Isolation, Culture, and Quality Assessment of Clinical-Grade Corneal Stromal Stem Cells.

The challenge of treating corneal scarring through keratoplasties lies in the limited availability of donor tissue. Various studies have shown the therapeutic use of cultivated corneal stromal stem cells (CSSCs) to mitigate tissue inflammation and suppress fibrosis and scar tissue formation in preclinical corneal wound models. To develop CSSC therapy for clinical trials on patients with corneal scarring, it is necessary to generate clinical-grade CSSCs in compliant to Good Manufacturing Practice (GMP) regulations. This chapter elucidates human CSSC isolation, culture, and cryopreservation under GMP-compliant conditions. It underscores quality assessment encompassing morphological traits, expression of stemness markers, anti-inflammatory activity, and keratocyte differentiation potency.

Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.

Trends and challenges in liquid-preserved boar semen production: From boar to product.

Boar semen production plays a pivotal role in modern swine breeding programmes, influencing the genetic progress and overall efficiency of the pork industry. This review explores the current challenges and emerging trends in liquid-preserved boar semen production, addressing key issues that impact the quality and quantity of boar semen. Advances in new reproductive technologies, boar selection, housing, semen processing, storage and transport, and the need for sustainable practices including the use of artificial intelligence are discussed to provide a comprehensive overview of the field.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Background: Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. Methods: PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. Results: All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. Conclusions: This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

The impact of intracytoplasmic sperm injection versus conventional in vitro fertilization on the reproductive outcomes of couples with non-male factor infertility and frozen-thawed embryo transfer cycles.

This study was aimed to investigate the impact of intracytoplasmic sperm injection (ICSI) on reproductive outcomes in couples with non-male factor infertility and frozen-thawed embryo transfer (FET) treatment. This retrospective cohort study involved a total of 10,143 cycles from 6206 couples who underwent FET at the Third Affiliated Hospital of Zhengzhou University between January 2016 and September 2022. Patients were categorized into two groups based on the insemination methods of transferred embryos. Clinical and neonatal outcomes were compared between ICSI and conventional in vitro fertilization (cIVF) groups. The results showed that ICSI was not associated with improved clinical outcomes compared to cIVF. However, ICSI was associated with lower birthweight when twins were born. In conclusion, although subgroup analysis showed that ICSI was associated with slightly improved live birth rate for infertile couples with non-male factor infertility compared to cIVF, the regression analysis showed that ICSI did not demonstrate any improvement of the reproductive outcomes. The infertile women with twin pregnancies should be further informed of the lower birthweight and lower birth length when their oocytes were inseminated with ICSI. The findings of this study provide valuable insights for clinicians when discussing the benefits and risks of ICSI in patients with non-male factor infertility.

The HSCT procedure (I): Mobilization, collection, manipulation, and cryopreservation of a HSC graft.

Most hematopoietic stem cell transplants performed for an autoimmune disease of the nervous system, use the patient's hematopoietic stem cells (HSCs). Obtaining an HSC graft is the first step of the process. This typically involves mobilization of bone marrow HSCs into the circulation using high-dose cyclophosphamide followed by filgrastim, a drug based on granulocyte colony-stimulating factor. Toxicity from these agents is usually manageable and adverse events are less severe and less frequent than those experienced during the hematopoietic stem cell transplant. Following mobilization, HSCs are collected from the circulation by leukapheresis. Some centers deplete the graft of lymphocytes using an ex vivo immunomagnetic selection procedure. HSC grafts are cryopreserved until required for the stem cell transplant. Quality testing of the graft ensures sterility and it contains sufficient HSCs and hematopoietic progenitors. The clinical and laboratory aspects of HSC graft mobilization, collection, and storage must meet standards set by national regulatory bodies and accredited by international professional standards organizations. Experienced stem cell transplant teams are important for minimizing procedural toxicity and enhancing successful collection.

Microfluidic chips in female reproduction: a systematic review of status, advances, and challenges.

The female reproductive system is essential to women's health, human reproduction and societal well-being. However, the clinical translation of traditional research models is restricted due to the uncertain effects and low efficiency. Emerging evidence shows that microfluidic chips provide valuable platforms for studying the female reproductive system, while no paper has ever comprehensively discussed the topic. Here, a total of 161 studies out of 14,669 records are identified in PubMed, Scopus, Web of Science, ScienceDirect and IEEE Xplore databases. Among these, 61 studies focus on oocytes, which further involves culture, cell surgeries (oocyte separation, rotation, enucleation, and denudation), evaluation and cryopreservation. Forty studies investigate embryo manipulation via microfluidic chips, covering in vitro fertilization, cryopreservation and functional evaluation. Forty-six studies reconstitute both the physiological and pathological statuses of in vivo organs, mostly involved in placenta and fetal membrane research. Fourteen studies perform drug screening and toxicity testing. In this review, we summarize the current application of microfluidic chips in studying the female reproductive system, the advancements in materials and methods, and discuss the future challenges. The present evidence suggests that microfluidic chips-assisted reproductive system reconstruction is promising and more studies are urgently needed.

Effect of different concentrations of inulin on ram sperm quality during cryopreservation.

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196 degree C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freeze-thawing with 28 mM inulin, compared to other treatment groups (P < 0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p <0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P < 0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P < 0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm. Doi.org/10.54680/fr24510110512.

Effects of vitrification on mitochondrial ultrastructure and membrane potential and its distribution in mouse oocytes.

BACKGROUND: Vitrification is commonly used for in vitro fertilization and has significant impact on gametes. OBJECTIVE: To investigate changes in ultrastructure, membrane potential and distribution of mitochondria in mouse oocytes after vitrification. MATERIALS AND METHODS: Mouse oocytes were divided into three groups: one group as fresh control, one group for the toxicity test (treated with cryoprotectant but without vitrification), and the other for vitrification. RESULTS: Most mitochondria in oocytes were damaged after cooling and warming, being rough and fuzzy in appearance, even swollen and broken. The membrane potential of the toxicity test group and the vitrification group was 0.320 +/-0.030 and 0.244 +/- 0.038, respectively, in comparison to the fresh group (0.398 +/- 0.043). The membrane potential of the vitrified oocytes was significantly lower than fresh oocytes and the toxicity test oocytes (P % 0.05), but there was no significant difference between fresh oocytes and the toxicity test oocytes (P > 0.05). Mitochondria in fresh oocytes were denser and strained stronger, with 59.5> distributed homogeneously and 36.4> polarized. The majority of mitochondria in the toxicity-tested oocytes were clustered (69.3>) and only a small portion were distributed homogeneously (19.6>), while mitochondria in vitrified oocytes were clustered (56.3>) and deficient (24.4>), and their fluorescent staining was weak and blurred. There was a significant disruption in mitochondrial function after vitrification. CONCLUSION: Vitrification alters the ultrastructure, membrane potential and distribution of mitochondria in oocytes, most likely caused by toxicity and mechanical injury. Doi.org/10.54680/fr24510110212.

In vitro dissolution of encapsulated or dispersed Vitamin e and cholesterol is correlated with preservation of refrigerated ovine sperm.

BACKGROUND: Vitamin E ( -tocopherol) and cholesterol are crucial components in cellular protection and physiological processes. Their uses in biological media face challenges due to their poor solubility and stability. OBJECTIVE: The study investigated the complex interactions of these bioactive compounds in various encapsulation systems of cyclodextrin and liposome, as well as dispersion in PEG-6000, in an attempt to improve the viability, motility, and preservation of ovine sperm cells. MATERIALS AND METHODS: The work explored the in vitro dissolution kinetics of vitamin E (d-tocopherol) and cholesterol using semi-empirical models. RESULTS: The release profiles of VitE and Chl varied considerably, depending on the specific carrier systems. For liposome-loaded VitE and Chl, the Korsmeyer-Peppas model gave the best fit; for CD/VitE and CD/Chl, the Higuchi model provided the best fit, whereas for PEG-6000 dispersions (VitE and Chl) both the Higuchi and Korsmeyer-Peppas models demonstrated the excellent fit. All systems indicated a Fickian diffusion mechanism dictated by the concentration gradient. The delivery of VitE and Chl with CD, liposome and PEG dispersion significantly increased sperm mobility and motility. The effect on the VCL parameter was the greatest by liposome-loaded VitE and Chl, followed by CD encapsulation and PEG-6000 dispersion. CONCLUSION: The dynamics of vitamin E and cholesterol within innovative delivery systems offers valuable insights into the development of advanced solutions in reproductive health, particularly on improving the viability, motility of refrigerated ovine sperm cells. Doi.org/10.54680/fr24510110712.

Immunofluorescence and biochemical investigation of the protective effects of naringin and diosmin on the freezability of merino ram semen.

BACKGROUND: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage. OBJECTIVE: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen. MATERIALS AND METHODS: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 10 7 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37 degree C for 25 s and analyzed in terms of spermatological parameters. RESULTS: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p < 0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p < 0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p < 0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells. CONCLUSION: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process. Doi.org/10.54680/fr24510110412.

Effect of cryoprotectant and concentration on the sperm quality of walking catfish, Clarias batrachus, post-cryopreservation.

BACKGROUND: Walking catfish, Clarias batrachus is one of the native and most popular freshwater catfish species in Indonesia. However, cultivation faces challenges, particularly due to the scarcity of larvae resulting from underdeveloped breeding technologies. Cryopreservation is a method of storing sperm to maintain viability for a long period and support the breeding technology of the fish. Cryoprotectant, in this context, plays an important role in determining the success of sperm cryopreservation. OBJECTIVE: To determine the best type and concentration of cryoprotectant for cryopreservation of walking catfish sperm. MATERIALS AND METHODS: A total of five different types of cryoprotectants, namely DMSO, glycerol, ethyl glycol, ethanol, and methanol, were tested at four concentration levels namely 0%, 5%, 10%, 15%, and 20%, each with four replications. RESULTS: The type and concentration of cryoprotectant had a significant effect on sperm motility and viability (P < 0.05). The best outcomes were obtained with 5% DMSO and ethyl glycol, 10% glycerol and methanol, as well as 15% ethanol. CONCLUSION: The highest motility and viability values were obtained with 5% DMSO, resulting in its recommendation for cryopreservation of walking catfish sperm. Doi.org/10.54680/fr24510110612.

Comparison of clinical outcomes and perinatal outcomes between natural cycle and hormone replacement therapy of frozen-thawed embryo transfer in patients with regular menstruation: a propensity score-matched analysis.

Purpose: To investigate potential differences in pregnancy outcomes among patients with regular menstruation who underwent frozen-thawed embryo transfer using natural cycle (NC) or hormone replacement therapy (HRT). Methods: This study retrospectively analyzed 2672 patients with regular menstruation who underwent FET from November 2015 to June 2021 at the single reproductive medical center. A one-to-one match was performed applying a 0.02 caliper with propensity score matching. Independent factors influencing the live birth and clinical pregnancy rates were screened and developed in the nomogram by logistic regression analysis. The efficacy of live birth rate and clinical pregnancy rate prediction models was assessed with the area under the ROC curve, and the live birth rate prediction model was internally validated within the bootstrap method. Results: The NC protocol outperformed the HRT protocol in terms of clinical pregnancy and live birth rates. The stratified analysis revealed consistently higher live birth and clinical pregnancy rates with the NC protocol across different variable strata compared to the HRT protocol. However, compared to the HRT treatment, perinatal outcomes indicated that the NC protocol was related to a higher probability of gestational diabetes. Multifactorial logistic regression analysis demonstrated independent risk factors for live birth rate and clinical pregnancy rate. To predict the two rates, nomogram prediction models were constructed based on these influencing factors. The receiver operating characteristic curve demonstrated moderate predictive ability with an area under curve (AUC) of 0.646 and 0.656 respectively. The internal validation of the model for live birth rate yielded an average AUC of 0.646 implying the stability of the nomogram model. Conclusion: This study highlighted that NC yielded higher live birth and clinical pregnancy rates in comparison to HRT in women with regular menstruation who achieved successful pregnancies through frozen-thawed embryo transfer. However, it might incur a higher risk of developing gestational diabetes.

Comprehensive fertility preservation can be offered in a timely manner around gonadotoxic therapy in children and adolescents.

BACKGROUND: Treatment for certain childhood cancers and nonmalignant conditions can lead to future infertility and gonadal failure. The risk of treatment delay must be considered when offering fertility preservation (FP) options. We examined the timeline from FP referral to return to treatment (RTT) in pediatric patients who underwent FP due to iatrogenic risk for infertility. METHODS: A retrospective review was performed of patients with FP consultation due to an increased risk of iatrogenic infertility at Ann & Robert H. Lurie Children's Hospital of Chicago from 2018 to 2022. Data on diagnosis, age, treatment characteristics, and procedure were collected. RESULTS: A total of 337 patients (n = 149 with ovaries, n = 188 with testes) had an FP consultation. Of patients with ovaries, 106 (71.1%) underwent ovarian tissue cryopreservation (OTC), 10 (6.7%) completed ovarian stimulation/egg retrieval (OSER), and 33 (22.1%) declined FP. Of the patients with testes, 98 (52.1%) underwent testicular tissue cryopreservation (TTC), 48 (25.5%) completed sperm banking (SB), and 42 (22.3%) declined FP. Median time from referral to FP consultation was short (ovaries: 2 days, range: 0-6; testes: 1 day, range: 0-5). OSER had a significantly longer RTT versus OTC and no FP (52.5 vs.19.5 vs. 12 days, p = .01). SB had a significantly quicker RTT compared to TTC or no FP (9.0 vs. 21.0 vs. 13.5 days; p = .008). For patients who underwent OTC/TTC and those who declined FP, there was no significant difference in time from consultation to treatment. CONCLUSIONS: It is feasible to promptly offer and complete FP with minimal delay to disease-directed treatment.

Callus induction, shoot regeneration, and conservation of in vitro grown date palm (Phoenix dactylifera).

Date palm (Phoenix dactylifera( cv. Medjool is a significant plant, grown in Jordan. In vitro propagation gives operative resources for the significant propagation of date palms. Maximum callus induction was achieved from MS media supplemented with benzyl amino purine (BA) and naphthalene acetic acid (NAA). The highest plant regeneration was recorded on MS medium supplemented with dichlorophenoxyacetic acid (2,4-D) at 3.0 mg/L, and BA at 2.0 mg/L. A significant positive impact on shoot formation was recorded on MS medium supplemented with 1.0 mg/L BA with 0.5 to 1.5 mg/L NAA in both liquid and solid MS medium. To maintain survival and regrowth capacity, sucrose could be used for medium-term conservation at lower concentrations (0.1 - 0.2 M). In addition, sorbitol might be used at 0.1 M to maintain the quality of explants. The vitrification technique for long-term preservation was experimented. Embryogenic callus was used as explants for conservation. The survival as well as regrowth percentages of non-cryopreserved and cryopreserved tissue cultures were affected by their duration of treatment with the vitrification solution plant vitrification solution 2 (PVS2) and modified plant vitrification solution 2 (MPVS2). Results showed that using PVS2 for 60 minutes for cryopreserved calli was more effective than other treatments. After storage in liquid nitrogen, the highest survival rate (65%) and regrowth rate (40%) were achieved.

Metformin alleviates cryoinjuries in porcine oocytes by reducing membrane fluidity through the suppression of mitochondrial activity.

Plasma membrane damage in vitrified oocytes is closely linked to mitochondrial dysfunction. However, the mechanism underlying mitochondria-regulated membrane stability is not elucidated. A growing body of evidence indicates that mitochondrial activity plays a pivotal role in cell adaptation. Since mitochondria work at a higher temperature than the constant external temperature of the cell, we hypothesize that suppressing mitochondrial activity would protect oocytes from extreme stimuli during vitrification. Here we show that metformin suppresses mitochondrial activity by reducing mitochondrial temperature. In addition, metformin affects the developmental potential of oocytes and improves the survival rate after vitrification. Transmission electron microscopy results show that mitochondrial abnormalities are markedly reduced in vitrified oocytes pretreated with metformin. Moreover, we find that metformin transiently inhibits mitochondrial activity. Interestingly, metformin pretreatment decreases cell membrane fluidity after vitrification. Furthermore, transcriptome results demonstrate that metformin pretreatment modulates the expression levels of genes involved in fatty acid elongation process, which is further verified by the increased long-chain saturated fatty acid contents in metformin-pretreated vitrified oocytes by lipidomic profile analysis. In summary, our study indicates that metformin alleviates cryoinjuries by reducing membrane fluidity via mitochondrial activity regulation.

Epigenetics modification among vitrified oocytes and early embryos derived from them: a narrative review.

Vitrification has important application in assisted reproductive technology (ART) and this technique has been widely used in the cryopreservation of oocytes and embryos. However, due to susceptibility of epigenetic modifications to environmental changes induced by cryopreservation procedures, there are concerns about the potential epigenetic consequences of oocyte and embryo vitrification. This review comprehensively summarized the effect of cryopreservation-especially the vitrification method in ART-on oocytes and embryos. Various studies have reported changes in different aspects of genomic status which directly affect the quality of fertilized embryos.  The objective of this review is to assess existing literature on the epigenetic modifications that occur in vitrified oocytes and early embryos resulting from oocyte vitrification, including DNA modifications, RNA methylation, histone modification and microRNAs related to ART.

Thawing of cryopreserved sperm from domestic animals: Impact of temperature, time, and addition of molecules to thawing/insemination medium.

In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses.

Mature oocyte found during ovarian tissue cryopreservation in an early adolescent female. / 青春初期女性行卵巢组织冷冻过程中发现成熟卵母细胞一例.

A 15-year-old female with Hodgkin's lymphoma underwent ovarian tissue cryopreservation for preserving fertility in Reproductive Department of Sir Run Run Shaw Hospital, Zhejiang University School of Medical after receiving one course of chemotherapy. During the ovarian tissue cryopreservation, one MⅡmature oocyte and three germinal vesicle oocytes were found. The three immature oocytes underwent in vitro maturation but failed. Ultimately, one mature oocyte and 12 ovarian cortex slices were cryopreserved using vitrification. This case indicates that for patients with established gonadal axis feedback, ovarian tissue cryopreservation may not be the only method for fertility preservation. It is advisable to consider ovarian stimulation and oocyte retrieval for oocyte cryopreservation. Alternatively, for individuals in the ovulation phase of their menstrual cycle, attempting oocyte retrieval before ovarian tissue cryopreservation to obtain mature oocytes from the natural cycle, followed by oocyte cryopreservation, may enhance the likelihood of successful fertility preservation.