE: To examine the ultrastructure changes and the effects of different sucrose concentrations on the developmental potential of human frozen-thawed oocytes at different maturity stages. METHODS: Oocytes at different maturity stages collected from polycystic ovarian syndrome patients were involved in the study. Different sucrose concentrations (0.1, 0.2 or 0.3 mol/L) were used to study the developmental potential of the frozen-thawed oocytes. Non-frozen and frozen-thawed different maturity oocytes were processed for transmission electron microscopy observation. RESULTS: The results revealed that the mitochondria and the vesicles in the immature oocytes cytoplasm were fewer than those in the mature oocytes without regular distribution. Electron density of the mitochondria and distribution of the vesicles in the mature oocytes changed with the cryopreservation. No remarkable change was produced in the immature oocytes between non-frozen and frozen-thawed oocytes. Cryopreserving with 0.2 mol/L sucrose resulted in perfect development potential for both mature and immature oocytes than that with 0.1 mol/L. Study involving 0.3 mol/L sucrose in the cryoprotectant resulted in higher survival rate and clinical pregnancies. CONCLUSION: The results suggest that sucrose concentration of 0.3 mol/L in the cryoprotectant solution is efficient in freezing oocytes with slow-freezing method. Cryopreservation with slow-freezing method could produce ultrastructure changes in the human oocytes.