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Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVPfreezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 +/- 4.1% vs. 20.7 +/- 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 +/- 4.2 vs. 54.5 +/- 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 +/- 2.5% vs. 38.8 +/- 3.0%, respectively) (P 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 +/- 7.8 vs. 82.2 +/- 6.2% (P 0.1 with tendency to increasing), 90.0 +/- 4.6 vs. 69.5 +/- 5.1% (P < 0.05), and 45.4 +/- 4.1% vs. 30.9 +/- 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopre

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryopmtectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 til of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (Delta Psi m) were determined after thawing at 42 degrees C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 +/- 1.7%, 34.5 +/- 2.8% and 34.0 +/- 1.4%, respectively (P-1-2,P-3<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 +/- 2.9% and 45.5 +/- 3.9%, respectively) was higher than in Group 1 (33.3 +/- 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 +/- 3.0%) and Group 2 (47.7 +/- 4.1%) were lower than in Group 1 (52.5 +/- 4.4%; P < 0.05). Delta Psi m rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.

Cryopreservation and re-transplantation of ovarian tissue after anticancer treatment is important medical technology. Today, during a pandemic, the risk of contamination of transplanted cells with SARS-CoV-2 virus is extremely high. Data about cryo-resistance (virulence and/or infectivity) of SARS-CoV-2 are limited. Analysis and systematization of literature data allow us to draw the following conclusions: 1) The cytoplasmic membrane of somatic cell, like envelope of corona viruses, consists of lipid bilayer and this membrane, like envelope of corona virus, contains membrane proteins. Thus, we can consider the cytoplasmic membrane of an ordinary somatic cell as a model of the envelope membrane of SARS-CoV-2. It is expected that the response of the virus to cryopreservation is similar to that of a somatic cell. SARS-CoV-2 is more poor-water and more protein-rich than somatic cell, and this virus is much more cryo-resistant. 2) The exposure of somatic cells at low positive temperatures increases a viability of these cells. The safety of the virus is also in direct proportion to the decrease in temperature: the positive effect of low temperatures on SARS-CoV-2 virus has been experimentally proven. 3) Resistance of SARS-CoV-2 to cryoprotectant-free cryopreservation is extremely high. The high viability rate of SARS-CoV-2 after freezing-drying confirms its high cryo-resistance. 4) The risk of SARS-CoV-2 infection after transplantation of cryopreserved ovarian tissues that have been contaminated with this virus, increases significantly. Our own experimental data on the increase in the viability of cancer cells after cryopreservation allow us to formulate a hypothesis about increasing of viability (virulence and/or infectivity) of SARS-CoV-2 virus after cryopreservation.

Cryopreservation and re-transplantation of ovarian tissue after anticancer treatment is important medical technology. Today, during a pandemic, the risk of contamination of transplanted cells with SARS-CoV-2 virus is extremely high. Data about cryo-resistance (virulence and/or infectivity) of SARS-CoV-2 are limited. Analysis and systematization of literature data allow us to draw the following conclusions: 1) The cytoplasmic membrane of somatic cell, like envelope of corona viruses, consists of lipid bilayer and this membrane, like envelope of corona virus, contains membrane proteins. Thus, we can consider the cytoplasmic membrane of an ordinary somatic cell as a model of the envelope membrane of SARS-CoV-2. It is expected that the response of the virus to cryopreservation is similar to that of a somatic cell. SARS-CoV-2 is more poor-water and more protein-rich than somatic cell, and this virus is much more cryo-resistant. 2) The exposure of somatic cells at low positive temperatures increases a viability of these cells. The safety of the virus is also in direct proportion to the decrease in temperature: the positive effect of low temperatures on SARS-CoV-2 virus has been experimentally proven. 3) Resistance of SARS-CoV-2 to cryoprotectant-free cryopreservation is extremely high. The high viability rate of SARS-CoV-2 after freezing-drying confirms its high cryo-resistance. 4) The risk of SARS-CoV-2 infection after transplantation of cryopreserved ovarian tissues that have been contaminated with this virus, increases significantly. Our own experimental data on the increase in the viability of cancer cells after cryopreservation allow us to formulate a hypothesis about increasing of viability (virulence and/or infectivity) of SARS-CoV-2 virus after cryopreservation.

Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVPfreezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 +/- 4.1% vs. 20.7 +/- 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 +/- 4.2 vs. 54.5 +/- 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 +/- 2.5% vs. 38.8 +/- 3.0%, respectively) (P 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 +/- 7.8 vs. 82.2 +/- 6.2% (P 0.1 with tendency to increasing), 90.0 +/- 4.6 vs. 69.5 +/- 5.1% (P < 0.05), and 45.4 +/- 4.1% vs. 30.9 +/- 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopre

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryopmtectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 til of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (Delta Psi m) were determined after thawing at 42 degrees C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 +/- 1.7%, 34.5 +/- 2.8% and 34.0 +/- 1.4%, respectively (P-1-2,P-3<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 +/- 2.9% and 45.5 +/- 3.9%, respectively) was higher than in Group 1 (33.3 +/- 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 +/- 3.0%) and Group 2 (47.7 +/- 4.1%) were lower than in Group 1 (52.5 +/- 4.4%; P < 0.05). Delta Psi m rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 degrees C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 degrees C, cooling from -9 to -34 degrees C at a rate of -0.3 degrees C/min and plunging at -34 degrees C into liquid nitrogen. After thawing in a 100 degrees C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 +/- 1.9% and 96.4 +/- 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 degrees C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 degrees C, cooling from -9 to -34 degrees C at a rate of -0.3 degrees C/min and plunging at -34 degrees C into liquid nitrogen. After thawing in a 100 degrees C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 +/- 1.9% and 96.4 +/- 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectantfree vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondria] membrane potential of frozen (5% glycerol, 10 C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin + 0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full -term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectantfree vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondria] membrane potential of frozen (5% glycerol, 10 C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin + 0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full -term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.

In fact, a full sterilization of commercially-produced liquid nitrogen contaminated with different pathogens is not possible. The aim of this study was to compare the viability of human pronuclear oocytes subjected to cooling by direct submerging of open carrier in liquid nitrogen versus submerging in clean liquid air (aseptic system). One- and three-pronuclei stage embryos (n = 444) were cryopreserved by direct plunging into liquid nitrogen (vitrified) in ethylene glycol (15%), dimethylsulphoxide (15%) and 0.2M sucrose. Oocytes were exposed in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature. Then first part of oocytes (n = 225) were directly plunged into liquid nitrogen, and second part of oocytes (n = 219) into liquid air. Oocytes were thawed rapidly at a speed of 20,000 degrees C/min and then subsequently were placed into a graded series of sucrose solutions (0.5, 0.25, 0.12 and 0.06M) at 2.5 min intervals and cultured in vitro for 3 days. In both groups, the rate of high-quality embryos (Grade 6A: 6 blastomeres, no fragmentation; Grade 8A: 8 blastomeres, no fragmentation; Grade 8A compacting: 8 blastomeres, beginning of compacting) was noted. The rates of high-quality embryos developed from one-pronuclear oocytes vitrified by cooling in liquid nitrogen and liquid air were: 39.4% +/- 0.6 and 38.7% +/- 0.8, respectively (P > 0.1). These rates for three-pronuclear oocytes were: 45.8 +/- 0.8% and 52.0 +/- 0.7%, respectively (P < 0.05). In conclusion, vitrification by direct submerging of oocytes in clean liquid air (aseptic system) is a good alternative for using of not sterile liquid nitrogen.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 +/- 6 years old), sized 20-210 mm(3) were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 +/- 2.4% vs. 90.4 +/- 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 +/- 3.4% vs. 89.7 +/- 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 +/- 3.4%; Group 2: 91.8 +/- 6.0%; Group 3: 90.5 +/- 6.9%; Group 4: 87.3 +/- 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 +/- 1.6%; Group 2: 4.4 +/- 1.6%; Group 3: 1.6 +/- 1.1%; Group 4: 2.4 +/- 1.5%), late apoptotic (Group 1: 2.7 +/- 2.4%; Group 2: 44.0 +/- 1.9%; Group 3: 3.1 +/- 1.1%; Group 4: 2.8 +/- 0.7% and necrotic (Group 1: 0.9% +/- 0.1%; Group 2: 2.9 +/- 0.8%; Group 3: 3.4 +/- 4.5%; Group 4: 1.1 +/- 0.6%) cells was low and was not sig

In fact, a full sterilization of commercially-produced liquid nitrogen contaminated with different pathogens is not possible. The aim of this study was to compare the viability of human pronuclear oocytes subjected to cooling by direct submerging of open carrier in liquid nitrogen versus submerging in clean liquid air (aseptic system). One- and three-pronuclei stage embryos (n = 444) were cryopreserved by direct plunging into liquid nitrogen (vitrified) in ethylene glycol (15%), dimethylsulphoxide (15%) and 0.2M sucrose. Oocytes were exposed in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature. Then first part of oocytes (n = 225) were directly plunged into liquid nitrogen, and second part of oocytes (n = 219) into liquid air. Oocytes were thawed rapidly at a speed of 20,000 degrees C/min and then subsequently were placed into a graded series of sucrose solutions (0.5, 0.25, 0.12 and 0.06M) at 2.5 min intervals and cultured in vitro for 3 days. In both groups, the rate of high-quality embryos (Grade 6A: 6 blastomeres, no fragmentation; Grade 8A: 8 blastomeres, no fragmentation; Grade 8A compacting: 8 blastomeres, beginning of compacting) was noted. The rates of high-quality embryos developed from one-pronuclear oocytes vitrified by cooling in liquid nitrogen and liquid air were: 39.4% +/- 0.6 and 38.7% +/- 0.8, respectively (P > 0.1). These rates for three-pronuclear oocytes were: 45.8 +/- 0.8% and 52.0 +/- 0.7%, respectively (P < 0.05). In conclusion, vitrification by direct submerging of oocytes in clean liquid air (aseptic system) is a good alternative for using of not sterile liquid nitrogen.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 +/- 6 years old), sized 20-210 mm(3) were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 +/- 2.4% vs. 90.4 +/- 3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 +/- 3.4% vs. 89.7 +/- 4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 +/- 3.4%; Group 2: 91.8 +/- 6.0%; Group 3: 90.5 +/- 6.9%; Group 4: 87.3 +/- 2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 +/- 1.6%; Group 2: 4.4 +/- 1.6%; Group 3: 1.6 +/- 1.1%; Group 4: 2.4 +/- 1.5%), late apoptotic (Group 1: 2.7 +/- 2.4%; Group 2: 44.0 +/- 1.9%; Group 3: 3.1 +/- 1.1%; Group 4: 2.8 +/- 0.7% and necrotic (Group 1: 0.9% +/- 0.1%; Group 2: 2.9 +/- 0.8%; Group 3: 3.4 +/- 4.5%; Group 4: 1.1 +/- 0.6%) cells was low and was not sig

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 degrees C) and (ii) incubated at 37 degrees C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (AWM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and Delta Psi M compared to the control, while the incubation at 37 degrees C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and Delta Psi M than at 37 degrees C.

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 degrees C) and (ii) incubated at 37 degrees C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (AWM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and Delta Psi M compared to the control, while the incubation at 37 degrees C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and Delta Psi M than at 37 degrees C.