FeTPPS, a Peroxynitrite Decomposition Catalyst, Ameliorates Nitrosative Stress in Human Spermatozoa.

Excessive levels of reactive nitrogen species (RNS), such as peroxynitrite, promote nitrosative stress, which is an important cause of impaired sperm function. The metalloporphyrin FeTPPS is highly effective in catalyzing the decomposition of peroxynitrite, reducing its toxic effects in vivo and in vitro. FeTPPS has significant therapeutic potential in peroxynitrite-related diseases; however, its effects on human spermatozoa under nitrosative stress have not been described. This work aimed to evaluate the in vitro effect of FeTPPS against peroxynitrite-mediated nitrosative stress in human spermatozoa. For this purpose, spermatozoa from normozoospermic donors were exposed to 3-morpholinosydnonimine, a molecule that generates peroxynitrite. First, the FeTPPS-mediated peroxynitrite decomposition catalysis was analyzed. Then, its individual effect on sperm quality parameters was evaluated. Finally, the effect of FeTPPS on ATP levels, motility, mitochondrial membrane potential, thiol oxidation, viability, and DNA fragmentation was analyzed in spermatozoa under nitrosative stress conditions. The results showed that FeTPPS effectively catalyzes the decomposition of peroxynitrite without affecting sperm viability at concentrations up to 50 µmol/L. Furthermore, FeTPPS mitigates the deleterious effects of nitrosative stress on all sperm parameters analyzed. These results highlight the therapeutic potential of FeTPPS in reducing the negative impact of nitrosative stress in semen samples with high RNS levels.

Biallelic mutations in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans and mice.

BACKGROUND: Asthenozoospermia is a major factor contributing to male infertility. The mitochondrial sheath (MS), an important organelle in the midpiece of spermatozoa, is crucial to sperm motility. ARMC12 is a mitochondrial peripheral membrane protein. Deletion of Armc12 impairs the arrangement of MS and causes infertility in mice. However, the role of ARMC12 in human asthenozoospermia remains unknown. OBJECTIVE: To study the genetic defects in patients with asthenozoospermia. METHODS: A total of 125 patients with asthenozoospermia and 120 men with proven fertility were recruited. Whole-exome sequencing and Sanger sequencing were performed for genetic analysis. Papanicolaou staining, HE staining, immunofluorescent staining, transmission electron microscopy and field emission scanning electron microscopy were employed to observe the morphological and structural defects of the spermatozoa and testes. Armc12-knockout mice were generated using the CRISPR-Cas9 system. Intracytoplasmic sperm injection was used to treat the patients. RESULTS: Biallelic ARMC12 mutations were identified in three patients, including homozygous mutations in two siblings from a consanguineous family and compound heterozygous mutations in one sporadic patient. ARMC12 is mainly expressed in the midpiece of elongated and late spermatids in the human testis. The patients' spermatozoa displayed multiple midpiece defects, including absent MS and central pair, scattered or forked axoneme and incomplete plasma membrane. Spermatozoa from Armc12-/- mice showed parallel defects in the midpiece. Moreover, two patients were treated with intracytoplasmic sperm injection and achieved good outcomes. CONCLUSION: Our findings prove for the first time that defects in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans.

Automatic Evaluation for Bioengineering of Human Artificial Ovary: A Model for Fertility Preservation for Prepubertal Female Patients with a Malignant Tumor.

INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects.

BACKGROUND: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. METHODS: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. RESULTS: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5' splice sites (A5SS), alternative 3' splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). CONCLUSIONS: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.

Artificial Ovary for Young Female Breast Cancer Patients.

In recent decades, there has been increasing attention toward the quality of life of breast cancer (BC) survivors. Meeting the growing expectations of fertility preservation and the generation of biological offspring remains a great challenge for these patients. Conventional strategies for fertility preservation such as oocyte and embryo cryopreservation are not suitable for prepubertal cancer patients or in patients who need immediate cancer therapy. Ovarian tissue cryopreservation (OTC) before anticancer therapy and autotransplantation is an alternative option for these specific indications but has a risk of retransplantation malignant cells. An emerging strategy to resolve these issues is by constructing an artificial ovary combined with stem cells, which can support follicle proliferation and ensure sex hormone secretion. This promising technique can meet both demands of improving the quality of life and meanwhile fulfilling their expectation of biological offspring without the risk of cancer recurrence.

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling.

INTRODUCTION: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. METHODS: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. RESULTS: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. CONCLUSION: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.

Cryo-banking of human spermatozoa by aseptic cryoprotectants-free vitrification in liquid air: Positive effect of elevated warming temperature.

Cryoprotectant-free vitrification is a common method for spermatozoa cryopreservation by direct plunging into liquid nitrogen. However, the commercial liquid nitrogen could be potentially contaminated by microorganisms. Warming temperature plays an essential role for quality of human spermatozoa after vitrification. This study aimed to evaluate comparatively a quality spermatozoa after vitrification in liquid nitrogen and clean liquid air as well as with two warming rates: at 42 °C and 45 °C. After performing of routine swim-up of normozoospermia samples, spermatozoa from the same ejaculate were divided into two groups: vitrified in liquid nitrogen (LN) and sterile liquid air (LA). Spermatozoa of LN group were warmed at 42 °C, and spermatozoa of LA groups were divided and warmed at 42 °C (LA42) and 45 °C (LA45). Then spermatozoa motility, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), reactive nitrogen species (RNS), and viability were assessed. It was no found significant differences in quality of spermatozoa from LN and LA groups in the motility, ROS, MMP, RNS rates after warming at 42 °C. A tendency to obtain better spermatozoa quality was found with using of warming by 42 °C in comparison with 45 °C. It was concluded that cryoprotectant-free vitrification by direct dropping of human spermatozoa into clean liquid air can be used as an alternative to cooling in liquid nitrogen. Warming of spermatozoa at 42 °C allows to preserve the spermatozoa physiological parameters.

Construction and cryopreservation of an artificial ovary in cancer patients as an element of cancer therapy and a promising approach to fertility restoration.

The proportion of cancer patients that survive is increasing because of improvements in cancer therapy. However, some cancer treatments, such as chemo- and radio-therapies, can cause considerable damage to reproductive function. The issue of fertility is paramount for women of childbearing age once they are cured from cancer. For those patients with prepubertal or haematogenous cancer, the possibilities of conventional fertility treatments, such as oocyte or embryo cryopreservation and transplantation, are limited. Moreover, ovarian tissue cryopreservation as an alternative to fertility preservation has limitations, with a risk of re-implanting malignant cells in patients who have recovered from potentially fatal malignant disease. One possible way to restore fertility in these patients is to mimic artificially the function of the natural organ, the ovary, by grafting isolated follicles embedded in a biological scaffold to their native environment. Construction and cryopreservation of an artificial ovary might offer a safer alternative option to restore fertility for those who cannot benefit from traditional fertility preservation techniques. This review considers the protocols for constructing an artificial ovary, summarises advances in the field with potential clinical application, and discusses future trends for cryopreservation of these artificial constructions.

Optimization of Follicle Isolation for Bioengineering of Human Artificial Ovary.

Background: A functional artificial ovary is a promising strategy to recover fertility and restore endocrine function in cancer patients. The aim of this study is to optimize the follicle isolation protocol for cryopreserved human ovarian tissues. Methods: Each of the cryopreserved human ovarian cortex pieces (OCPs) from 10 patients was cut into two equal parts and randomly distributed into two treatment groups. Group 1: OCPs digested with Tumor Dissociation Enzyme (TDE); Group 2: OCPs digested with Liberase Dispase High (DH). The efficiency of both groups were evaluated in terms of yield, viability, morphology, and a short-term in vitro culture (IVC) in alginate scaffolds. Results: The TDE can isolate more primordial follicles and smaller diameter of follicles than Liberase DH. The TDE also enabled the isolation of more bright red follicles, higher percent of viable follicles, more morphologically normal follicles, and lower oxidative stress levels compared with Liberase DH. After eight days of IVC, follicles in the TDE group had a higher growth rate from Day 0 to Day 8, and higher viability on Day 8 than the Liberase DH Group. Conclusion: The TDE can be considered an alternative to Liberase DH, enables the isolation of a higher number of healthy follicles from human OCPs, and improves follicle survival after IVC in contrast to Liberase DH.

Influence of Risk Factors for Male Infertility on Sperm Protein Composition.

Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.

New method for cryoprotectant-free freezing of human oligoasthenoteratozoospremic spermatozoa with high-molecular polymer.

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) (PVP-freezing). 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 cryopreservation of human oligoasthenoteratozoospremic spermatozoa.

High cryo-resistance of SARS-CoV-2 virus: Increased risk of re-contamination at transplantation of cryopreserved ovarian tissue after COVID-19 pandemic.

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.

Pathogenic Variants in ACTRT1 Cause Acephalic Spermatozoa Syndrome.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia, but its pathogenesis is largely unknown. Here, we performed whole-exome sequencing for 34 patients with acephalic spermatozoa syndrome and identified pathogenic variants in the X-linked gene, ACTRT1, in two patients. Sanger sequencing confirmed the pathogenic variants of ACTRT1 in the patients. Both pathogenic variants of ACTRT1 were highly conserved, and in silico analysis revealed that they were deleterious and rare. Actrt1-knockout mice exhibited a similar acephalic spermatozoa phenotype. Therefore, we speculated that mutations in ACTRT1 account for acephalic spermatozoa syndrome. Moreover, the patients in this study conceived their children through artificial insemination. This study provides further insights for clinicians and researchers regarding the genetic etiology and therapeutic strategies for acephalic spermatozoa patients with pathogenic variants in ACTRT1.

Unraveling Subcellular and Ultrastructural Changes During Vitrification of Human Spermatozoa: Effect of a Mitochondria-Targeted Antioxidant and a Permeable Cryoprotectant.

Mitochondria-targeted antioxidants have great potential to counterbalance the generated reactive oxygen species (ROS) because they cross the inner membrane of the mitochondria. Still, their use was not reported in vitrified human spermatozoa. Our laboratory has successfully vitrified spermatozoa without the use of permeable cryoprotectants, but subcellular-level evidence was missing. Therefore, this study aimed to improve spermatozoa vitrification using a mitochondria-targeted antioxidant (mitoquinone, MitoQ), reveal ultrastructural changes in the spermatozoa due to the use of a permeable cryoprotectant, and report alterations of functional proteins during the spermatozoa vitrification process. For this, each of 20 swim-up-prepared ejaculates was divided into seven aliquots and diluted with a vitrification medium supplemented with varying concentrations of MitoQ (0.02 and 0.2 µM), glycerol (1, 4, and 6%), and a combination of MitoQ and glycerol. All aliquots were vitrified by the aseptic capillary method developed in our laboratory. The spermatozoa function assays revealed that the addition of either MitoQ (0.02 µM), glycerol (1%), or a combination of MitoQ (0.02 µM) and glycerol (1%) in the vitrification medium results in better or equivalent spermatozoa quality relative to the control. Transmission electron microscopy revealed that MitoQ protects the spermatozoa from undergoing ultrastructural alterations, but glycerol induced ultrastructural alterations during the vitrification process. Next, we performed label-free quantitative proteomics and identified 1,759 proteins, of which 69, 60, 90, and 81 were altered in the basal medium, 0.02 µM MitoQ, 1% glycerol, and Mito-glycerol groups, respectively. Actin, tubulins, and outer dense fiber proteins were not affected during the vitrification process. Some of the identified ubiquitinating enzymes were affected during spermatozoa vitrification. Only a few proteins responsible for phosphorylation were altered during vitrification. Similarly, several proteins involved in spermatozoa-egg fusion and fertilization (IZUMO1 and Tektin) were not affected during the vitrification process. In conclusion, MitoQ attenuates the vitrification-induced ultrastructural changes and alterations in the key proteins involved in spermatozoa functions and fertilization.

Biallelic mutations in KATNAL2 cause male infertility due to oligo-astheno-teratozoospermia.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, and most of idiopathic OAT patients are thought to be caused by genetic defects. Here, we recruited 38 primary infertile patients with the OAT phenotype and 40 adult men with proven fertility for genetic analysis and identified biallelic mutations of KATNAL2 by whole-exome sequencing in two cases. F013/II:1, from a consanguineous family, carried the KATNAL2 c.328C > T:p.Arg110X homozygous mutations. The other carried c.55A > G: p.Lys19Glu and c.169C > T: p Arg57Trp biallelic mutations. None of the KATNAL2 variants were found in the 40 adult men with proven fertility. The spermatozoa from patients with KATNAL2 biallelic mutations exhibited conspicuous defects in maturation, head morphology, and the structure of mitochondrial sheaths and flagella. KATNAL2 was mainly expressed in the pericentriolar material and mitochondrial sheath of the spermatozoa from control subjects, but it was undetectable in the spermatozoa from the patients. Furthermore, Katnal2 null male mice were infertile and displayed an OAT phenotype. Our results proved that the biallelic mutations in KATNAL2 cause male infertility and OAT in humans for the first time, to our knowledge, which could enrich the genetic defect spectrum of OAT and be beneficial for its accurate genetic screening and clinical diagnosis.

Emergency Fertility Preservation in a Young Woman With Non-Hodgkin Lymphoma.

A nulliparous woman, age 25 years, had received a diagnosis of non-Hodgkin lymphoma (NHL) and now presented with stage IIA diffuse large B-cell lymphoma (DLBCL). According to her hematological oncologist's treatment plan, chemotherapy had to start immediately (within 1 week), with the patient receiving 6 courses of the standard R-CHOEP21 regimen (rituximab 375 mg/m², cyclophosphamide 750 mg/m², hydroxydaunorubicin 50 mg/m², vincristine 1.4 mg/m², etoposide 100 mg/m², prednisone 40 mg/m²). Due to potential risks of chemotherapy-induced gonadotoxicity and subsequent iatrogenic premature ovarian failure (POF) and fertility loss, the patient was referred to the reproductive medicine department for fertility preservation counseling and further management.

In vitro activation of cryopreserved ovarian tissue: A single-arm meta-analysis and systematic review.

OBJECTIVE: Primordial follicles in premature ovarian failure (POF) patients are very difficult to be activated spontaneously, so that mature oocytes are difficult to be obtained for in vitro fertilization. The aim of our review is to analyze and to systematize the published data regarding effectiveness of different strategies for in vitro activation of cryopreserved ovarian tissue. STUDY DESIGN: According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a review of the literature was performed for all relevant full-text articles published in PubMed in English. Meta-analysis conducted using STATA 14.0. The random-effects model was used to combine 8 study results because the examination of heterogeneity was minimal. RESULTS: One hundred and seventy seven patients after in vitro activation treatment (IVA) of ovarian tissue had accumulatively 26 pregnancies through IVF or natural pregnancy and then produced 18 live births. The random-effects model showed that the total clinical pregnancy and baby born rates reported in 8 studies evidence about effectiveness of IVA. CONCLUSION: In vitro activation of primordial follicles as a new potential treatment for ovarian disorder patients, can be a promising option for fertility preservation. Drug-free activation of ovarian tissue in comparison with drug-included activation seemed to be more efficient.

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation.

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n = 16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n = 16) 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. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

Aseptic capillary vitrification of human spermatozoa: Cryoprotectant-free vs. cryoprotectant-included technologies.

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 cryoprotectant-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 µl 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 (ΔΨm) were determined after thawing at 42 °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 (P1-2,3<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). ΔΨm 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.

Aseptic Cryoprotectant-Free Vitrification of Human Spermatozoa by Direct Dropping into a Cooling Agent.

Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice crystallization and the detrimental effects of high salt concentrations. One of the problems with this technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid air with the same temperature as liquid nitrogen (-195.7 °C). This has been used to develop aseptic technology for cryoprotectant-free vitrification of human spermatozoa.