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Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [Ctrl C ]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (Ctrl N ). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity ( p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to Ctrl C . All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa ( p < 0.001) than Ctrl C . Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR ( p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 ( p < 0.01), while all TBR doses stabilized the levels of PKC ( p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

Testicular organoids have great potential for maintaining male fertility and even restoring male infertility. However, existing studies on generating organoids with testis-specific structure and function are scarce and come with many limitations. Research on cryopreservation of testicular organoids is even more limited, and inappropriate cryopreservation methods may result in the loss of properties in resuscitated or regenerated organoids, rendering them unsuitable for clinical or research needs. In this paper, we investigated the effects of mouse age and cell number on the self-aggregation of testicular cells into spheres in low-adsorption plates. Various media compositions, culture systems, and cell numbers were used to culture cell spheres for 14 days to form testicular organoids, and the self-organization of the organoids was assessed by histological and immunofluorescence staining. We determined the appropriate cryopreservation conditions for testicular cells, cell spheres, and tissues. Subsequently, organoids derived from cryopreserved testicular tissues, testicular cells, and testicular cell spheres were compared and evaluated by histological and immunofluorescence staining. The results indicate that testicular cell spheres consisting of 30 × 10 4 testicular cells from 2-week-old mice were able to form organoids highly similar to the luminal structure and cell distribution of natural mouse testicular tissues. This transformation occurred over 14 days of incubation in α-MEM medium containing 10 % knockout serum replacer (KSR) using an agarose hydrogel culture system. Additionally, the Sertoli cells were tightly connected to form a blood-testis barrier. The relative rates of tubular area, germ cells, Sertoli cells, and peritubular myoid cells were 36.985 % ± 0.695, 13.347 % ± 3.102, 47.570 % ± 0.379, and 27.406 % ± 1.832, respectively. The optimal cryopreservation protocol for primary testicular cells involved slow freezing with a cryoprotectant consisting of α-MEM with 10 % dimethyl sulfoxide (DMSO). Slow freezing with cryoprotectants containing 5 % DMSO and 5 % ethylene glycol (EG) was optimal for all different volumes of testicular cell spheres. Compared to testicular organoids generated from frozen testicular tissue and cell spheres, freezing testicular cells proved most effective in maintaining organoid differentiation characteristics and cell-cell interactions. The findings of this study contribute to a "universal" testicular organoid in vitro culture protocol with promising applications for fertility preservation and restoration in prepubertal cancer patients and adult infertile patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cryopreservation of rat embryos is useful for efficiently archiving rat resources in bioresource repositories. The cryopreserved fertilized oocytes can be quickly reanimated to rats with homozygous mutations using embryo transfer. In addition, cryopreserved rat fertilized oocytes are easier to transport than live animals. Before cryopreservation, fertilized oocytes are obtained by mating or in vitro fertilization. However, it is not clear which fertilized oocytes are most suited to cryopreservation. In this study, we performed a systematic comparison of the fertilizing ability, cryotolerance, and developmental ability of cryopreserved fertilized oocytes at the pronuclear stage produced either by mating (in vivo) or in vitro fertilization (in vitro) in SD and F344 rats. In vivo-fertilized oocytes had higher cryotolerance and developmental ability than in vitro-fertilized oocytes in SD and F344 rats. Furthermore, the fertilization ability, cryotolerance, and developmental ability of vitrified-warmed fertilized oocytes differed between SD and F344 rats. In conclusion, our study suggests that in vivo-fertilized rat oocytes were more suitable for cryopreservation. Our protocol provides an optimized system for the management of rat colonies using fertilized oocytes cryopreservation and contributes to the 3Rs principle by reducing the number of animals used for research., (© 2024. The Author(s).)

After cryopreservation, reactive oxygen species (ROS) can damage sperm. Antioxidants are the primary defense against oxidative damage. Berberine is a bioactive alkaloid found in Berberis vulgaris, Curcuma longa, and Ergon grape, and is a potent antioxidant. Due to the negative effects of free radicals in oxidative stress processes, antioxidant chemicals are required to protect sperm. However, berberine has low bioavailability, making it less effective. Loading techniques on nanoparticles and nanotechnology can help overcome this limitation. Selenium nanoparticles were synthesized with barberry extract, and berberine was loaded on them. Berberine nanoparticles were then synthesized using anti-solvent precipitation with a syringe pump technique. The synthesis of nanoparticles was confirmed by EDX, UV-visible, FE-SEM, Zeta-Potential, and FTIR tests. In this experiment, we aim to investigate the impact of nano-berberine and berberine loaded on Se-NPs on goat sperm parameters after freeze-thawing. We assessed the generation of reactive oxygen species (ROS), in vitro fertility, and the subsequent embryo development of zygote with treated sperm after determining the optimal concentration of various chemicals on sperm parameters. The study found that all treatments had significant differences from the control group in terms of motility, viability, DNA and membrane integrity, ROS level, lipid peroxidation, in vitro fertility ability, and the capacity to develop inseminated oocytes (p < 0.05). The most significant outcomes were observed with berberine loaded on Se-NPs and the combination of selenium nanoparticles with berberine nanoparticles., (© 2024. The Author(s).)

Background: The impact of high body mass index (BMI) on embryo and pregnancy outcomes in women using the PPOS (progestin-primed ovarian stimulation) protocol during their first frozen embryo transfer (FET) cycles is not clear. This study is to investigate the impact of BMI on oocyte, embryo, and pregnancy outcomes in patients who underwent the PPOS protocol., Methods: This retrospective study included the first FET cycle of 22,392 patients following the PPOS protocol. The impact of BMI on oocyte and pregnancy outcomes was assessed across different BMI groups, using direct acyclic graph to determine covariates, followed by the application of multiple linear and logistic regressions to further validate this influence., Results: The high BMI groups exhibited a higher number of oocytes; however, no significant differences were observed in good-quality embryos, clinical pregnancy rate, and implantation rate. Nevertheless, the high BMI groups demonstrated a significantly elevated miscarriage rate (9.9% vs. 12.2% vs. 15.7% vs. 18.3%, P < 0.001), particularly in late miscarriages, resulting in lower live birth rates (LBR, 41.1% vs. 40.2% vs. 37.3% vs. 36.2%, P = 0.001). These findings were further confirmed through multiple liner and logistic regression analyses. Additionally, several maternal factors showed significant associations with adjusted odds ratios for early miscarriage. However, women with a BMI ≥ 24 who underwent hormone replacement cycle or hMG late stimulation protocol for endometrial preparation experienced an increased risk of late miscarriage., Conclusions: By utilizing the PPOS protocol, women with a high BMI exhibit comparable outcomes in terms of embryo and clinical pregnancies. However, an elevated BMI is associated with an increased risk of miscarriage, leading to a lower LBR. Adopting appropriate endometrial preparation protocols such as natural cycles and letrozole stimulation cycles may potentially offer benefits in reducing miscarriages., (© 2024. The Author(s).)

As interest in deep space travel grows exponentially, understanding human adaptation in becoming an interplanetary species is crucial. This includes the prospect of reproduction. This review summarizes recent updates and innovations in assisted reproductive technologies (ART) on Earth, while also discussing current challenges and areas for improvement in adapting ART studies to the space environment. We discuss the critical components of ART - gamete handling and preparation, fertilization, embryo culture, and cryopreservation - from the daily practice perspective of clinical embryologists and reproductive endocrinologists and lay out the complicated path ahead.In vitro embryo development in low Earth orbit and beyond remains questionable due to synergetic effects of microgravity and radiation-induced damage observed in simulated and actual in-space mammalian studies. Cryopreservation and long-term storage of frozen samples face substantial obstacles - temperature limitations, lack of trained personnel, and absence of adapted cosmic engineering options. We touch on recent innovations, which may offer potential solutions, such as microfluidic devices and automated systems. Lastly, we stress the necessity for intensive studies and the importance of an interdisciplinary approach to address numerous practical challenges in advancing reproductive medicine in space, with possible implications for both space exploration and terrestrial fertility treatments., (© 2024. The Author(s).)

Mesenchymal stromal cell (MSC) apoptosis is required for in vivo immunosuppression. However, the induction of apoptosis is heavily dependent on the recipient's immune system. In graft-versus-host disease (GvHD), patients who fail to respond to MSCs are in fact those whose immune cells are unable to induce MSC apoptosis ex vivo. The information is critical to explain why responses in clinical trials vary even though the same sources of MSC products are infused. More importantly, it highlights the need for an alternative MSC treatment for the nonresponders. By using a mouse model of ovalbumin (OVA)-induced allergic inflammation, we demonstrated that we could generate apoptotic MSCs (ApoMSCs) in vitro and use them to successfully reduce allergic airway inflammation. In order to address the logistics of their potential future clinical application, we have shown that ApoMSCs could be cryopreserved without impairing efficacy compared to freshly generated ApoMSCs. We have also highlighted that MSCs need to undergo complete apoptosis before cryopreservation to retain their immunosuppressive activity. The cryopreserved ApoMSCs could serve as a potential future off-the-shelf cellular product, in particular for patients who suffer from inflammatory conditions yet do not harbor the immune capacity to induce MSC apoptosis in vivo. Our data provide proof-of-concept that under laboratory conditions, ApoMSCs can be successfully frozen and thawed without affecting their anti-inflammatory activity, as tested in a murine model of allergic inflammation., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Currently, there is no consensus on the optimal management of women with low prognosis in ART. In this Delphi consensus, a panel of international experts provided real-world clinical perspectives on a series of literature-supported consensus statements regarding the overall relevance of the POSEIDON criteria for women with low prognosis in ART., Methods: Using a Delphi-consensus framework, twelve experts plus two Scientific Coordinators discussed and amended statements and supporting references proposed by the Scientific Coordinators (Round 1). Statements were distributed via an online survey to an extended panel of 53 experts, of whom 36 who voted anonymously on their level of agreement or disagreement with each statement using a six-point Likert-type scale (1 = Absolutely agree; 2 = More than agree; 3 = Agree; 4 = Disagree; 5 = More than disagree; 6 = Absolutely disagree) (Round 2). Consensus was reached if > 66% of participants agreed or disagreed., Results: The extended panel voted on seventeen statements and subcategorized them according to relevance. All but one statement reached consensus during the first round; the remaining statement reached consensus after rewording. Statements were categorized according to impact, low-prognosis validation, outcomes and patient management. The POSEIDON criteria are timely and clinically sound. The preferred success measure is cumulative live birth and key management strategies include the use of recombinant FSH preparations, supplementation with r-hLH, dose increases and oocyte/embryo accumulation through vitrification. Tools such as the ART Calculator and Follicle-to-Oocyte Index may be considered. Validation data from large, prospective studies in each POSEIDON group are now needed to corroborate existing retrospective data., Conclusions: This Delphi consensus provides an overview of expert opinion on the clinical implications of the POSEIDON criteria for women with low prognosis to ovarian stimulation., (© 2024. The Author(s).)

Background: Frozen embryo transfer (FET) is usually recommended for women with polycystic ovary syndrome (PCOS) undergoing In vitro fertilization (IVF). While there is no consensus as to the optimal protocol of endometrial preparation for FET. The effect of gonadotropin-releasing hormone agonist (GnRH-a) pretreatment for FET among women with PCOS remains controversial., Purpose: We intend to explore whether GnRH-a pretreatment could improve clinical outcomes for women with PCOS undergoing FET., Methods: PubMed, Embase, ClinicalTrials.gov, Cochrane Library, and Web of Science were searched up to May 16, 2024. Eligible studies involved patients with PCOS undergoing FET and receiving GnRH-a pretreatment for endometrial preparation, with artificial cycle (AC) as the control therapy. Only randomized controlled trials (RCTs) published in Chinese and English were included. Data extraction was performed independently by two authors. Effect was quantified using odd ratios (ORs) with 95% confidence intervals (CIs) using random-effect models with the Mantel-Hansel (M-H) method in Revman software. Quality of outcomes was evaluated using the GRADEpro system. Primary outcomes contained the clinical pregnancy rate, miscarriage rate, and live birth rate. Secondary outcomes included the incidence of preterm labor and gestational diabetes mellitus (GDM)., Results: Ninety-seven records were initially retrieved, with 21 duplicates and 65 articles excluded after title and abstract screening. Seven studies were excluded due to retrospective design, leaving three RCTs with 709 participants. Among them, 353 received GnRH-a pretreatment as the intervention group and 356 received AC as the control group. No significant differences were observed in the clinical pregnancy rate (OR 1.09, 95% CI 0.75 to 1.56, P = 0.66), miscarriage rate (OR 0.73, 95% CI 0.28 to 1.90, P = 0.52), live birth rate (OR 0.87, 95% CI 0.61 to 1.25, P = 0.46), and the risk of preterm labor (OR 1.45, 95% CI 0.79 to 2.65, P = 0.23) and GDM (OR 0.73, 95% CI 0.37 to 1.48, P = 0.39) between the two groups., Conclusions: In this meta-analysis, GnRH-a pretreatment does not confer any advantages and appears unnecessary for women with PCOS undergoing FET. Additional RCTs should focus on maternal complications and the health of offspring., (© 2024. The Author(s).)

Background: Infertility affects one in six couples worldwide, with advanced maternal age (AMA) posing unique challenges due to diminished ovarian reserve and reduced oocyte quality. Single vitrified-warmed blastocyst transfer (SVBT) has shown promise in assisted reproductive technology (ART), but success rates in AMA patients remain suboptimal. This study aimed to identify and refine predictive factors for live birth following SVBT in AMA patients, with the goal of enhancing clinical decision-making and enabling personalized treatment strategies., Methods: This retrospective cohort study analyzed 1,168 SVBT cycles conducted between June 2016 and December 2022 at the First Affiliated Hospital of Guangxi Medical University and Nanning Maternity and Child Health Hospital. Nineteen machine-learning models were applied to identify key predictive factors for live birth. Feature selection and 10-fold cross-validation were employed to validate the models., Results: The most significant predictors of live birth included inner cell mass quality, trophectoderm quality, number of oocytes retrieved, endometrial thickness, and the presence of 8-cell blastomeres on day 3. The stacking model demonstrated the best predictive performance (AUC: 0.791), followed by Extra Trees (AUC: 0.784) and Random Forest (AUC: 0.768). These models outperformed traditional methods, achieving superior accuracy, sensitivity, and specificity., Conclusion: Leveraging advanced machine-learning models and identifying critical predictive factors can improve the accuracy of live birth outcome predictions for AMA patients undergoing SVBT. These findings offer valuable insights for enhancing clinical decision-making and managing patient expectations. Further research is needed to validate these results in larger, multi-center cohorts and to explore additional factors, including fresh embryo transfers, to broaden the applicability of these models in clinical practice., (© 2024. The Author(s).)

Preimplantation embryos can experience stress from laboratory interventions and a sub-optimal culture environment. Though research has demonstrated advanced maternal age impairs oocyte quality, the response of embryos derived from such oocytes to vitrification-thawing and culture in a high oxygen (O 2 ) environment in the assisted reproductive technology laboratory is unknown. Therefore, in this study, embryos produced by in vitro fertilization (IVF) using oocytes from two- and eight-month-old Swiss albino mice were vitrified and thawed during their 6-8 cell stage. and cultured at low oxygen (5%) tension (LOT) and high oxygen (20%) tension (HOT). Embryo development, apoptosis, inner cell mass (ICM) outgrowth proliferation ability in vitro and pluripotency were assessed. Embryos from advanced maternal age cultured at HOT showed reduced fertilizing ability (p < 0.05), poor survival post-thawing (p < 0.05), and increased apoptosis (p < 0.01) in comparison to sibling embryos cultured at LOT. Importantly, the extended culture of vitrified-thawed embryos from advanced maternal age led to a significant (p < 0.001) reduction in complete ICM outgrowth formation at HOT in comparison to the LOT environment. The findings of this study suggest that HOT is detrimental to embryos from advanced maternal age, and importantly, vitrified-thawed embryos are more susceptible to stress, which could have negative implications, especially during the peri-implantation developmental period., (© 2024. The Author(s).)

Oxidative stress, caused by both endogenous and exogenous factors, affects sperm function by damaging morphology and reducing metabolic activity, leading to reduced fertilization ability. The purpose of this study was to investigate the effects of oxidative stress on bull sperm and to evaluate the efficacy of targeted antioxidants in mitigating these detrimental effects. Fresh bull semen samples were subjected to hydrogen peroxide (H 2 O 2 ) and antimycin treatments to induce oxidative stress, and the antioxidants PQQ, ergothioneine, and vitamin C were applied to counteract the induced stress. Sperm motility, viability, and reactive oxygen species (ROS) levels in the cytoplasm and mitochondria of sperm were assessed using computer-assisted sperm analysis (CASA) and flow cytometry. The treatment with H 2 O 2 rapidly decreased sperm viability, and antimycin-induced mitochondrial ROS mainly decreased sperm motility; PQQ and vitamin C effectively reduced mitochondrial ROS, while ergothioneine and vitamin C reduced cytosolic ROS. In frozen-thawed sperm, oxidative stress was elevated in both cytoplasm and mitochondria, and all three antioxidants improved sperm motility by inhibiting ROS production. Furthermore, the localization of oxidized lipids (4-hydroxynonenal) in sperm was detected using immunofluorescence, indicating that oxidative stress affects the head and midpiece of sperm. These findings highlight the potential of targeted antioxidants to mitigate the detrimental effects of oxidative stress on bull sperm and provide valuable insights to improve semen quality and optimize the use of antioxidants in artificial insemination., (© 2024. The Author(s).)

Ovarian tissue cryopreservation (OTC) is increasingly offered globally as a fertility preservation strategy for both postpubertal women and prepubertal girls, with subsequent reimplantation of cryopreserved ovarian cortex resulting in a rapidly growing number of live births. There remains very limited evidence of efficacy from tissue stored when the patient was prepubertal or from conditions affecting the ovary directly, e.g., Turner syndrome. Although OTC is becoming a more established practice, several clinical dilemmas remain from a practical and ethical standpoint. This review discusses the challenges regarding optimal patient selection for the procedure, the use of OTC in patients with a poor prognosis, the potential of reimplantation of tissue contaminated with malignant cells, and the role of OTC in those with an intrinsic ovarian disorder., Competing Interests: Declaration of Interests C.E.D. has nothing to disclose. R.A.A. has received consulting fees from Roche Diagnostics outside the submitted work., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

The low resistance of boar sperm to cryopreservation dictates that addition antioxidants and energetic substances to the diluent to improve sperm quality is necessary. This study evaluated the effect of spermidine and phosphocreatine in combination on the quality, antioxidant capacity, and antiapoptotic-like changes capacity of cryopreserved boar sperm based on previous reports. The results showed that the combined application of spermidine and phosphocreatine significantly enhanced the motility, average path velocity, straight-line velocity, curvilinear velocity, beat cross frequency, acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity compared with the control group (p < 0.05). In addition, the combined application of spermidine and phosphocreatine significantly enhanced the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity while significantly decreasing malondialdehyde content and hydrogen peroxide content (p < 0.05). Western Blot analysis further showed that spermidine and phosphocreatine significantly decreased the expression of CASP3 and BAX and significantly enhanced the expression of BCL2 (p < 0.05); therefore, the combination of spermidine and phosphocreatine has potentially positive implications for improving the quality of cryopreserved boar sperm., (© 2024 Wiley Periodicals LLC.)

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen., Competing Interests: Declaration of competing interest A number of products used in this study are proprietary (Botupharma) and Drs. Marco A. Alvarenga and Frederico O. Papa are co-owners of Botupharma., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction., Competing Interests: Declaration of Competing Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cancer cell lines are important tools to investigate the biology of cancer and test hypotheses to improve cancer treatments. A major challenge in establishing epithelial cancer cell lines is the removal of cancer-associated fibroblasts (CAFs). CAFs are abundant within the tumor microenvironment. CAFs generally proliferate faster than epithelial cancer cells in culture. CAFs can be mistakenly identified as cancer cells, especially when cancer cells display spindle-shaped morphology. Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Here, we describe protocols for establishing epithelial cancer cell lines from mouse models of PDAC and verifying that they are not CAFs. The approach is cost-effective and can be used for other types of cancer. If needed, CAF cell lines can also be established and preserved using this protocol. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of cells from tumors Basic Protocol 2: Isolation and cryopreservation of cancer cell clones Basic Protocol 3: Assessment of the identity of cancer cell lines and CAFs by western blotting., (© 2024 Wiley Periodicals LLC.)

This study aimed to compare the effectiveness, in terms of viability and quality post-warming, when vitrifying in vitro-produced (IVP) pig blastocysts with either a modified Cryotop (n = 161; 20 blastocysts/device) or the conventional Superfine Open Pulled Straw (SOPS; n=160; 5-6 blastocysts/device systems. Blastocyst viability, morphology, and apoptosis parameters were evaluated after 24 h of in vitro culture. The Cryotop system yields better results in terms of higher embryo viability and total cell numbers (p < .05) and lower apoptosis (p < .05) parameters than the SOPS procedure, defining a high effectiveness to simultaneously vitrify 20 pig IVP blastocysts at one time, thus increasing the yield of IVP blastocysts readily available for embryo transfer., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

The generation of stem cell-derived β-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human β-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries., (© 2024 by the American Diabetes Association.)

Purpose: This retrospective cohort study aims to investigate whether high-normal fasting blood glucose (FBG) affects assisted reproductive technology (ART) outcomes undergoing single blastocyst frozen-thawed embryo transfer (FET) cycles in women with normal body mass index (BMI)., Methods: 944 women with normal BMI and FBG levels undergoing single blastocyst FET cycles were enrolled. Based on the median of FBG (4.97 mmol/L, 1 mmol/L = 18 mg/dL), the subjects were categorized into the low-normal group (3.90 ≤ FBG ≤ 4.97 mmol/L, n = 472) and the high-normal group (4.97 < FBG < 6.10 mmol/L, n = 472). Multivariable logistic regression and receiver operating characteristic (ROC) were used to analyze the relationship between high-normal FBG and ART outcomes., Primary Outcome: live birth rate (LBR)., Results: LBR was significantly lower in the high-normal group than in the low-normal group (36.8% vs. 45.1%, p = 0.010), and the miscarriage rate was considerably higher than that in the low-normal group (23.9% vs. 16.5%, p = 0.041). High-normal FBG of female was an independent predictor of live birth (adjusted OR:0.747, 95% CI: 0.541-0.963, p = 0.027) and miscarriage (adjusted OR:1.610, 95% CI: 1.018-2.547, p = 0.042). ROC analyses showed that the cut-off values of FBG (endpoints: live birth and miscarriage) were 5.07 mmol/L, and 5.01 mmol/L, respectively., Conclusions: In women with normal BMI, high-normal FBG is an independent risk factor for lower LBR and higher miscarriage rate in single blastocyst FET cycles. Attention to preconception FBG monitoring in this particular population may allow early intervention to improve ART outcomes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In the last decade, the evolution of oncofertility has sparked a resurgence of interest in in vitro maturation (IVM) due to its suitability in certain oncological scenarios where controlled ovarian hyperstimulation may not be feasible. The retrieval of immature cumulus-oocyte complexes from small antral follicles, regardless of the menstrual cycle phase, presents a swift opportunity to vitrify mature oocytes or embryos post-IVM in urgent situations or when stimulation is not advisable. Harvesting immature cumulus-oocyte complexes and immature oocytes can be achieved transvaginally or directly in the laboratory from extracorporeal ovarian tissue. Although IVM has transitioned from an experimental status due to safety validations, it relies on the intricate process of oocyte maturation. Despite successful live births resulting from IVM in fertility preservation contexts, the comparatively lower developmental competence of in vitro matured oocytes highlights the necessity to enhance IVM culture systems. Recent advancements in IVM systems hold promise in bolstering oocyte competence post-IVM, thereby narrowing the gap between IVM and outcomes from ovarian stimulation. Additionally, for optimizing the chances of conception in cancer survivors, the combination of IVM and ovarian tissue cryopreservation stands as the favored choice when ovarian stimulation is unfeasible.

Background: In vitro culture of fibroblasts is a technique based on cell isolation, physiological characterization, and cryopreservation. This technique has not been described for Galea spixii, therefore, it can be used to learn about its cellular biology and genetic diversity., Objective: We established fibroblast lines of six G. spixii individuals from several passages (second, fifth, eighth, and tenth) and cryopreserved them., Methods: Fibroblasts recovered from skin biopsies were identified based on morphology, immunocytochemistry, and karyotyping. The cells were analyzed for morphology, ultrastructure, viability, proliferation, metabolism, oxidative stress, bioenergetic potential, and apoptosis before and after cryopreservation., Results: After the eighth passage, the fibroblasts showed morphological and karyotypic changes, although their viability, metabolism, and proliferation did not change. An increase in oxidative stress and bioenergetic potential from the fifth to the eighth passages were also observed. Post cryopreservation, cell damage with respect to the ultrastructure, viability, proliferative rate, apoptotic levels, oxidative stress, and bioenergetic potential were verified., Conclusion: Fibroblasts up to the tenth passage could be cultured in vitro. However, cells at the fifth passage were of better quality to be used for reproductive techniques. Additionally, optimization of the cryopreservation protocol is essential to improve the physiological parameters of these cells., Competing Interests: Declaration of Competing Interest The authors declare having no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.

Objective: Intracytoplasmic sperm injection (ICSI) is commonly employed in preimplantation genetic testing (PGT) to minimize the risk of foreign sperm DNA contamination. Cryopreserved embryos from patients with recurrent miscarriage or repeated implantation failure, who have undergone conventional in vitro fertilization (IVF), can be thawed and biopsied for PGT. Therefore, we aimed to assess the accuracy and effectiveness of preimplantation genetic testing for aneuploidy (PGT-A) on frozen embryos using conventional IVF (c-IVF) insemination methods., Methods: From January 2021 to November 2023, our center conducted 107 thawed cryopreserved embryo biopsy cycles to screen for PGT-A. Among them, 58 cycles used c-IVF insemination, and 49 used ICSI insemination. Basic patient clinical information, laboratory data, PGT test results, and clinical outcome data were collected. To minimize the confounding effects of patient characteristics and embryo quality on PGT-A outcomes, clinical outcomes, and contamination assessment, these variables were included in the analysis. We then evaluated the blastocyst euploidy rate, clinical outcomes, and accuracy of PGT-A results between the two groups and analyzed potential contamination in the c-IVF insemination group., Results: A total of 320 blastocysts underwent PGT-A testing, with 179 blastocysts from c-IVF insemination and 141 from ICSI insemination. Considering participants' baseline characteristics and embryological outcomes, no significant differences were found between the two groups regarding infertility type, average age, body mass index, percentage of PGT-A indications, or quality of embryonic development. Regarding PGT-A results, all 320 biopsy samples were successfully analyzed, showing no statistical variance in chromosomal euploidy, abnormality, or mosaicism rates between the two insemination methods. No parental contamination was detected in the c-IVF insemination group. When assessing clinical outcomes, parameters such as biochemical pregnancy, clinical pregnancy, and miscarriage rates did not exhibit significant discrepancies between the two groups, and no misdiagnoses were reported during the study period., Conclusion: Embryo transfer and PGT-A results are not affected by potential parental contamination in frozen-thawed embryos conceived via c-IVF. PGT-A guided embryo transfer in thawed embryos conceived by c-IVF is a viable and clinically effective approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wen, Li, Cheng, Huo, Yu, Ou, Liu, Li, Fang and Lin.)

L-carnitine has an important role in the control of oxidative stress and lipid β-oxidation during in vitro culture and cryopreservation of ovarian follicles, oocytes and embryos. This substance balances the acetyl-CoA/CoA ratio, maintains glucose metabolism and increases energy production in mitochondria. It also plays a key role in reducing endoplasmic reticulum stress, by transferring palmitate to mitochondria or eliminating it to avoid toxicity. By eliminating reactive oxygen species, L-carnitine increases the percentages of mature oocytes with uniform mitochondrial distribution and improves embryo post-thaw cryotolerance. Therefore, L-carnitine controls lipid β-oxidation and oxidative stress during in vitro culture of ovarian follicles, oocyte maturation, embryonic development and cryopreservation.

This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated for motility and kinetics at different time points. Sperm viability, plasma membrane, acrosome, DNA integrity, mitochondrial membrane potential, apoptosis, and intracellular reactive oxygen species (ROS) concentrations were evaluated. The results revealed that MitoQ at concentrations of 25, 50, and 100 nM improved (P< 0.01) the total sperm motility after 30 minutes of incubation. In addition, 25 nM MitoQ improved the sperm amplitude of lateral head displacement values (P< 0.01) after 30 minutes of incubation. Conversely, negative effects on sperm motility, kinetics, and viability were observed with the highest tested concentration of MitoQ (200 nM). The various concentrations of MitoQ did not affect the plasma membrane, acrosome, and DNA integrity, or the mitochondrial membrane potential and intracellular ROS concentrations. In conclusion, supplementation of MitoQ during cryopreservation, had a mild positive effect on sperm motility and kinetics especially at a concentration of 25 nM, while the highest concentration (200nM) has a detrimental effect on motility and viability parameters of frozen-thawed stallion sperm., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024. Published by Elsevier Inc.)

The ability to cryopreserve oocytes without ultrastructural injury has been a concern in the development and use of methods to preserve female reproduction. The stability of the cell membrane must be preserved to reduce the damage caused by ice crystals during vitrification. One approach that has been explored is the use of static magnetic fields (SMFs), which are believed to influence cell membrane stability. In this study, the in vitro effects of SMF that range between 20-80 mT on the vitrification of mice germinal vesicle (GV) oocytes were studied. The viability and mitochondrial (Mt) membrane potential of both vitrified and nonvitrified oocytes were assessed using Trypan blue and JC1 staining. The high in vitro maturation (IVM) rate and high Mt membrane potential in metaphase II (MII) oocytes were taken into account to determine the optimal magnetic field intensity, that is, 20 mT. None of the SMF conditions resulted in intact spindles in MII oocytes. The study also explored the expression of store-operated calcium entry ( Stim1 , Orai1 , and Ip3r ) and meiosis resumption ( Ccnb , Cdk ) genes in GV and MII oocytes of both vitrified and control groups. The results show that the expressions of Orai1 and Ccnb genes in Vit-MII-SMF oocytes were considerably increased. However, no significant difference in Stim1 expression was observed between the groups. The Vit-MII-SMF group exhibited a significantly higher Ccnb expression compared to other groups. In vitro fertilization (IVF) was performed to evaluate the 2 pronuclear (2PN) rates. The findings demonstrated that using 20 mT SMF improved 2PN rates compared to the nonvitrified groups. This study provides a deeper understanding of the effects of moderate SMF and vitrification on the expression of calcium channel genes in GV and MII oocytes. The results suggest that applying a 20 mT SMF can help prevent cryoinjury and enhance the characteristics of vitrified-warmed oocytes.

1. This study investigated the potential use of glycerol as a cryoprotectant for quail sperm cells. Its role in maintaining sperm fertilising ability in vivo and in vitro quality parameters, such as motility, was assessed.2. The data showed that the presence of glycerol in semen samples was associated with infertility, which suggested that removal prior to insemination is mandatory. Removal through serial dilution centrifugation was associated with fewer than 5% of motile sperm cells and resulted in no fertility.3. In conclusion, glycerol alone is not suitable for quail semen cryopreservation, and other approaches need to be investigated to develop cryobanking programmes for this species.

Individuals with a disease or treatment that increases their risk of premature gonadal insufficiency may opt to undergo fertility preservation. Those who are postpubertal can often cryopreserve gametes, sperm, or eggs to expand their biologic family using assisted reproductive technologies. Ovarian tissue cryopreservation (OTC) and testicular tissue cryopreservation may be an option for individuals who are unable to use standard fertility preservation techniques. The development of OTC was critical for many patients, including prepubertal children with ovaries that do not yet produce eggs, adolescents who make few good-quality eggs, and adult women with ovaries who cannot undergo ovarian stimulation. The only option to restore fertility and hormone production after OTC is through ovarian tissue transplantation (OTT). Ovarian tissue cryopreservation and OTT have been successful for some patients. Although OTC is no longer considered experimental by the American Society for Reproductive Medicine, the process is far from standardized. Significant research needs to be done, especially at the point of OTT, to improve the success and longevity of ovarian tissue function. This article lists the main steps from surgical procurement of the ovarian tissue to transplantation and restoration of function. Our pediatric hospital program has had to decide which options in procurement, processing, cryopreservation, and warming will be used in our clinical laboratory. The options and limitations within the research and analyses are briefly discussed. Literature focusing on techniques to improve OTT effectiveness and longevity was reviewed. Ovarian tissue transplantation studies that performed xenograft experiments after pretreatment of the tissue graft by a ligand or drug, treatment of the host, or encapsulation of the ovarian tissue were identified. The intended effects of the treatments include increasing vascularization, reducing apoptosis, and directing activation or suppression of primordial follicles. Robust research in this area must continue with rigorous analyses to make strides in improving fertility preservation and restoration options for patients., Competing Interests: Declaration of Interests L.M.G. reports funding from NIH/NICHD R01HD104683 and NIH/NICHD R21HD108710 for the submitted work; funding from NIH/NICHD R01HD114210 and NIH/NICHD U01HD110336, outside the submitted work. C.J. reports funding from NIH/NICHD R01HD104683 and NIH/NICHD R21HD108710 for the submitted work; funding from NIH/NICHD R01HD114210 and NIH/NICHD U01HD110336, outside the submitted work. M.M.L. reports funding from NIH/NICHD R01HD104683, Gesualdo Family Foundation, and NIH/NICHD R21HD108710 for the submitted work; funding from NIH/NICHD R01HD114210 and NIH/NICHD U01HD110336, outside the submitted work., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the "immortal" nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model., Methods: Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus ., Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus ., Conclusion: Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.

Cryopreservation of bovine oocytes and embryos is essential for long-term preservation and widespread distribution of genetic material, particularly in bovine in vitro embryo production, which has witnessed substantial growth in the past decade due to advancements in reproductive biotechnologies. Among current cryopreservation methods, vitrification has emerged as the preferred cryopreservation technique over slow freezing for preserving oocytes and in vitro-produced (IVP) embryos, as it effectively addresses membrane chilling injury and ice crystal formation. Nonetheless, challenges remain and a simple and robust vitrification protocol that guarantees the efficiency and viability after warming has not yet been developed. Furthermore, although slow cooling can easily be adapted for direct transfer, an easier and more practical vitrification protocol for IVP embryos is required to allow the transfer of IVP embryos on farms using in-straw dilution. In addition, the susceptibility of bovine oocytes and embryos to cryoinjuries highlights the need for novel strategies to improve their cryotolerance. This manuscript examines various methodological approaches for increasing the viability of bovine oocytes and IVP embryos during vitrification. Strategies such as modifying lipid content or mitigating oxidative damage have shown promise in improving cryotolerance. Additionally, mathematical modelling of oocyte and embryo membrane permeability has facilitated the rational design of cryopreservation protocols, optimizing the exposure time and concentration of cryoprotectants to reduce cytotoxicity., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Objective: The study investigated factors associated with successful intra-operative oocyte retrieval for fertility preservation during transabdominal gynecologic surgery., Study Design: A total of 29 patients who underwent intraoperative oocyte retrieval during staging surgery at a single academic hospital from May 2014 to August 2022 were enrolled in this study, and their outcomes were analyzed., Results: Among 29 patients who underwent intra-operative oocyte retrieval during staging surgery, oocytes were obtained in 24 patients, representing 82.8 % of the retrieval rate (24/29), and two patients returned to use cryopreserved oocytes (6.9 %). Among 24 women who succeeded in obtaining oocytes, 20 patients succeeded in oocyte cryopreservation, and two patients proceeded to embryo cryopreservation. The cryopreservation rate was 91.7 % (22/24). All patients with failed oocyte retrieval (n = 5) and cryopreservation (n = 7) were diagnosed with malignancy. AMH of those with successful cryopreservation oocytes was higher than those without cryopreservation (4.10 ng/mL vs. 1.18 ng/mL, p = 0.003). A higher portion of the unstimulated cycle was observed in those with failed cryopreservation (8.3 % vs. 40.0 %, p = 0.01). No complications were noted., Conclusion: For women planning to undergo open pelvic surgery, intra-operative oocyte retrieval is a feasible option. High serum AMH and ovarian stimulation before surgery may predict successful oocyte cryopreservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Premature acrosomal exocytosis in cryopreserved semen is one of the reasons attributed to low fertility among livestock. In the present study, we attempted to enhance the cryopreserved semen quality by selective removal of acrosome-reacted spermatozoa using FITC-PNA conjugated iron magnetic nanoparticles (MNPs). Further, the effect of nano purification on other sperm functional attributes was also assessed. Iron MNPs were prepared using co-precipitation method and dextran-coated MNPs were conjugated with FITC-PNA (0.04 mg/mL). A preliminary experiment was conducted to standardise the dose of FITC-PNA conjugated iron MNPs (0.02, 0.05, 0.1, 0.15, 0.2, 0.4 and 0.6 mg). Among the different doses used, 0.6 mg FITC-PNA conjugated iron MNPs significantly (p < 0.05) removed higher acrosomal reacted spermatozoa from the semen, and therefore, this dose was used in further experiments. Cryopreserved semen from Holstein Friesian breeding bulls (n = 6) were thawed and washed using Sperm-TALP to remove residual extender. Washed spermatozoa (2 × 10 6 ) were exposed to 0.6 mg of FITC-PNA conjugated iron MNPs for 10 min at 37°C. The nano purified semen was assessed for various vital sperm parameters viz., viability, intracellular calcium, apoptosis, mitochondrial ROS and mitochondrial membrane potential using flow cytometry. We found that nanopurification using FITC-PNA conjugated iron MNPs significantly (p < 0.05) improved the sperm quality. The proportion of viable non-apoptotic spermatozoa with low intracellular calcium levels was significantly (p < 0.05) enriched in nano purified semen. Nano purification did not affect sperm mitochondrial membrane potential and ROS production. In conclusion, these preliminary findings indicate that FITC-PNA coated iron MNPs effectively removed acrosome reacted spermatozoa and significantly improved sperm functional attributes in the purified fraction., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Cryopreservation is a common way for long-term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane-impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo-injury and are easily removed post-thaw, are promising candidates with biocompatibility and feasibility. Water-soluble hydroxyl-containing polymers such as poly(vinyl alcohol) and polyol-based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio-inspired (glyco)polypeptides can significantly increase post-thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells., (© 2024 Wiley‐VCH GmbH.)

Purpose: To evaluate changes of hydroxyproline concentration and its influencing factors of small incision lenticule extraction (SMILE)-derived corneal stromal lenticules with different preservation methods., Methods: A total of 390 corneal stromal lenticules of 195 patients were derived from SMILE surgeries. Thirty of the lenticules were classified as the fresh (control) group, and the rest were randomly and evenly divided and stored in anhydrous glycerol, silicone oil, Optisol, and cryopreservation for 1 day, 1 week, or 1 month. A hydroxyproline assay kit (ab222941, Abcam) was used to measure the hydroxyproline concentration in each preservation method. Concentrations of MMP-2, TIMP-2, TNFα, TGFβ2, and reactive oxygen species were also evaluated., Results: In the anhydrous glycerol group, the concentration of hydroxyproline decreased within 1 week (fresh: 1 dΔ = 0.229, P < 0.001*; 1 d - 1 wΔ = 0.055, P < 0.001*) while that in the silicone oil group remained stable in 1 week (1 d - 1 wΔ = -0.005, P = 0.929) and decreased significantly in 1 m (1 m - 1 wΔ = -0.041, P = 0.003*). The sequence of hydroxyproline concentration in the Optisol group was 1 m > 1 day > 1 week. Hydroxyproline concentration in the cryopreservation group decreased within 1 m. Hydroxyproline concentration was highest in the Optisol group and lowest in the anhydrous glycerol group under the same preservation time. Hydroxyproline concentration was negatively correlated with MMP-2 (r = -0.16, P = 0.421) and TIMP-2 (r = -0.56, P = 0.002*) while MMP-2 and TNFα (r = 0.17, P = 0.242), TIMP-2 and TGFβ2 (r = 0.21, P = 0.207), and TNFα and reactive oxygen species (r = 0.52, P = 0.007*) were positively correlated., Conclusions: More collagen was retained in SMILE lenticules preserved in Optisol under the same preservation time. The mechanism of the changes of collagen in preserved SMILE-derived lenticules and oxidative stress requires additional investigation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Is it safe and effective to perform controlled ovarian stimulation (COS) and oocyte retrieval (OR) in prepubertal and peripubertal patients?, Methods: In this retrospective cohort study, data of 20 pre-/peripubertal patients who underwent COS and OR for the purpose of oocyte cryopreservation (OC) between 2008 and 2023 were reviewed. Following COS, all OR procedures were performed transabdominally using a vaginal ultrasound probe. Ovarian reserve was assessed by serum FSH, LH, estradiol, AMH, and antral follicle counts (AFC) in all subjects. All mature oocytes were vitrified., Results: Mean age of the patients was 15.05 ± 1.87, mean AMH was 0.84 ± 0.8 ng/ml, mean FSH was 6.39 ± 3.95 IU/L, mean estradiol was 61.6 ± 51.9 pg/ml, mean LH was 4.69 ± 3.46 IU/L, and mean AFC was 5.5 ± 5.82. Among the patients, 12 had regular menstrual cycle, 5 had irregular menstrual cycle, whereas 3 patients still did not have their menarche yet. The indications for OC were as follows: primary ovarian insufficiency (n = 7), ovarian surgery for ovarian tumors (n = 5) or ovarian torsion (n = 1), mosaic Turner syndrome (n = 2), acute lymphoblastic leukemia (n = 1) anaplastic B-cell lymphoma (n = 1), Ewing's sarcoma (n = 1), Noonan syndrome (n = 1), and Thalassemia (n = 1). The mean number of oocytes retrieved, MII oocytes frozen, and maturation rate were 5.11 ± 5.0, 3.92 ± 4.48, and 75.1 ± 25.6%, respectively. Stepwise linear regression analysis demonstrated a positive correlation between AFC and number of total oocytes retrieved and number of MII oocytes. In the case diagnosed with Noonan syndrome, all 7 retrieved oocytes were MI and all frozen at MI phase. No patient had any complication related to COS or OR., Conclusion: Even though number of the enrolled subjects is limited and mean AMH is lower in our cohort, we demonstrated that performing COS and OR is safe in pre-/peripubertal patients. If required, transabdominal route can be performed in this age group for OR. AFC appears as a prognostic factor for stimulation outcome in this age group. Pediatric patients or young adolescents at risk for primary ovarian insufficiency should not be discouraged from utilizing OC., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5 mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26 days post hatching) and grow-out periods (from June to October: between 26 and 105 days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106 µm s -1 ), LIN (75%), and ALH (1.84 µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61 µm s -1 ) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company)., Competing Interests: Declarations Ethics approval The experiments were carried out in accordance with the Government Decree 40/2013 (II. 14.) on Animal Experiments and Act XXVIII of the 1998 Hungarian Parliament on the protection and humane treatment of animals. Furthermore, the study was performed with the knowledge and permission of the Institutional Animal Welfare Committee of the Hungarian University of Agricultural and Life Sciences, Szent István Campus (Certification number: MATE-SZIC/1742–1/2022). Consent to participate All co-authors agreed to participate to the submission. Consent for publication All co-authors agreed to publish this study. Competing interests The authors declare no competing interests., (© 2023. The Author(s).)

The present study compares two protocols for the cryopreservation of chicken semen. Both protocols had an initial low cooling rate in the first step, followed by higher cooling rates around ice nucleation (Protocol 1) or following the dissipation of the latent heat of fusion (Protocol 2) in the second step. Semen ejaculates obtained from 12 roosters were diluted with Rootex with 6% dimethylformamide and frozen following either Protocol 1 (from +5°C to -10°C at 5°C/min and from -10°C to -130°C at 60°C/min) or Protocol 2 (from +5°C to -35°C at 7°C/min and from -35°C to -140°C at 60°C/min). Compared with fresh semen, following both protocols, cryopreservation resulted in reduced post-thaw sperm quality (p < .001). Post-thaw percentage of sperm with an intact plasma membrane was greater using Protocol 2 than Protocol 1 (p < .05). The results suggest that high cooling rates around the time of ice nucleation are not recommendable., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Background: Awareness of the age-related decline in fertility potential has increased the popularity of planned oocyte cryopreservation (POC). However, data regarding outcomes of POC, including rates of women returning to thaw oocytes, as well as pregnancy and live birth rates, are scarce and based mostly on small case series., Objective and Rationale: POC was defined as cryopreservation exclusively for prevention of future age-related fertility loss. The primary outcome was live birth rate per patient. The secondary outcomes included the return to thaw rate and laboratory outcomes. A meta-regression analysis examining the association between live birth and age above 40 or below 35 was conducted., Search Methods: We conducted a systematic database search from inception to August 2022. The search included PubMed (MEDLINE) and EMBASE. Our search strategies employed a combination of index terms (Mesh) and free text words to compile relevant concepts. The systematic review and meta-regression were undertaken following registration of systematic review (PROSPERO registration number CRD42022361791) and were reported following guidelines of Preferred Reporting Items for Systematic Review and Meta-Analyses 2020 (PRISMA 2020)., Outcomes: The database search yielded 3847 records. After the selection process, 10 studies, conducted from 1999 to 2020, were included. Overall, 8750 women underwent POC, with a mean cryopreservation age of 37.2 (±0.8). Of those, 1517 women returned to use their oocytes with a return rate of 11.1% (± 4.7%). The mean age at the time of cryopreservation for women who returned to use their oocytes was 38.1 (±0.4), with an average of 12.6 (±3.6) cryopreserved oocytes per woman. In a meta-analysis, the oocyte survival rate was 78.5% with a 95% CI of 0.74-0.83 (I2 = 93%). The live birth rate per patient was 28% with a 95% CI of 0.24-0.33 (I2 = 92%). Overall, 447 live births were reported. In a sub-group analysis, women who underwent cryopreservation at age ≥40 achieved a live birth rate per patient of 19% (95% CI 0.13-0.29, I2 = 6%), while women aged ≤35 years old or younger had a higher live birth rate per patient of 52% (95% CI 0.41-0.63, I2 = 7%)., Wider Implications: POC emerges as a feasible option for women aiming to improve their chances of conceiving at a later reproductive age. Nonetheless, it must be acknowledged that the overall success rates of POC are limited and that the likelihood of successful live birth declines as the age at cryopreservation rises. With increasing interest in POC, the collation of comprehensive and high-quality data is imperative to clearly define the outcomes for various age groups., Registration Number: CRD42022361791., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Vitrification of reproductive cells is definitely essential and integral in animal breeding, as well as in assisted reproduction. However, issues accompanied with this technology such as decreased oocyte competency and relatively low embryo survival rates appear to be a tough conundrum that has long perplexed us. As significant organelles in cell metabolism, mitochondria play pivotal roles in numerous pathways. Nonetheless, extensive evidence has demonstrated that vitrification can seriously impair mitochondrial function in mammalian oocytes. Thus, in this article, we summarize the current progress in oocyte vitrification and particularly outline the common mitochondrial abnormalities alongside subsequent injury cascades seen in mammalian oocytes following vitrification. Based on existing literature, we tentatively come up with the potential mechanisms related to mitochondrial dysfunction and generalize efficacious ways which have been recommended to restore mitochondrial function.

The technological necessity that has driven the field of ovarian tissue cryopreservation arose from the urgency of saving the reproductive potential of children with cancer facing gonadotoxic therapies. The field has expanded rapidly, with many centers offering this service, yet there are few outcomes reported because of the time lag between tissue freezing and its utilization. Questions about the best practices for patient selection, the best techniques for freezing and thawing, and the large loss of oocytes that accompanies the freezing and thawing process remain. These problems will need to be solved before this important clinical advance can be said to fully live up to its promise., Competing Interests: Declaration of Interests N.S. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: Fertility issues are of great concern for young women undergoing treatment for breast cancer (BC). Fertility preservation (FP) protocols using controlled ovarian stimulation (COS) with letrozole have been widely used with overall good results. However, letrozole cannot be used in every country in this context. This study aimed to assess the efficacy of tamoxifen for COS in women with early BC undergoing FP., Methods: This multicentric prospective study included patients aged 18-40, diagnosed with stage I, II and III invasive BC, undergoing tamoxifen-COS before adjuvant or neoadjuvant chemotherapy (NAC). The primary endpoint was the efficacy of tamoxifen-COS protocol evaluated by the number of oocytes collected and vitrified. Secondary endpoints included the time interval before chemotherapy, breast cancer (BC) recurrence rates, and reproductive outcomes., Results: Ninety-five patients were included between 2014 and 2017, aged 31.5 ± 4 years on average. 37.9 % received NAC and 62.1 % received adjuvant chemotherapy. FP procedure was successful in 89.5 % of the cycles. The mean number of collected and vitrified oocytes was 12.8 ± 7.9 and 9.8 ± 6.2, respectively. The mean duration of COS was 10.4 ± 1.9 days. Median time before chemotherapy initiation was 3.6 weeks (IQR 3.1; 4.1) for women receiving NAC. Five-year relapse-free and overall survival rates were in-line with those expected in this population. Twenty-one women had spontaneous full-term pregnancies, while 5 underwent IVF cycles with frozen-thawed oocytes, without pregnancy., Conclusion: Tamoxifen-COS protocols appear to be feasible before adjuvant or NAC treatment in young BC patients and efficient in terms of oocyte yield., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:None of the authors have any conflicts of interest related to this study. Florence Leperlier reports honoraria for lectures and presentations from Ferring Pharmaceuticals, Gedeon Richter, and IBSA, unrelated to this work. Pierre-Emmanuel Bouet reports travel and meeting support from Merck, Gedeon-Richter, Theramex, Ferring, Organon and IBSA. P-E.B. have undertaken consultancy work for Merck and Gedeon-Richter and have received research grants from Theramex, Ferring, MSD, and Genevrier, not related to the present work. Thomas Freour has undertaken consultancy work for Vitrolife France and Gedeon-Richter. T.F. reports travel and meeting support from Gedeon-Richter, Theramex, Ferring and IBSA. T.F. reports honoraria for lectures and presentations from Ferring Pharmaceuticals, Theramex, Gedeon Richter and Merck Serono, unrelated to this work. Marie Robert reports travel fees and congress fees from AstraZeneca, Gilead and Creafirst, and has undertaken consultancy work for AstraZeneca and Lilly. Mario Campone is on the advisory board of AstraZeneca, Novartis, Sanofi, Lilly, Pfizer, Seagen, Gilead and Daiichi-Sankyo. M.C. has undertaken consultancy work for AstraZeneca, Novartis, Daiichi-Sankyo, PET-Therapy, Menarini and DIACCURATE, and he is a speaker for Novartis, Lilly and Amgen. He reports travel support from Pfizer, Novartis, Roche, AstraZeneca and Daiichi-Sankyo., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Meat quality (MQ) is unstable during cold chain logistics (CCL). Different technologies have been developed to enhance MQ during the CCL process, while most of them cannot cover all the links of the cold chain because of complex environment (especially transportation and distribution), compatibility issues, and their single effect. Electric fields (EFs) have been explored as a novel treatment for different food processing. The effects and potential advantages of EFs for biological cryopreservation have been reported in many publications and some commercial applications in CCL have been realized. However, there is still a lack of a systematic review on the effects of EFs on their quality attributes in meat and its applications in CCL. In this review, the potential mechanisms of EFs on meat physicochemical properties (heat and mass transfer and ice formation and melting) and MQ attributes during different CCL links (freezing, thawing, and refrigeration processes) were summarized. The current applications and limitations of EFs for cryopreserving meat were also discussed. Although high intensity EFs have some detrimental effects on the quality attributes in meat due to electroporation and electro-breakdown effect, EFs present good applicability opportunities in most CCL scenes that have been realized in some commercial applications. Future studies should focus on the biochemical reactions of meat to the different EFs parameters, and break the limitations on equipment, so as to make EFs techniques closer to usability in the production environment and realize cost-effective large-scale application of EFs on CCL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Study Question: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos?, Summary Answer: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another., What Is Known Already: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence., Study Design, Size, Duration: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls., Participants/materials, Setting, Methods: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC)., Main Results and the Role of Chance: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673)., Limitations, Reasons for Caution: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial., Wider Implications of the Findings: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required., Study Funding/competing Interests: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Allogeneic hematopoietic stem cell transplant (alloHSCT) is a mainstay of treatment for hematologic malignancies such as acute leukemias and aggressive lymphomas. Historically, fresh hematopoietic progenitor cell (HPC) products have been preferred to cryopreserved products (cryo-HPC) due to concerns of loss of stem cell viability and number with the cryopreservation procedure., Objective: We aimed to analyze the outcomes of patients who received cryo-HPCs during the COVID-19 pandemic and compare this against historical cohorts that received fresh HPC., Study Design: A retrospective chart review was conducted on all adult patients who received a peripheral blood alloHSCT in British Columbia, Canada between June 2017 and November 2021. Baseline characteristics, Kaplan-Meier (KM) overall survival (OS), engraftment, and incidences of acute and chronic graft versus host disease were compared between patients who received cryo-HPCs and fresh HPCs. Univariable analysis followed by multivariable analysis was performed using a backward stepwise selection procedure to generate predictors of OS, cumulative incidence of relapse (CIR), nonrelapse mortality (NRM), and primary and secondary graft failure., Results: Three hundred eighty-three patients were included in the analysis, with cryo-HPC representing 40%. Median viability was higher in the fresh-HPC group at 99.2% (IQR 98.3-99.5) versus cryo-HPCs at 97.0% (96.0, 98.6) (P < 0.01). The 12-month actuarial survivals were 77% in the fresh HPC and 75% in the cryo-HPC groups (P = 0.21). There were no differences between cryo-HPCs and fresh HPCs on univariable analysis of OS, CIR, or NRM. There was a shorter median time to platelet engraftment in patients receiving fresh HPC at 17 days (IQR 16, 20) versus cryo-HPC at 21 days (IQR 18, 29), P < 0.001. There was a shorter median time to neutrophil engraftment in the fresh HPC group at 17 days (IQR 14, 20) versus 20 days (17, 23), P < 0.001. Cryo-HPC accounted for 5 out of 6 cases of primary graft failure (P = 0.04), and 3 out of five cases of secondary graft failure (P = 0.39). There were no significant differences in acute GVHD between the fresh HPC and cryo-HPC groups (P = 0.34). The incidence of moderate or severe chronic GVHD was 32% in the fresh-HPC group and 17% in the cryo-HPC group (P < 0.001). In multivariable analysis, cryopreservation did not emerge as an independent predictor of OS, CIR, NRM, primary GF or secondary GF. However, viability <90% on arrival at our center was a significant predictor of OS (HR 5.3, 2.3-12.3, P < 0.01), primary graft failure (OR 36.3, 5.4-210.2, P < 0.01), and secondary graft failure (OR 18.4, 1.7-121.1, P < 0.01)., Conclusions: Patients who received cryo-HPCs had similar OS and relapse rates to those who received fresh-HPCs but typically took 2-3 days longer to achieve engraftment of platelets or neutrophils and were associated increased primary graft failure. However, after accounting for multiple variables, cryopreservation was no longer a significant predictor of survival or engraftment while viability <90% emerged as an important predictor of OS, primary graft failure, and secondary graft failure. If confirmed, this suggests that viability on arrival at the infusion center may be a good quality control indicator used to identify HPC products that may warrant recollection if the risk of graft failure is sufficiently increased., Competing Interests: Declaration of Competing Interest The following authors have financial disclosures as outlined in the following: David Sanford, Membership on an entity's Board of Directors or advisory committees (Abbvie, Astellas); Kevin Song, honoraria (Janssen, Sanofi, BMS, Forus, Amgen, GSK, Gilead, Novartis); Shanee Chung, Consultancy & honoraria (Takeda), Honoraria (Astella Pharma, Novartis, Paladin, Pfizer); Ryan Stubbins, Honoraria (AbbVie, Pfizer, Jazz, Takeda), Advisory board (AbbVie), Research funding (Jazz); Heather Sutherland, Honoraria (Amgen, Forus, BMS); Kevin Hay, research funding (Janssen), Honoraria (BMS, Kite/Gilead, Novartis). The remaining authors have no competing interests or financial disclosures., (Copyright © 2024 International Society for Cell & Gene Therapy. All rights reserved.)