Research progress on mechanism of follicle injury after frozen ovarian tissue transplantation and protective strategies. / 移植冷冻卵巢组织卵泡损伤机制及保护策略.

The iatrogenic ovarian dysfunction caused by cancer treatment have been increasing, along with the age at onset of malignant tumors getting younger, the survival of cancer patients being longer, as well as the delayed childbearing age for females; therefore it becomes a major clinical challenge to preserve the fertility of these patients. Ovarian tissue cryopreservation is the only solution for female cancer patients in prepubertal ages and those who cannot delay gonadotoxic therapy. However, the successful rate of cryopreservation and transplantation of ovarian tissue is still low at present due to the risk of ischemia and hypoxia of grafted tissues. Abnormal activation of primordial follicle and ischemia-reperfusion injury after blood supply recovery also cause massive loss of follicles in grafted ovarian tissues. It has been tried in various studies to reduce the damage of follicles during freezing and transplantation by adding certain drugs, and extend the duration of endocrine and reproductive function in patients with ovarian transplantation. For example, melatonin, N-acetylcysteine, erythropoietin or other antioxidants are used to reduce oxidative stress; mesenchymal stem cells derived from different tissues, basic fibroblast growth factor, vascular endothelial growth factor, angiopoietin 2 and gonadotropin are used to promote revascularization; anti-Müllerian hormone and rapamycin are used to reduce abnormal activation of primordial follicles. This article reviews the research progress on the main mechanisms of follicle loss after ovarian tissue transplantation, including hypoxia, ischemia-reperfusion injury and associated cell death, and abnormal activation of follicles; and explores the methods of reducing graft follicle loss to provide reference for improving the efficiency of ovarian tissue cryopreservation and transplantation.

A retrospective study of ovarian tissue cryopreservation in female patients with hematological diseases for fertility preservation.

PURPOSES: To investigate the effect and safety of ovarian tissue cryopreservation (OTC) for fertility preservation in female patients with hematological diseases. METHODS: We designed a retrospective study. The clinical data of patients with hematological diseases undergoing OTC admitted to Peking University People's Hospital from April 2017 to January 2023 were analyzed and summarized. RESULTS: A total of 24 patients were included in the study, including 19 patients with malignant hematological diseases and 5 patients with non-malignant hematological diseases. The former included 14 patients with acute leukemia, 1 patient with chronic leukemia, and 4 patients with myelodysplastic syndrome, while the latter 5 patients were aplastic anemia (AA). 16 patients had received chemotherapy before OTC. The average age of 24 patients was 22.80 ± 6.81 years. The average anti-Mullerian hormone (AMH) was 1.97 ± 2.12 ng/mL, and the average follicle-stimulating hormone (FSH) was 7.01 ± 4.24 IU/L in examination before OTC. FSH was greater than 10.0 IU/L in 4 cases. The pre-OTC laboratory tests showed that the average white blood cell (WBC) count was (3.33 ± 1.35) × 109/L, the average hemoglobin was 91.42 ± 22.84 g/L, and the average platelet was (147.38 ± 114.46) × 109/L. After injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF), blood transfusion, and iron supplementation in pre-OTC treatment, the average WBC count was (4.91 ± 3.07) × 109/L, the average hemoglobin was 98.67 ± 15.43 g/L, and the average platelet was (156.38 ± 103.22) × 109/L. Of the 24 patients, 22 underwent laparoscopic bilateral partial oophorectomy and oophoroplasty, and 2 underwent laparoscopic unilateral oophorectomy. The average duration of OTC was 59.54 ± 17.58 min, and the average blood loss was 32.1 ± 41.6 mL. The maximum blood loss was 200 mL. There was no significant difference in WBC count and hemoglobin concentration after OTC compared to pre-OTC period. Only the platelet count after OTC surgery was significantly different from that before surgery ([134.54 ± 80.84 vs. 156.38 ± 103.22] × 109/L, p < 0.05). None of the 24 patients had serious complications after OTC. 2 patients had mild infection symptoms, but both recovered well. 23 patients underwent hematopoietic stem cell transplantation (HSCT) after OTC. The median and interquartile range from OTC to the pretreatment of HSCT was 33 (57) days, and the median and interquartile range from OTC to HSCT was 41 (57) days. Seven of them began pretreatment of HSCT within 20 days and began HSCT within 30 days after OTC. All patients were followed up. Of the 23 patients who underwent HSCT after surgery, 22 presented with amenorrhea and 1 with scanty menstrual episodes. Seven patients underwent hormone replacement therapy (HRT) after HSCT. A patient with AA underwent ovarian tissue transplantation (OTT) 3 years after HSCT and resumed regular menstruation 6 months after OTT. CONCLUSIONS: Ovarian tissue cryopreservation has a promising future in fertility protection in patients with hematological diseases. However, patients with hematological malignancies often have received gonadotoxic therapy before OTC, which may be accompanied by myelosuppression while patients with non-malignant hematological diseases often present with severe hemocytopenia. So perioperative complete blood count of patients should be paid attention to. There was no significant difference in the WBC count and hemoglobin concentration in patients with hematological diseases before and after OTC surgery, and the platelet count decreased slightly within the normal range. Infection is the most common post-OTC complication, and HSCT pretreatment can be accepted as early as the 10th day after OTC. OTC has no adverse effects on patients with hematological diseases and does not delay HSCT treatment. For young patients with hematological diseases, OTC is an effective method of fertility preservation.

Fertility preservation in hematological cancer patients.

Among adolescents and young adults, hematological malignancies are the most common malignancies. Although the survival rate of hematological malignancies in young patients has been dramatically improved, due to the continuous improvement and development of tumor diagnosis and treatment options, cytotoxic therapies can significantly reduce a patient's reproductive capacity and cause irreversible infertility. The most two established solutions are embryo cryopreservation and oocyte cryopreservation which can be considered in single female. Sperm or testicular tissue cryopreservation in adult male are feasible approaches that must be considered before gonadotoxic therapy. A comprehensive consultation with reproductive specialists when once diagnosed is a significantly issue which would help those survivors who want to have children. In this article, we review germ cell toxicity, which happens during the treatment of hematological malignancies, and aims to propose safety, efficacy fertility preservation methods in younger patients with hematological malignancies.

Standardized Reproductive Endocrinology and Infertility Consultation for Pediatric and Adolescent Oncology Patients.

Purpose: To investigate the role of formal reproductive endocrinology and infertility (REI) consultation in fertility preservation counseling in a pediatric/adolescent oncology patient population. Methods: Retrospective chart review was performed at an academic adult hospital from 2021 to 2022. Pre- and postpubertal patients admitted to the pediatric/adolescent oncology service with cancer diagnoses and imminent gonadotoxic chemotherapy plans were included. Baseline characteristics were collected, including patient age, sex, race, language, insurance, and cancer diagnosis. Primary outcomes were formal REI consultation and fertility preservation election. Results: Nineteen of 58 eligible patients received a formal REI consultation. Patients were more likely to elect fertility preservation if they received a consult. Females were more likely to receive a consult than males and more likely to elect fertility preservation. Patients of age ≥16 years were more likely to receive consultation than younger patients. However, all patients of age <16 years who received a consult elected fertility preservation. There was no difference in consultation based on race, language, or insurance. Thirteen of 19 patients who received an REI consultation elected fertility preservation. Ten of 11 female elections were ovarian suppression, an unproven method of fertility preservation. The two male elections were semen cryopreservation. Conclusion: Underutilization of formal REI consults and a relative lack of proven fertility preservation elections may shed light on a need for increased fertility preservation awareness among young oncology patients and the providers who care for them. A streamlined process that automates formal REI consultation for all eligible patients may maximize the potential for comprehensive counseling and improve patient participation in fertility preservation.

The beneficial effects of quinoa seed extract supplementation on ram sperm quality following cryopreservation.

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500-1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H2O2 levels than the control group (P<0.001). The expressions of SOD1, CAT, GABPB1, and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene.

Comparison of different cryoprotectants for freezing donkey (Equus asinus) semen.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7%) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n=8; r=3) were divided into 4 extenders: BotuSemen Gold with 5% or 7% MF and 5% or 7% DMF, all containing 11% lactose, 20% egg-yolk and 0.5% Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P>0.05). However, samples with 5% DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5%: 53.67±22.01; 7%: 33.92±23.4; MF: 5%: 44.5±20.46; 7%: 38.75±27.4) (Data: mean ± SD; P>0.05). Hence, thirty mares were inseminated: 15 with 5% DMF and 15 with 7% DMF. The pregnancy rate was 46% (7/15) and 0% (0/15) using the extender with 5% or 7% DMF, respectively (P=0.003). To conclude, the use of 5% or 7% of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5% DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Effect of exogenous melatonin implant on post-thaw semen quality of buffalo bulls.

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.

Assessing Attitudes and Understanding After Ovarian Tissue Cryopreservation: A Follow-Up Telephone Interview Survey.

Purpose: Assessing patient and guardian experiences regarding their history of ovarian tissue cryopreservation (OTC) years after initial procedure. Methods: Cross-sectional follow-up telephone survey. A questionnaire developed by The Pediatric Initiative Network of the Oncofertility Consortium, modified to assess intent and attitudes regarding OTC, tissue access knowledge, financial burden of tissue storage, and intent to use tissue, was utilized. Interviews were conducted for those who underwent OTC at a metropolitan children's hospital between 2013 and 2022. Results: Of 60 eligible patients, 39 interviews were completed. Contacted patients were 3-28 years old, with minors accompanied by guardians. Average age at OTC was 8.5 years old, and 5.1% (2/39) were deceased at the time of contact. All interviewees underwent OTC for fertility preservation before gonadotoxic treatment. Seventy percent of patients (7/10) and 48.1% (13/27) of guardians stated they would use frozen tissue for pregnancy, with 50% (5/10) of patients and 59.3% (16/27) of guardians not understanding tissue access. Regret occurred in 10% (1/10) of patients and 3.4% (1/29) of guardians. It was associated with 10.8% (4/37) of tissue discard due to failed storage payments. Financial concerns occurred in 29.7% (11/37) of interviewees. Overall, 92.3% (36/39) would recommend OTC, and 94.9% (37/39) would repeat their choice to undergo OTC. Conclusion: Follow-up after OTC is essential to patient understanding of tissue status, access, and payments. Most do not regret OTC, except in cases of financial burden leading to tissue discard. Follow-up should be sequentially scheduled and include counseling on financial assistance programs.

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

STUDY QUESTION: Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? SUMMARY ANSWER: LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. WHAT IS KNOWN ALREADY: Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. STUDY DESIGN, SIZE, DURATION: Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. MAIN RESULTS AND THE ROLE OF CHANCE: The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3m protein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. LIMITATIONS, REASONS FOR CAUTION: The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. WIDER IMPLICATIONS OF THE FINDINGS: LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Successful ovarian tissue cryopreservation with transvaginal natural orifice transluminal endoscopic surgery: A case report.

Chemotherapy and radiation therapy can cause gonadal dysfunction in women of reproductive age. Ovarian tissue cryopreservation is performed to restore fertility by allowing transplantation of the patient's frozen-thawed ovarian tissue or through future in vitro maturation and in vitro fertilization of frozen-thawed oocytes. Herein, we describe our initial experience with vaginal natural orifice transluminal endoscopic surgery for ovarian tissue preservation in a young woman with malignant tumor. A 23-year-old woman with anaplastic lymphoma kinase-positive malignant lymphoma was scheduled for hematopoietic stem cell transplantation after experiencing relapse following R-cyclophosphamide, doxorubicin, vincristine, and prednisolone therapy. Ovarian tissue cryopreservation was selected as only MII2 oocytes were collected. Vaginal natural orifice transluminal endoscopic surgery was performed to excise the left ovary. Ovarian tissues were frozen using the vitrification method. The operative time was 37 min, and blood loss was minimal. Pathological examination revealed no metastatic findings of malignant lymphoma and no thermal damage to the ovarian tissue due to bipolar disorder. The patient was discharged on the first day postoperatively, and her postoperative course was uneventful. The vaginal natural orifice transluminal endoscopic surgery technique can provide a safe and effective alternative to laparoscopy or laparotomy for the cryopreservation of ovarian tissue in young patients with cancer. We believe this method has potential application in sexually mature female cancer survivors.

Ovarian tissue cryopreservation with vaginal natural orifice transluminal endoscopic surgeryChemotherapy and radiotherapy can affect a woman's ability to have children by reducing ovarian function. This can make it hard to conceive even with fertility treatments. Freezing healthy ovaries before these treatments can help restore fertility. This can be done by freezing and later transplanting ovarian tissue or by fertilizing frozen eggs in a lab. Traditional surgery to remove ovaries can cause cosmetic issues and pain. But now, a new method called vaginal spontaneous opening transperitoneal endoscopic surgery is becoming more common. This surgery is less invasive, quicker, and causes less bleeding. We recently used this method to preserve ovarian tissue in young women with cancer. The surgery was successful with minimal complications. This new approach could offer a safer option for preserving fertility in female cancer survivors.

Safety evaluation of single-sperm cryopreservation technique applied in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is a technique that directly injects a single sperm into the cytoplasm of mature oocytes. Here, we explored the safety of single-sperm cryopreservation applied in ICSI. This retrospective study enrolled 186 couples undergoing ICSI-assisted pregnancy. Subjects were allocated to the fresh sperm (group A)/single-sperm cryopreservation (group B) groups based on sperm type, with their clinical baseline/pathological data documented. We used ICSI-compliant sperm for subsequent in vitro fertilization and followed up on all subjects. The recovery rate/cryosurvival rate/sperm motility of both groups, the pregnancy/outcome of women receiving embryo transfer, and the delivery mode/neonatal-related information of women with successful deliveries were recorded. The clinical pregnancy rate, cumulative clinical pregnancy rate, abortion rate, ectopic pregnancy rate, premature delivery rate, live birth delivery rate, neonatal birth defect rate, and average birth weight were analyzed. The two groups showed no significant differences in age, body mass index, ovulation induction regimen, sex hormone [anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH)/luteinizing hormone (LH)] levels, or oocyte retrieval cycles. The sperm recovery rate (51.72%-100.00%) and resuscitation rate (62.09% ± 16.67%) in group B were higher; the sperm motility in the two groups demonstrated no significant difference and met the ICSI requirements. Group B exhibited an increased fertilization rate, decreased abortion rate, and increased safety versus group A. Compared with fresh sperm, the application of single-sperm cryopreservation in ICSI sensibly improved the fertilization rate and reduced the abortion rate, showing higher safety.

Predicting the cryotolerance of boar sperm through antioxidant stress.

High sperm cryotolerance is crucial to the successful cryopreservation of boar sperm. Evaluating the cryotolerance of boar sperm by using a rapid and convenient technique can enhance the commercial viability of these sperm. This study investigated the correlation between sperm parameters for three sample subsets-fresh sperm, sperm with H2O2-induced oxidative damage (hereinafter referred to as H2O2-induced sperm), and frozen-thawed sperm-to identify the potential of these correlations to predict cryotolerance. A total of 64 sperm samples were obtained from 64 Duroc boars. The sperm parameters of the three subsets, where the frozen-thawed sperm were analysed at 30 or 180 min after thawing, were determined, and the coefficients of correlation between these parameters were calculated. The results indicated that H2O2-induced oxidative stress resulted in decreases in various sperm parameters-including total motility (TM), viability (VIA), mitochondrial membrane potential (MMP), and live sperm with MMP (LMP)-but increased their coefficients of variation. Receiver operating characteristic (ROC) curve analysis revealed that the kinematic parameters of the H2O2-induced sperm effectively predicted those of the frozen-thawed boar sperm at 30 min after thawing; the corresponding area under the ROC curve (AUC) was 0.8667 for TM and 0.8733 for progressive motility in the H2O2-induced sperm. For measurement at 180 min after thawing, the sperm membrane and mitochondrial parameters of the H2O2-induced sperm effectively predicted the LMP of the frozen-thawed boar sperm; the corresponding AUC was 0.8489 for VIA, 0.8289 for MMP, and 0.8444 for LMP. To our knowledge, this is the first study to directly establish a strong correlation between post-thaw boar sperm quality and H2O2-induced oxidative stress before freezing. Our proposed technique can serve as a valuable reference for the development of practical applications aimed at enhancing techniques for cryopreserving boar sperm.

Scaling Insulin-Producing Cells by Multiple Strategies.

In the quest to combat insulin-dependent diabetes mellitus (IDDM), allogenic pancreatic islet cell therapy sourced from deceased donors represents a significant therapeutic advance. However, the applicability of this approach is hampered by donor scarcity and the demand for sustained immunosuppression. Human induced pluripotent stem cells are a game-changing resource for generating synthetic functional insulin-producing ß cells. In addition, novel methodologies allow the direct expansion of pancreatic progenitors and mature ß cells, thereby circumventing prolonged differentiation. Nevertheless, achieving practical reproducibility and scalability presents a substantial challenge for this technology. As these innovative approaches become more prominent, it is crucial to thoroughly evaluate existing expansion techniques with an emphasis on their optimization and scalability. This manuscript delineates these cutting-edge advancements, offers a critical analysis of the prevailing strategies, and underscores pivotal challenges, including cost-efficiency and logistical issues. Our insights provide a roadmap, elucidating both the promises and the imperatives in harnessing the potential of these cellular therapies for IDDM.

A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm.

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.

Paternal Age Amplifies Cryopreservation-Induced Stress in Human Spermatozoa.

The global fall in male fertility is a complicated process driven by a variety of factors, including environmental exposure, lifestyle, obesity, stress, and aging. The availability of assisted reproductive technology (ART) has allowed older couples to conceive, increasing the average paternal age at first childbirth. Advanced paternal age (APA), most often considered male age ≥40, has been described to impact several aspects of male reproductive physiology. In this prospective cohort study including 200 normozoospermic patients, 105 of whom were ≤35 years (non-APA), and 95 of whom were ≥42 years (APA), we assessed the impact of paternal age on different endpoints representative of sperm quality and cryopreservation tolerance. Non-APA patients had superior fresh semen quality; DNA fragmentation was notably increased in APA as compared to non-APA individuals (21.7% vs. 15.4%). Cryopreservation further increased the DNA fragmentation index in APA (26.7%) but not in non-APA patients. Additionally, APA was associated with increased mtDNAcn in both fresh and frozen/thawed sperm, which is indicative of poorer mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, as indicated by reduced incidences of unreacted acrosome in relation to fresh counterparts in non-APA (from 71.5% to 57.7%) and APA patients (from 75% to 63%). Finally, cryopreservation significantly reduced the phosphorylation status of proteins containing tyrosine residues in sperm from young males. Therefore, the present findings shed light on the effects of paternal age and cryopreservation on sperm quality and serve as valuable new parameters to improve our understanding of the mechanisms underlying sperm developmental competence that are under threat in current ART practice.

27 MHz constant field dielectric warming of kidneys cryopreserved by vitrification.

Organs cryopreserved by vitrification are exposed to the lowest possible concentration of cryoprotectants for the least time necessary to successfully avoid ice formation. Faster cooling and warming rates enable lower concentrations and perfusion times, reducing toxicity. Since warming rates necessary to avoid ice formation during recovery from vitrification are typically faster than cooling rates necessary for vitrification, warming speed is a major determining factor for successful vitrification. Dielectric warming uses an oscillating electric field to directly heat water and cryoprotectant molecules inside organs to achieve warming that's faster and more uniform than can be achieved by heat conduction from the organ surface. This work studied 27 MHz dielectric warming of rabbit kidneys perfused with M22 vitrification solution. The 27 MHz frequency was chosen because its long wavelength and penetration depth are suitable for human organs, because it had an anticipated favorable temperature of maximum dielectric absorption in M22, and because it's an allocated frequency for industrial and amateur use with inexpensive amplifiers available. Previously vitrified kidneys were warmed from -100 °C by placement in a 27 MHz electric field formed between parallel capacitor plates in a resonant circuit. Power was varied during warming to maintain constant electric field amplitude between the plates. Maximum power absorption occurred near -70 °C, with a peak warming rate near 150 °C/min in 50 mL total volume with approximately 500 W power. After some optimization, it was possible to warm ∼13 g vitrified kidneys with unprecedentedly little injury from medullary ice formation and a favorable serum creatinine trend after transplant. Distinct behaviors of power absorption and system tuning observed as a function of temperature during warming are promising for non-invasive thermometry and future automated control of the warming process at even faster rates with user-defined temperature dependence.

Utilization of sperm cryopreservation in patients with testicular cancer.

PURPOSE: We assessed factors that affect the utilization of sperm cryopreservation before 2021, when patients covered expenses, and the influence on quality of life. METHODS: Between 2011 and 2021, testicular cancer survivors (TCS) at our clinic completed a questionnaire, including EORTC QLQ-TC26, covering sperm cryopreservation, sociodemographic details, post-treatment births, and artificial insemination. RESULTS: After 5.7 ± 3.0 years, 279 participants (64%) responded to the questionnaire. Among them, 33% (91/279) of testicular cancer survivors chose sperm cryopreservation prior to treatment, with 11% (10/91) using it for insemination. Conversely, 2% (3/188) without cryopreservation reported unfulfilled desire to have children. Univariate analysis showed TCS with cryopreservation were younger (30.6 ± 7.1 (35 (21-59)) vs. 42.4 ± 10.9 (48 (22-81)) years; p = 0.001), had a lower BMI (24.2 ± 3.3 vs. 26.6 ± 4.6 kg/m2; p = 0.009) and a lower Charlson Score (> 3: 36% vs. 60%; p < 0.001). Multivariate analysis revealed older age (≥ 37 years: OR 13.1 (5.5-31.2), p < 0.001) and lower education (middle school or less: OR 3.3 (1.6-6.9), p = 0.001) as independent factors associated with not undergoing cryopreservation. Regarding quality of life, multivariate analysis identified a lower infertility anxiety score (OR 4.3 (2.0-9.0), p < 0.001) and higher age (≥ 44 years: OR 5.4 (2.6-11.3); p < 0.001) as predictors for the absence of prior cryopreservation. CONCLUSIONS: Age and education seem to impact the choice of undergoing paid sperm cryopreservation. Urologists should inform testicular cancer patients about costs and coverage. Importantly, the occurrence of unmet desires for parenthood is minimal among those who forego cryopreservation.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.