Correction: Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0129108.].

Cryoprotectant-free vitrification of spermatozoa: Fish as a model of human.

Post-thawing motility of spermatozoon, which is directly correlated with the integrity of mitochondrion, is the main parameter for evaluation of respective cryopreservation treatments. In this review, we describe our model of mitochondrial apparatus of spermatozoa and behaviour of this apparatus during cryopreservation. This model shows why a priori the mitochondrial apparatus of the human spermatozoon is expected to be more cryo-stable than the mitochondrial apparatus of the fish spermatozoon. Negative changes of mitochondrial membrane potential are a good indicator of the functional normality of mammalian and fish spermatozoa. It is concluded that the cryostability of mitochondrial membranes of fish spermatozoa is lower than that of human spermatozoa, and protocols for effective cryopreservation of fish spermatozoa can be extrapolated to human spermatozoa. It is also provided a biological explanation for why cryoprotectant-free vitrification for human ejaculates is better than conventional freezing and vitrification with cryoprotectants. This review also includes a description of the various technologies of vitrification of human and fish spermatozoa. For cryobiological investigations, we propose to evaluate the fish spermatozoon as a suitable representative model of the human spermatozoon.

Vitrification of human pronuclear oocytes by direct plunging into cooling agent: Non sterile liquid nitrogen vs. sterile liquid air.

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 °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.

"Naturalization" of Routine Assisted Reproductive Technologies by In Vitro Culture of Embryos with Microvibration: Sex Ratio, Body Length, and Weight of 2,456 Live-Birth Deliveries after Transfer of 9,624 Embryos In Vitro Cultured in Static System and with Microvibration.

Aim was to determine whether there is any difference in the sex ratio, body length, and body weight of 2,456 deliveries after transfer of 9,624 embryos derived using in vitro culture under static and mechanical microvibration conditions. Pronuclear embryos from 4435 patients were cultured in vitro under two different conditions: without (n = 4821) and with mechanical agitation (n = 4803). Sex ratio, body length, and weight of 2,456 live-birth deliveries after transfer of 9,624 embryos were noted. The proportion of males at birth was significantly associated with mode of in vitro culture of embryos only among women aged 40 years and older. The rate "body length" was significantly associated with mode of in vitro culture of embryos only among women aged 29 and younger. In the same time, among twins, this ratio positively associated with in vitro culture of embryos under microvibration only among women aged 30-34 years as well as ≥40 years and negatively among women aged 35-39 years. It was concluded that birth weight of infants was positively associated with mode of in vitro culture of embryos under microvibration among women of all age groups. This trial registration number is ISRCTN13773904, registered 6 April 2016.

In Vitro Microvibration Increases Implantation Rate After Embryonic Cell Transplantation.

In natural conditions the oocyte and embryo are subjected to ever-changing dynamic processes. However, the routine assisted reproductive technologies today involve the use of static in vitro culture systems. The objective was to determine whether there is any difference in the viability of embryos after in vitro culture under static and mechanical microvibration conditions. The viability of embryonic cells (9,624 embryos) generated from 4,436 couples after in vitro culture was evaluated. For groups ≤29, 30-34, 35-39, and ≥40 years, the following rates of high-quality embryos without fragmentation (two to four blastomeres on day 2; six to eight blastomeres and compacting morula on day 3; blastocyst, expanded and hatching blastocyst on day 5) were detected (static vs. vibration, respectively): 65% versus 71%, 44% versus 69%, 67% versus 76% (for statistically significant differences between respective rates in these three groups, p < 0.05), and 67% versus 66% (p > 0.1). The following baby-take-home rates were determined for groups ≤29, 30-34, 35-39, and ≥40 years (static vs. vibration, respectively): 30% versus 31% (p > 0.1, increasing only on the level of tendency), 28% versus 37%, 23% versus 29%, and 9% versus 15% (differences between respective rates in these three groups with p < 0.05). It was concluded that in vitro culture of embryos under microvibration (with a mimic of conditions in nature whereby oviductal fluid is mechanically agitated by the epithelial cilia) significantly increases the baby-take-home rate for patients 30 years and older.

Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate.

BACKGROUND: Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments. METHODS: Ovarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5-3.0 × 1.5-3.0 × 0.5-0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5-3.0 × 1.5-3.0 × 1.5-2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). RESULTS: For Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative). CONCLUSION: The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

OBJECTIVES: To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. MATERIALS AND METHODS: Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). RESULTS: For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). CONCLUSIONS: Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.

Rapid evaluation of soluble HLA-G levels in supernatants of in vitro fertilized embryos.

Human leukocyte antigen G (HLA-G) molecules are crucial for the maternal tolerance against the fetus during pregnancy. Thus, the presence of soluble HLA-G (sHLA-G) in embryo cultures is thought to be correlated to a successful pregnancy after assisted reproductive techniques (ART). Here, we established a rapid detection assay based on Luminex technology, which can be integrated into ART proceedings, allowing sHLA-G quantification in sample volumes of only 10 microl within 1.5 hours. Using this method, sHLA-G levels of 526 single-embryo cultures, 47 two-embryo cultures, and 15 three-embryo cultures were analyzed corresponding to 313 ART cycles. In 117 embryo cultures, sHLA-G was detectable. In single-embryo cultures, the sHLA-G levels were positively correlated to embryo quality (p = 0.048, r = 0.20, n = 100). The presence of sHLA-G in embryo cultures was significantly (p < 0.0001) associated with clinical pregnancy after intracytoplasmatic sperm injections (ICSI), especially in couples with male factor infertility, but not after in vitro fertilization (IVF) or in couples with female infertility. Importantly, in sHLA-G negative embryos, the abortion rate was increased threefold (p = 0.04). In conclusion, the results obtained by our novel method support strongly the diagnostic relevance of sHLA-G for predicting pregnancy outcome after ART. The ultimate conditions for this prediction have to be further investigated in a multicenter study.