ObjectiveThis study was performed to compare the efficiency of slow freezing and vitrification based on survival, development to blastocysts, and cell numbers of blastocysts. Changes in embryonic gene expression in fresh and frozen-thawed embryos were also examined.MethodsEight-cell stage embryos were collected from superovulated female BDF1 mice. The collected embryos were randomly divided into three groups. One group was maintained as fresh controls (n=42), one was frozen by slow freezing (n=43), and one was cooled by vitrification (n=43). After thawing or cooling, survival rates, development to blastocyst, and cell numbers and inner cell mass (ICM) cell numbers of blastocysts were compared with those of the control group. The expressions of eight genes (Rbm3, Birc5, Sod1, Sod2, Cirbp, Caspase3, Trp53, Hsp70.1) were examined by real time-quantitative polymerase chain reaction in the fresh and frozen-thawed embryos.ResultsThere were no significant differences in the slow freezing and vitrification groups' survival rate after thawing (88.4% vs. 88.4%), development to blastocyst (100% vs. 97.4%), cell numbers (107.0±21.0 vs. 115.0±19.7), or ICM cell numbers of blastocysts (11.3±5.2 vs. 11.1±3.7). Cell numbers of blastocysts were significantly (p<0.05) lower in the frozen-thawed embryos than the fresh embryos. There were no significant differences in the slow freezing and the vitrification groups' expressions of the eight genes. The expressions of CirbP and Hsp70.1 were higher in the frozen-thawed embryos than in the fresh embryos but there were no significant differences.ConclusionThese results suggest that there were no significant differences between embryos that underwent slow freezing and vitrification.

ObjectiveThis study was performed to evaluate testicular sperm chromatin condensation using aniline blue-eosin (AB-E) staining and its effects on IVF-ET.MethodsChromatin condensation was analyzed using AB-E staining in 27 cases of testicular sperm extraction. There were 19 cases of obstructive azoospermia (OA) and 8 cases of non-obstructive azoospermia (NOA) in IVF-ET. Mature sperm heads were stained red-pink whereas immature sperm heads were stained dark blue. The percentage of sperm chromatin condensation was calculated from the ratio of the number of red-pink sperm to the total number of sperm analyzed.ResultsThe overall percentages of chromatin condensation in OA and NOA were 31.1±11.2% and 26.3±14.4%, respectively. The fertilization rate was significant higher in OA than NOA (p<0.05); however, the rates of good embryos and clinical pregnancy did not show statistical differences. In OA and NOA, statistical differences were not observed in the rate of chromatin condensation, fertilization, good embryos, and clinical pregnancy between the pregnant group and non-pregnant group.ConclusionChromatin condensation is less stable than OA and showed a low fertilization rate in NOA. While there were no significant differences in chromatin condensation results between NOA and OA, we propose that a pattern of decreased chromatin condensation in NOA is one of the factors of low fertilization results requiring further study.

Purpose To evaluate the viability of frozen embryos generated by intracytoplasmic sperm injection (ICSI) with frozen testicular spermatozoa. Methods A total of 68 fresh embryo transfer (ET) cycles and 85 subsequent frozen-thawed ET (FET) cycles were grouped according to the source of spermatozoa: fresh testicular spermatozoa (TESE) or frozen-thawed testicular spermatozoa (t-TESE). Results There were no significant differences in the age of female patients, number of oocytes, or fertilization rates in fresh ET cycles with TESE (TESE-fresh ET) versus t-TESE (t-TESE-fresh ET). The rate of embryo survival after thawing (95.7 % vs. 94.0 %) was similar in frozen ET cycles (FET) with TESE (TESE-FET) and with t-TESE (t-TESE-FET). While there were significant differences in the proportion of good quality embryos, no statistical differences were found in the pregnancy or clinical abortion rates between the two groups. Moreover, delivery rates were not significantly different. Conclusions Although the proportion of good quality embryos was affected by cryopreservation of testicular tissue, embryo survival rate was not. As well, subsequent pregnancy could be achieved successfully via t-TESE-FET cycles. Therefore, FET is not affected by the cryopreservation of testicular tissue, and avoids further oocyte retrieval and TESE procedures.