Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential.

Sometimes, for medical reasons, when a frozen tissue has already thawed, an operation by re-transplantation may be cancelled, and ovarian tissues should be re-frozen for transplantation next time. Research about the repeated cryopreservation of ovarian cells is rarely reported. It has been published that there is no difference in the follicle densities, proportions of proliferation of early preantral follicles, appearance of atretic follicles, or ultrastructural quality of frozen-thawed and re-frozen-rethawed tissue. However, the molecular mechanisms of a repeated cryopreservation effect on the developmental potential of ovarian cells are unknown. The aim of our experiments was to investigate the effect of re-freezing and re-thawing ovarian tissue on gene expression, gene function annotation, and protein-protein interactions. The morphological and biological activity of primordial, primary, and secondary follicles, aimed at using these follicles for the formation of artificial ovaries, was also detected. Second-generation mRNA sequencing technology with a high throughput and accuracy was adopted to determine the different transcriptome profiles in the cells of four groups: one-time cryopreserved (frozen and thawed) cells (Group 1), two-time cryopreserved (re-frozen and re-thawed after first cryopreservation) cells (Group 2), one-time cryopreserved (frozen and thawed) and in vitro cultured cells (Group 3), and two times cryopreserved (re-frozen and re-thawed after first cryopreservation) and in vitro cultured cells (Group 4). Some minor changes in the primordial, primary, and secondary follicles in terms of the morphology and biological activity were detected, and finally, the availability of these follicles for the formation of artificial ovaries was explored. It was established that during cryopreservation, the CEBPB/CYP19A1 pathway may be involved in regulating estrogen activity and CD44 is crucial for the development of ovarian cells. An analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not significantly affect the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

Comparative Transcriptomic Analyses for the Optimization of Thawing Regimes during Conventional Cryopreservation of Mature and Immature Human Testicular Tissue.

Cryopreservation of human testicular tissue, as a key element of anticancer therapy, includes the following stages: saturation with cryoprotectants, freezing, thawing, and removal of cryoprotectants. According to the point of view existing in "classical" cryobiology, the thawing mode is the most important consideration in the entire process of cryopreservation of any type of cells, including cells of testicular tissue. The existing postulate in cryobiology states that any frozen types of cells must be thawed as quickly as possible. The technologically maximum possible thawing temperature is 100 °C, which is used in our technology for the cryopreservation of testicular tissue. However, there are other points of view on the rate of cell thawing, according to how thawing should be carried out at physiological temperatures. In fact, there are morphological and functional differences between immature (from prepubertal patients) and mature testicular tissue. Accordingly, the question of the influence of thawing temperature on both types of tissues is relevant. The purpose of this study is to explore the transcriptomic differences of cryopreserved mature and immature testicular tissue subjected to different thawing methods by RNA sequencing. Collected and frozen testicular tissue samples were divided into four groups: quickly (in boiling water at 100 °C) thawed cryopreserved mature testicular tissue (group 1), slowly (by a physiological temperature of 37 °C) thawed mature testicular tissue (group 2), quickly thawed immature testicular tissue (group 3), and slowly thawed immature testicular tissue (group 4). Transcriptomic differences were assessed using differentially expressed genes (DEG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and protein-protein interaction (PPI) analyses. No fundamental differences in the quality of cells of mature and immature testicular tissue after cryopreservation were found. Generally, thawing of mature and immature testicular tissue was more effective at 100 °C. The greatest difference in the intensity of gene expression was observed in ribosomes of cells thawed at 100 °C in comparison with cells thawed at 37 °C. In conclusion, an elevated speed of thawing is beneficial for frozen testicular tissue.

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.

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.

Hysteroscopic management of intramural ectopic pregnancy.

The uterine myometrium is the rarest location for an ectopic pregnancy resulting in the so-called 'intramural or intramyometrial ectopic pregnancy'. It presents a particular diagnostic and therapeutic challenge for the treating physician. If passed undiagnosed can lead to life-threatening uterine rupture, which may warrant hysterectomy, leaving the woman with irreversible infertility. Different treatment modalities have been proposed for the management of this condition. In this case report, we are describing a rare case of intramural ectopic pregnancy and reporting the use of hysteroscopy for the surgical management of this case for the first time in the literature.

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.

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.

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.

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.

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 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.

Patient with ovarian insufficiency: baby born after anticancer therapy and re-transplantation of cryopreserved ovarian tissue.

BACKGROUND: The second major cause of death is cancer. In fact, the effectiveness of anticancer treatments and positive long-term prognosis for young women has increased. However, the problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic and lead to the functional death of ovaries. There is potential key solution to this problem: cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence. Data regarding cryopreservation and re-transplantation of ovarian tissue from patients with ovarian insufficiency is limited. The aim of this treatment was the re-transplantation of cryopreserved ovarian tissue after anticancer therapy of patient with ovarian insufficiency (56 IU/l FSH, 8 ng/l ß-estradiol, < 1.1 ng/ml anti-Mullerian hormone, 1 primary follicle per 10mm3). CASE PRESENTATION: After the operation, four tissue fragments (10-16 × 8-13 × 1.0-1.2 mm) were cooled to 5 °C in the freezing medium (culture medium+ 6% ethylene glycol+ 6% dimethyl sulfoxide+ 0.15 M sucrose) for 24 h, frozen and thawed. Freezing was performed in four standard 5 ml cryo-vials with ice formation at - 9 °C, cooling from - 9 to - 34 °C at a rate of - 0.3 °C/min and plunging at - 34 °C into liquid nitrogen. After thawing in a 100 °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. After thawing of one cryo-vial, part (5 mm3) of experimental ovarian tissue after 7 day in vitro culture was histological evaluated and two ovarian fragments (8 × 7 × 1.0 mm and 7 × 6 × 1.0 mm) were re-transplanted. The quantity of follicles after cryopreservation and in vitro culture was not increased (P > 0.1): it was found 1 primordial follicle in 5 mm3 of tissue. Thirty seven days after the re-transplantation of ovarian tissue, the restoration of the menstrual cycle of Patient W. was noted. Three months after the transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. CONCLUSIONS: Described protocol of conventional cryopreservation of ovarian tissue can be used for treatment of patients with ovarian insufficiency.

Increasing of malignancy of breast cancer cells after cryopreservation: molecular detection and activation of angiogenesis after CAM-xenotransplantation.

BACKGROUND: Ovarian tissue cryopreservation has a wide range of cancerous indications. Avoiding relapse becomes a specific concern that clinicians frequently encounter. The data about the comparative viability of cancer cells after cryopreservation are limited. This study aimed to evaluate the effect of cryopreservation on breast cancer cells. METHODS: We used in-vitro cultured ZR-75-1 and MDA-MB-231 cell lines. Cell samples of each lineage were distributed into the non-intervened and cryopreserved groups. The cryopreservation procedures comprised programmed slow freezing followed by thawing at 100 °C, 60 s. Biological phenotypes and the related protein markers were compared between the two groups. The EVOS FL Auto 2 Cell Image System was used to monitor cell morphology. Cell proliferation, motility, and penetration were characterized by CCK-8, wound-healing, and transmembrane assay, respectively. The expression of Ki-67, P53, GATA3, E-cadherin, Vimentin, and F-Actin was captured by immunofluorescent staining and western blotting as the proxy measurements of the related properties. The chorioallantoic membrane (CAM) xenotransplantation was conducted to explore angiogenesis induced by cancer cells. RESULTS: After 5 days in vitro culture, the cell concentration of cryopreserved and non-intervened groups was 15.7 × 104 vs. 14.4 × 104cells/ml, (ZR-75-1, p > 0.05), and 25.1 × 104 vs. 26.6 × 104 cells/ml (MDA-MB-231, p > 0.05). Some cryopreserved ZR-75-1 cells presented spindle shape with filopodia and lamellipodia and dissociated from the cell cluster after cryopreservation. Both cell lines demonstrated increased cell migrating capability and invasion after cryopreservation. The expression of Ki-67 and P53 did not differ between the cryopreserved and non-intervened groups. E-cadherin and GATA3 expression downregulated in the cryopreserved ZR-75-1 cells. Vimentin and F-actin exhibited an upregulated level in cryopreserved ZR-75-1 and MDA-MB-231 cells. The cryopreserved MDA-MB-231 cells induced significant angiogenesis around the grafts on CAM with the vascular density 0.313 ± 0.03 and 0.342 ± 0.04, compared with that of non-intervened cells of 0.238 ± 0.05 and 0.244 ± 0.03, p < 0.0001. CONCLUSIONS: Cryopreservation promotes breast cancer cells in terms of epithelial-mesenchymal transition and angiogenesis induction, thus increasing metastasis risk.