A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells

<p>Abstract</p> <p>Background</p> <p>The finding of human umbilical cord blood as one of the most likely sources of hematopoietic stem cells offers a less invasive alternative for the need of hematopoietic stem cell transplantation. Due to the once-in-a-life time chance of collecting it, an optimum cryopreservation method that can preserve the life and function of the cells contained is critically needed.</p> <p>Methods</p> <p>Until now, slow-cooling has been the routine method of cryopreservation; however, rapid-cooling offers a simple, efficient, and harmless method for preserving the life and function of the desired cells. Therefore, this study was conducted to compare the effectiveness of slow- and rapid-cooling to preserve umbilical cord blood of mononucleated cells suspected of containing hematopoietic stem cells. The parameters used in this study were differences in cell viability, malondialdehyde content, and apoptosis level. The identification of hematopoietic stem cells themselves was carried out by enumerating CD34⁺in a flow cytometer.</p> <p>Results</p> <p>Our results showed that mononucleated cell viability after rapid-cooling (91.9%) was significantly higher than that after slow-cooling (75.5%), with a <it>p </it>value = 0.003. Interestingly, the malondialdehyde level in the mononucleated cell population after rapid-cooling (56.45 μM) was also significantly higher than that after slow-cooling (33.25 μM), with a <it>p </it>value < 0.001. The apoptosis level in rapid-cooling population (5.18%) was not significantly different from that of the mononucleated cell population that underwent slow-cooling (3.81%), with a <it>p </it>value = 0.138. However, CD34⁺enumeration was much higher in the population that underwent slow-cooling (23.32 cell/μl) than in the one that underwent rapid-cooling (2.47 cell/μl), with a <it>p </it>value = 0.001.</p> <p>Conclusions</p> <p>Rapid-cooling is a potential cryopreservation method to be used to preserve the umbilical cord blood of mononucleated cells, although further optimization of the number of CD34⁺cells after rapid-cooling is critically needed.</p

Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia

The aim of the present study was to describe a simplified and inexpensive method of testicular tissue freezing to assess the cumulative clinical pregnancy rate (CPR) by this technique, and to provide useful information for counselling couple with non-obstructive azoospermia. One hundred and sixty-five couples with non-obstructive azoospermic males pursuing assisted conception, from December 1995 to December 2002, were included. In all cases, the testicular tissue retrieved by open multiple-biopsy (both sides, by testicular sperm extraction) was frozen using a simple liquid nitrogen vapour freezing technique and was stored in liquid nitrogen thereafter. Only mature spermatozoa were used for intracytoplasmic sperm injection (ICSI) after thawing. Expected CPR were calculated using the Kaplan-Meier survival analysis. A total of 281 cycles were performed resulting in 53 clinical pregnancies. Crude and expected CPR (95% confidence intervals) after three cycles were 32.1 (25.7-40.1) and 55.7% (37.0-74.4) respectively. In conclusion, this simplified method for freezing testicular tissue resulted in a satisfactory outcome after ICSI in cases of non-obstructive azoospermia

Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia

There is a lack of data regarding variables affecting the treatment outcome for non-obsructive azoospermia when spermatozoa from cryopreserved testicular specimens are utilized for ICSI. The objective of the present retrospective analysis was to investigate the effect of various parameters on treatment outcome it such cases. One hundred and sixty-five couples with non-obstructive azoospermic males undergoing a total of 297 cycles were included. In all cases the testicular tissue retrieved by multiple open-biopsy testicular sperm extraction was stored in liquid nitrogen and, after thawing, only, mature spermatozoa were used for ICSI. When no motile spermatozoa were recovered, immotile spermatoa were used. In 159 cycles, motile spermatozoa were utilized for ICSI, while in 138 cycles immotile spermatozoa were utilized. Higher normal fertilization rate (60.4. ± 3.1 versus 51.3 ± 1.6%, P < 0.05), number of emberyos transffered (2.8 ± 0.06 versus 2.6 ± 0.04, P < 0.05), modified cumulative embryo score (31.2 ± 2.6 versus 0.04, P < 0.05), modified cumulative embryo score 31.2 ± 2.6% ± spermatozoa injected (67.8 versus 49.8%, P < 0.05) were observed in cycles that resulted in clinical pregnancies. Binary logistic regression analysis showed that sperm motility (odds ratio 2.06, 95% CI 1.1-3.9, P < 0.05), but not woman's age, number of treatment cycle, type of GnRH-analogue used for pituitary suppression, number of oocytes retrieved or number of embryos transferred was a significant determinant of the likelihood of clinical pregnancy. It conclusion, sperm motility after freeze/thawing of testicular tissue is the major determinant of the success of ICSI in non-obstructive azoospermia