Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue.

Recently, there has been increasing emphasis on the gonadotoxic effects of cancer therapy in prepubertal boys. As advances in oncology treatments continue to enhance survival rates for prepubertal boys, the need for preserving their functional testicular tissue for future reproduction becomes increasingly vital. Therefore, we explore cutting-edge strategies in fertility preservation, focusing on the cryopreservation and transplantation of immature testicular tissue as a promising avenue. The evolution of cryopreservation techniques, from controlled slow freezing to more recent advancements in vitrification, with an assessment of their strengths and limitations was exhibited. Detailed analysis of cryoprotectants, exposure times, and protocols underscores their impact on immature testicular tissue viability. In transplantation strategy, studies have revealed that the scrotal site may be the preferred location for immature testicular tissue grafting in both autotransplantation and xenotransplantation scenarios. Moreover, the use of biomaterial scaffolds during graft transplantation has shown promise in enhancing graft survival and stimulating spermatogenesis in immature testicular tissue over time. This comprehensive review provides a holistic approach to optimize the preservation strategy of human immature testicular tissue in the future.

First report on successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe.

RESEARCH QUESTION: Cryopreservation of ovarian tissue is one feasible option to preserve female fertility prior to cancer treatment. The slow freezing protocol represents the current standard approach, while vitrification has been suggested as a promising alternative. This paper reports the follow-up and first successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe. DESIGN: After the patient received a diagnosis of breast cancer, ovarian tissue was removed laparoscopically and sent via overnight transportation to University Hospital Bonn for vitrification on site. The patient was treated with chemotherapy, leading to ovarian failure. After 2 years, retransplantation of the vitrified, rapid warmed tissue was conducted on site. RESULTS: Two months after grafting, the patient reported regular menstrual cycles. After 1 further month a clinical pregnancy occurred, which ended in a spontaneous abortion at the 8th week of pregnancy. Six months after grafting, another naturally conceived pregnancy was determined, resulting in the birth of a healthy boy 14 months after retransplantation of the ovarian tissue. CONCLUSIONS: Complementing the successful deliveries reported by the groups of Suzuki (Japan) and Silber (USA) regarding vitrified tissue, the current results confirm the high potential of this cryopreservation method in a clinical routine setting as an alternative approach to the widespread slow freezing method.

The application of mean number of DNA breakpoints in sperm cryopreservation.

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17 × 105) and conventional slow freezing (10.80 × 105), compared to the DNA breakpoints of fresh semen samples (5.19 × 105). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

Comprehensive characterisation and cryopreservation optimisation of buffalo (Bubalus bubalis) adipose tissue-derived mesenchymal stem cells.

Over half of the world's buffalo (Bubalus bubalis) inhabit India, and buffaloes frequently encounter health challenges that resist conventional treatments, prompting the exploration of alternative therapeutic strategies. One promising approach is stem cell therapy, particularly multipotent mesenchymal/stromal stem cells (MSCs). These cells have shown significant efficacy in addressing various diseases in livestock that exhibit resistance to conventional therapies. Adipose tissue-derived MSCs (ADSCs) have garnered attention due to their accessibility and robust expansion potential. The current study comprehensively characterises buffalo ADSCs (bADSCs), confirming their identity as MSCs capable of differentiating into diverse cell lineages-the identified characteristics position bADSCs as promising candidates for applications in regenerative medicine, applicable in veterinary contexts. Notably, the study established that a cryoprotective solution comprising 10 % dimethyl sulfoxide and 90 % fetal bovine serum is optimal for preserving bADSCs. This cryoprotective solution maintains vital parameters, including viability, apoptosis, senescence, cell adherence, adherent cell viability, metabolic and clonogenic efficiency, and the activity of reactive oxygen species and trilineage differentiation potential following thawing. These findings lay the foundation for developing a cryo-banking system for bADSCs. Subsequent research efforts are focused on exploring the therapeutic potential of bADSCs in specific disease models and clinical settings. The outcomes of such investigations may pave the way for innovative and effective treatments, further enhancing our understanding of the regenerative capabilities of bADSCs.

Is post-hypertonic lysis of human red blood cells caused by excessive cell volume regulation?

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

Comparison of liquid nitrogen-free slow freezing protocols toward enabling a practical option for centralized cryobanking of ovarian tissue.

Geographically distributed ovarian tissue cryobanks remain limited due to the high facility and staff costs, and cold transportation to centers is associated with ischemia-induced tissue damage that increases with transport distance. It is ideal to perform the cryopreservation procedure at a tissue removal site or local hospital before shipment to cost-effective centralized cryobanks. However, conventional liquid nitrogen-based freezers are not portable and require expensive infrastructure. To study the possibility of an ovarian tissue cryopreservation network not dependent on liquid nitrogen, we cryopreserved bovine ovarian tissue using three cooling techniques: a controlled rate freezer using liquid nitrogen, a liquid nitrogen-free controlled rate freezer, and liquid nitrogen-free passive cooling. Upon thawing, we evaluated a panel of viability metrics in frozen and fresh groups to examine the potency of the portable liquid nitrogen-free controlled and uncontrolled rate freezers in preserving the ovarian tissue compared to the non-portable conventional controlled rate freezer. We found similar outcomes for reactive oxygen species (ROS), total antioxidant capacity (TAC), follicular morphology, tissue viability, and fibrosis in the controlled rate freezer groups. However, passive slow cooling was associated with the lowest tissue viability, follicle morphology, and TAC, and the highest tissue fibrosis and ROS levels compared to all other groups. A stronger correlation was found between follicle morphology, ovarian tissue viability, and fibrosis with the TAC/ROS ratio compared to ROS and TAC alone. The current study undergirds the possibility of centralized cryobanks using a controlled rate liquid nitrogen-free freezer to prevent ischemia-induced damage during ovarian tissue shipment.

Carnosine supplementation in cryopreservation solution improved frozen-thawed bovine embryo viability.

Cryopreservation adversely affects embryo quality and viability in vitro.We investigated the effects of cryopreservation solutions supplemented with the antioxidant carnosine on frozen-thawed bovine embryo viability. Bovine blastocysts were produced in vitro and cryopreserved using slow freezing. The rates of re-expanded and hatched blastocysts in the 50 µg/ml carnosine-supplemented group at 4, 24, and 48 h after thawing were higher than those in the control (P< 0.05) group. In frozen-thawed embryos, cryopreservation solution supplemented with carnosine (50 µg/ml) significantly reduced reactive oxygen species (ROS) production(P < 0.05), decreased TUNEL-positive apoptotic cells (P< 0.05), and increased the mRNA expression of BCL2 (P< 0.05), an apoptosis suppressor gene. The expression of translocase of outer mitochondrial membrane 20 (TOMM20), which is involved in protein mitochondrial transport, in the carnosine (50 µg/ml)-treated embryos was significantly higher than that in the control group (P < 0.05). ATP production in frozen-thawed embryos in the 50 µg/ml carnosine-supplemented group was significantly higher than that in the control group (P< 0.05), however no significant difference in the total number of cells per embryo among the groups was observed. These results suggest that supplementing the cryopreservation solution with carnosine can improve the viability of frozen-thawed bovine embryos by reducing oxidative damage.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Sperm Cryopreservation Today: Approaches, Efficiency, and Pitfalls.

The cryopreservation of human spermatozoa has been an option for patients undergoing chemo or radiotherapies since the late 1950s. Presently, there are different techniques for the cryopreservation of spermatozoa. The most commonly used techniques are programmable slow freezing and freezing on liquid nitrogen vapors, while the use of vitrification is still not accepted as clinically relevant. Although there have been many improvements, the ideal technique for achieving better post-thaw sperm quality continues to be a mystery. A major obstacle during cryopreservation is the formation of intracellular ice crystals. Cryodamage generated by cryopreservation causes structural and molecular alterations in spermatozoa. Injuries can happen because of oxidative stress, temperature stress, and osmotic stress, which then result in changes in the plasma membrane fluidity, motility, viability, and DNA integrity of the spermatozoa. To prevent cryodamage as much as possible, cryoprotectants are added, and in some clinical trial cases, even antioxidants that may improve post-thaw sperm quality are added. This review discusses cryopreservation techniques, cryodamage on molecular and structural levels, and cryoprotectants. It provides a comparison of cryopreservation techniques and describes recent advances in those techniques.

Sperm cryopreservation does not affect live birth rate in normozoospermic men: analysis of 7969 oocyte donation cycles.

STUDY QUESTION: Does sperm cryopreservation influence the reproductive outcomes of normozoospermic patients in oocyte donation cycles? SUMMARY ANSWER: After controlling for confounders, the use of cryopreserved semen from normozoospermic patients does not affect pregnancy and live birth rates after elective ICSI. WHAT IS KNOWN ALREADY: Sperm cryopreservation by slow freezing is a common practice in ART. While frozen-thawed semen typically presents reduced motility and vitality, its use for ICSI is generally considered adequate in terms of reproductive outcomes. Nevertheless, most studies comparing reproductive outcomes between fresh and cryopreserved sperm include patients with severe male factor (testicular sperm, oligo-, and/or asthenozoospermia) or women of advanced maternal age, where the altered quality of the gametes can partially mask the full effect of freezing/thawing. STUDY DESIGN, SIZE, DURATION: The study included a retrospective cohort of 7969 couples undergoing their first oocyte donation cycle between January 2013 and December 2019 in one large clinic, using normozoospermic semen from the male partner. All cycles involved elective ICSI, fresh oocytes, and a fresh embryo transfer, either at cleavage or blastocyst stage. Two study groups were established based on the sperm status: fresh (n = 2865) and cryopreserved (n = 5104). PARTICIPANTS/MATERIALS, SETTING, METHODS: A slow freezing protocol was used for all sperm cryopreservation. Sperm washing, capacitation, and selection prior to ICSI were performed identically for fresh and frozen-thawed samples, using pellet swim-up. Fertilization rate (FR), pregnancy (biochemical and ongoing), and live birth rates were compared between study groups using univariate and multivariate regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Male and female age, sperm concentration and motility after ejaculation, and number of oocytes inseminated were similar between cycles using fresh or cryopreserved sperm. Analysis by Student's t-test did not indicate a significant difference in FR between fresh and cryopreserved sperm (P = 0.0591); however, after adjusting for confounders, this difference reached statistical significance: 74.65% FR for fresh (CI 95%: 73.92-75.38) versus 73.66% for cryopreserved sperm (CI 95%: 73.11-74.20), P = 0.0334. The adjusted regression analysis revealed higher odds of biochemical pregnancy when using fresh sperm (odds ratio (OR): 1.143, P = 0.0175), but no significant effects of sperm cryopreservation were observed for ongoing pregnancy (OR: 1.101, P = 0.0983) and live birth (OR: 1.082, P = 0.1805). LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when extrapolating these results to different protocols for sperm cryopreservation and selection, or to IVM, advanced maternal age and classical IVF cycles, which were excluded from analysis. Owing to the retrospective nature of the study, some uncontrolled for variables may affect the results. WIDER IMPLICATIONS OF THE FINDINGS: Sperm cryopreservation does not affect pregnancy and live birth rates in normozoospermic patients, and although it may lower FR s slightly, this would not be clinically relevant. In line with previous studies that included patients with an apparent male or female factor, sperm cryopreservation is a safe and convenient technique. STUDY FUNDING/COMPETING INTEREST(S): The study received no external funding and all authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant.

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in "pearls" and in "mini straws" using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

Ultrastructure of human ovarian tissues and risk of cancer cells re-implantation after transplantation to chick embryo chorioallantois membrane (CAM) following vitrification or slow freezing.

Ovarian follicle depletion and premature ovarian failure are significant challenges in cancer patients subjected to radio- or chemotherapy. Ovarian tissue (OT) cryopreservation would be an option when other fertility preservation methods are not accessible. This study aimed to analyze the structure and ultrastructure of human OTs transplanted onto chick embryo chorioallantois membrane (CAM) after cryopreservation by vitrification or slow freezing. OTs from 10 cancer patients underwent cryopreservation. CAM transplantation was done on fresh and cryopreserved OTs, to assign samples to nine study groups as follows: 1) FI-FIII = fresh, 5- and 10-days post-CAM transplantation groups; 2) VI-VIII = vitrified, 5- and 10-days post-transplantation vitrified groups; 3) SFI-SFIII: slow frozen, 5- and 10-days post-transplantation slow freezing groups. Proliferation ability, folliculogenesis, and structural and ultrastructure were analyzed. The density of primordial follicles did not change after both freezing methods, but reduced after 5 (P ≥ 0.05) and 10 days (P ≤ 0.05) post-CAM transplantation. The follicular grade significantly decreased in all transplanted tissues (P ≤ 0.0). The proliferation marker increased after cryopreservation, but reduced after transplantation (P ≤ 0.05). TEM evaluation showed better follicular ultrastructure in the fresh group, after transplantation. Stromal ultrastructure appeared more preserved after vitrification compared with slow freezing. There was no sign of malignant cell contamination after transplantation. Some follicular TEM abnormalities were found in both methods of freezing, with a better transplantation rate after vitrification. Also, enhanced follicular activation resulted in faster follicular depletion in this method. The information regarding post grafting events would improve our knowledge for longer OTs' lifespans.

Use of fine capillaries for cryopreservation of Caenorhabditis elegans by vitrification.

Caenorhabditis elegans is an exceptional model organism. More than twenty thousand different strains have been developed, increasing knowledge on countless topics. However, the traditional method to cryopreserve this nematode, based on slow freezing, usually reaches recovery rates of around 35% for the L1 and L2 larval stages. Here, we propose two alternative methods to cryopreserve this nematode based on vitrification that are applicable in common laboratories and allow the selective individual cryopreservation of this organism. These new methods require ultra-high warming rates, which are achieved by employing very thin capillaries as the nematode container, and a very low final concentration of cryoprotectants, which, as compared to slow freezing, reduce toxicity damage. The recovery rate was 98.5% for larvae (L1 - L4) and 84.3% for adults. Given these results, our procedures offer an alternative to cryopreserve this nematode (larvae and adults) with higher recovery rates, avoiding expensive requirements. Indeed, it only needed a container with liquid nitrogen and a warming bath for water at 37 °C. The high performance of this approach has been revealed by preserving the long-term memory and, probably, the connectome of this nematode.

Review of human oocyte cryopreservation in ART programs: Current challenges and opportunities.

Oocyte cryopreservation has notably increased in recent times, to become an essential part of clinical infertility treatment. Since the 1980s, many improvements in oocyte cryopreservation (OC) have been adopted, including the great advance with the application of vitrification. The commonly used vitrification protocol applies different cryoprotectants (Ethylene glycol and/or DMSO and/or PROH and sucrose and/or Trehalose) and two different steps: firstly, exposure in equilibration solution for 5-15 min, followed by a vitrification solution for 60-90 s at room temperature. The warming method includes a first step for 1 min at 37 °C and 3 subsequent steps at room temperature to remove the cryoprotectant for a total of 9-12 min. In addition, biosafety is a critical aspect to mention, and it is related to devices used during the vitrification, mainly in terms of whether the biological vitrified material comes in direct contact with liquid nitrogen (open vitrification) or not (closed vitrification), where LN2 may contain potentially contaminating viruses or pathogens. Furthermore, during early development major waves of epigenetic reprogramming take place. Recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, including osmotic shock, temperature, rapid changes of pH and toxicity of cryoprotectants. It is, therefore, important to better understand the potential perturbations of epigenetic modifications that may be associated with the globally used vitrification methods. Therefore, we here discuss the benefits and efficiency of human oocyte vitrification; we also review the evidence surrounding oocyte cryopreservation-related epigenetic modifications and potential epigenetic dysregulations, together with long-term consequences for offspring health.

Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken.

Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 µg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Exploring the effect of cryopreservation in assisted reproductive technology and potential epigenetic risk.

Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.

Vitrification versus slow freezing of human ovarian tissue: a systematic review and meta-analysis of histological outcomes.

A systematic review and meta-analysis of pertinent literature published from 2006 to January 2022 were conducted to study and compare vitrification and slow freezing, the two prominent methods of ovarian tissue cryopreservation. The primary outcome measures for this study were (1) proportion of intact primordial follicles, (2) proportion of intact stromal cells, (3) proportion of DNA fragmentation in primordial follicles, and (4) mean primordial follicle density. This meta-analysis of 19 studies revealed a significantly greater proportion of intact stromal cells in vitrified tissue versus slow-frozen tissue. No significant differences upon pooled analyses were observed between the two cryopreservation methods with respect to the proportion of intact primordial follicles, proportion of DNA fragmentation, or mean primordial follicle density. Due to differences seen in stromal cell viability, vitrification may be a preferred option to preserve histology of tissue. However, more work should be done to compare the two freezing techniques with less heterogeneity caused by patients, samples, and protocols.

The effect of high-throughput vitrification of human ovarian cortex tissue on follicular viability: a promising alternative to conventional slow freezing?

BACKGROUND: The standard procedure most frequently used for ovarian tissue cryopreservation (OTC) is slow freezing, while vitrification has been proposed as promising alternative and has built an impressive catalog of success in fertility laboratories regarding cryopreservation of oocytes and embryos. METHODS: We developed and evaluated a high-throughput protocol for vitrification of human ovarian tissue suitable for clinical processing. Follicular viability was assessed via calcein staining prior and after cryopreservation analyzing ovarian tissue of a cohort of 30 patients. RESULTS: We found no significant differences regarding follicular viability between slow frozen and vitrified cortex tissue samples 24 h after thawing and rapid warming. Follicular viability of thawed and rapid warmed samples was not significantly different in comparison to fresh samples, indicating high proportions of follicular survival rates with both methods. CONCLUSIONS: High-throughput vitrification is a promising option in a clinical setting. More research is required to determine the status of other tissue-specific quality indicators potentially influencing on autotransplantation.

Cryopreservation of ovarian tissue as a method for fertility preservation in women.

OBJECTIVE: To summarize the current possibilities of ovarian tissue cryopreservation as one of the other possible methods for fertility preservation in women. METHODS: Literature review obtained from studies and literature related to ovarian tissue cryopreservation. CONCLUSION: Cryopreservation of ovarian tissue and its subsequent transplantation has a significant potential for preserving fertility not only for prepubertal and oncological patients, but also for patients with various medical indications leading to premature ovarian insufficiency. In order to maintain the best possible quality of oocytes in cryopreserved ovarian tissue, it is necessary to constantly optimize, standardize and compare both cryopreservation protocols, procedures and strategies, as well as the process of thawing ovarian tissue with its subsequent transplantation.