Analysis Of The Clinical Outcome After Rescue Icsi And The Spindle And Chromosome In Unfertilized Oocytes

The average fertilization rate after conventional IVF is about 60%–70%; however, in case of normospermia fertilization failure and low fertilization (defined as lower than 25% fertilization) occur in 5%–15%, and 20%, respectively, of the couples undergoing IVF, with a recurrence rate of about 30%–50%. Intracytoplasmic sperm injection (ICSI) can successfully avert fertilization failure outcomes in most IVF cycles and consequently when conventional IVF is not successful, re-insemination of those fertilization failed IVF oocytes can be considered. But attempts to rescue unfertilized oocytes by ICSI when they are ~1 day old have yielded poor results. We summarized clinical outcome by retrospective clinical study to demonstrate that it is has no value for rescue intracytoplasmic sperm injection of non-fertilized oocytes after conventional in vitro fertilization for treatment of non-male infertility because of oocyte defects. Oocyte-borne failures of conventional IVF are also known, and the corresponding oocyte abnormalities are even more difficult to detect, especially without access to relevant animal models to explain. Spindle and chromosome were the microstructure related to the quality of oocytes and which were the main reason which caused fertilization failure, or low good embryo and pregnancy rate after rescue ICSI (late rescue ICSI and early rescue ICSI). So we performed experiments to analyze oocyte structure by laser scanning confocal microscope (LSCM) and we established control groups for the first time to further confirm the oocyte defects which affected conventional IVF for non-male infertility. The 1st chapter Clinical analysis of Intracytoplasmic sperm injection to fertilization failure after conventional in vitro fertilization in non-male infertilityObjective:To analyze the clinical outcome of intracytoplasmic sperm injection (ICSI) in patients with rescue and previous fertilization failure after conventional IVF.Methods:Data from 10 rescue-ICSI cycles with the normospermia fertilization failure and low fertilization (defined as lower than 25% fertilization) after conventional IVF and Data from 19 subsequent-ICSI cycles with the previous complete failure of fertilization or with fertilization rate≤25% between January 2006 and December 2008 were retrospectively analyzed. The control group consisted of 133 ICSI cycles for male factor infertility in 2007.Results:There was significantly lower fertilization rate, delivery rate (P<0.05) and good quality embryo rate, implantation rate, and pregnancy rate (P<0.01) in rescue ICSI group than those in ICSI group (P<0.05). Implantation rate, pregnancy rate and delivery rate in the subsequent ICSI group was 8.8%, 21.1%, 15.8%, respectively, which were significantly different from ICSI group of 21.2%, 45.1%, 39.1% (P<0.05), fertilization rate was 49.5%, 63.5%, respectively, which was significantly different (P<0.01). Good quality embryo rate in the subsequent ICSI group was lower than control group, but there were not significant differences.Conclusion:(1) ICSI can overcome fertilization failure with conventional in vitro fertilization in non-male infertility, but fertilization rate and pregnancy outcome were still affected by the potential oocyte abnormality.(2) Because of the risk of ICSI, late ICSI should be used only for diagnostic purpose and not for therapeutic treatment. (3) Rescue ICSI (16–18 hours after insemination) was not recommended because of lack of pregnancy outcome.The 2nd chapter Analysis of the spindle and chromosome in MII oocytes after conventional in vitro fertilization failure for non-male infertilityObjective:To study the morphology of microtubules and spindle, chromosome alignment, as well as activation failure of oocytes which failed to fertilized in vitro fertilization (IVF) on the day 2 after retrieval for non-male infertility. So as to explore whether abnormal spindle and chromosome including spindle morphology abnormality, chromosome incorrectly linked to the spindle, abnormal microtubule arrangement, are involved in IVF fertilization failure (IVF FF).Methods:We collected denuded oocytes which is lower than 25% fertilization in IVF (IVF FF group) on the day 2 (non-fertilization oocytes mean no pronuclei and no second polar body, no cleavage) after retrieval as experiment group. We also collected oocytes which failed to unfertilized after intracytoplasmic sperm injection (ICSI NF group) on the day 2 and the in vitro matured MII oocytes (IVM group) from ICSI for non-male infertility as control groups. The localization of tubulin and chromatin revealed by FITC- anti-α-tubulin antibody and propidium fluorescence among three groups was assessed with a laser-scanning confocal microscope. We also compared the differences of oocytes activation between IVF FF group and ICSI NF group.Results:A total of 164 unfertilized oocytes (93 oocytes in IVF and 71 oocytes in ICSI) and 56 oocytes in vitro maturation were available for this study. The abnormality of spindle and chromosomes was significantly higher in IVF FF group than that in ICSI NF group or IVM group (abnormal spindle rates were 80.6%, 64.8% and 57.1%, respectively; abnormal chromosome rates were 91.4%, 80.3% and 75%, respectively). By contrast, abnormal spindle and chromosome have no significant differences between ICSI NF group and IVM group. Activation failure occurred in 16.1% of the IVF FF cases and 49.3% of ICSI NF oocytes respectively, and activation failure have significant differences between IVF FF and ICSI NF.Conclusion:(1) The internal defects of oocytes played an important role on the fertilization in IVF.(2) Spindle and chromosome were the microstructure related to the quality of oocytes and which were the main reason which caused fertilization failure, or low good embryo and pregnancy rate after rescue ICSI (late rescue ICSI and early rescue ICSI).(3) The abnormality of MII oocytes'spindle and chromosome in IVF FF group were significantly higher than ICSI NF group (P<0.05), which showed that oocytes'quality was an important factor. It also illustrated the reason of the significantly reduced implantation and pregnancy rate per transfer in the ICSI group with previously failed IVF attempts compared to the ICSI group in male factor infertility.(4) spindle and Chromosome abnormality had no significant differences between ICSI NF group and IVM group (P>0.05), but oocytes activation failure was significantly higher in ICSI NF group than in IVF FF group, indicating that oocytes activation failure was the main reason caused fertilization failure after ICSI for male infertility.