Optimizing Vitrification Equilibration Methods: Toward a Better Protocol for the Recovery and Subsequent Development of Mature Oocytes from Ovarian Cancer Patients

Optimizing Vitrification Equilibration Methods: Toward a Better Protocol for the Recovery and Subsequent Development of Mature Oocytes from Ovarian Cancer Patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing Vitrification Equilibration Methods: Toward a Better Protocol for the Recovery and Subsequent Development of Mature Oocytes from Ovarian Cancer Patients Hao Wang, Qi Jiang, Qiaoyun Wu, Qiaodan Li, Xue Sun, Xiao Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8975751/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Ovarian cancer affects a significant number of women of reproductive age, and while advances in treatment have dramatically improved survival rates, the gonadotoxic effects of chemotherapy and radiotherapy pose a major threat to future fertility. For these patients facing an urgent need to begin cancer therapy, oocyte cryopreservation via vitrification is the standard method for fertility preservation. The success of vitrification hinges on the equilibration step, where oocytes are exposed to cryoprotectants. While standard protocols use a gradual approach, a fast vitrification (FV) method with a shortened equilibration time has been developed. Objectives To evaluate whether the shortened equilibration time in fast vitrification (FV) achieves high oocyte survival and maintains functional integrity post-fertilization, thereby supporting its application for fertility preservation in ovarian cancer patients. Methods To compare standard vitrification (SV) and fast vitrification (FV) procedures in mouse oocyte survival after vitrification-warming (VW) and subsequent early embryonic development following fertilization. For SV procedure was as that the oocytes were equilibrated in V1 (7.5% dimethyl sulphoxide, DMSO and 7.5% ethylene glycol, EG) solution for 5 minutes and then transferred the oocytes to V2 (15% DMSO and 15% EG) for 1 minute, and the oocytes were immediately loaded onto a carrier for cooling directly plunging into LN 2 ; For FV procedure was as that oocytes were equilibrated in V1 solution for 1 minute and then transferred to V2 solution for 1 minute, and the oocytes immediately loaded onto a carrier for cooling by directly plunging into LN 2 . For warming, one-step warming (OW) procedure was applied in both groups. The survival rates and early embryonic development potentials of the warmed oocytes were compared following insemination by piezo-ICSI. Spindle integrity of the warmed oocytes was measured by α-tubulin/Hoechst staining, and mitochondrial status was assessed by Mitotracker/JC-1 staining and the mitochondrial damages were analyzed by confocal microscope. Additionally, the apoptosis status of the formed blastocysts in each group was compared via TUNEL assay. Results With shortened equilibration time, the FV procedure maintained comparable oocyte survival and fertilization as well as early embryonic development rates to SV procedure. Mechanistic measures revealed that there were no differences between the oocytes vitrified by SV and FV procedures in terms of spindle integrity, mitochondrial status and damages as well as apoptosis status in the formed blastocysts. Conclusion By reducing exposure time to cryoprotectants and limiting time under suboptimal temperature and osmotic conditions, fast vitrification (FV) offers an efficient oocyte cryopreservation method. The improved workflow afforded by FV supports its application in fertility preservation for ovarian cancer patients. Ovarian Cancer Fertility preservation Oocyte Fast vitrification One-step warming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction For young women diagnosed with ovarian cancer, fertility preservation has become an integral component of comprehensive oncological care [ 1 ]. Although fertility-sparing surgery may be feasible for select patients with early-stage disease, adjuvant chemotherapy is often required and poses a significant threat to future fertility due to the well-established gonadotoxic effects of platinum-based agents and alkylating agents [ 2 ]. Oocyte cryopreservation following controlled ovarian stimulation represents the standard approach for fertility preservation in post-pubertal women facing urgent cancer treatment [ 3 ]. Since the introduction of vitrification, there has been a significant improvement in the efficacy of oocyte cryopreservation [ 4 ]. Vitrification achieves rapid cooling, transforming intracellular water into a glass-like state, thereby preventing ice crystal formation and reducing oocyte damage [ 5 , 6 ]. The standard vitrification (SV) procedure, which has been widely established, relies on the use of high concentrations of cryoprotectants during an equilibration phase that allows cryoprotectants to penetrate the cell membrane and replace intracellular water over a few minutes (with longer durations potentially required for blastocysts) [ 7 ]. This is followed by rapid cooling, which facilitates the transition of intracellular water into a glass-like state and avoids structural cellular damage. The SV procedure has significantly improved oocyte survival, fertilization, and pregnancy rates following embryo transfer [ 8 ]. Recently, novel alternatives to the SV procedure, such as ultra-fast vitrification (UFV) and one-step warming (OW), have been reported to achieve high survival rates for both oocytes and embryos. Most clinical reports have demonstrated that OW for embryos following the SV procedure, particularly for blastocysts, achieves comparable survival and higher pregnancy rates [ 9 – 13 ]. However, reports on the application of UFV and OW specifically for oocytes remain limited [ 14 – 17 ]. Gallardo et al. reported that oocyte dehydration upon exposure to standard cryoprotectant solutions occurs very rapidly, with the minimum volume of the shrink-swell curve reached within 60 seconds [ 14 ]. At this point, the ejection of intracellular water is complete, and coupled with the permeation of low molecular weight cryoprotectants, results in similar intracellular and extracellular solute concentrations. This finding suggests that prolonging exposure to cryoprotectant solutions does not enhance the cytosolic glass-forming tendency and may be unnecessary. It implies that short exposures to increasingly hypertonic solutions could represent a more time-efficient strategy for preparing human oocytes and embryos for vitrification. Indeed, in clinical practice, the SV procedure is time-consuming, with most of the duration dedicated to a long equilibration phase (minimum of 5–8 minutes) in a lower concentration of cryoprotectants, followed by exposure to a higher concentration vitrification solution (minimum of 1 minute). Reducing the duration of vitrification and warming procedures is desirable to improve workflow in a busy IVF setting and to minimize exposure to suboptimal temperatures and osmolarity, as well as the potential toxicity of cryoprotectants. Although it has been reported that ultra-fast vitrification (FV) appears more effective for oocyte cryopreservation due to efficient cytoplasmic water extraction, reduced osmotic stress, and minimization of cell contraction and expansion amplitude [ 15 ], the mechanistic functions of the FV procedure remain unclear. This is particularly true concerning the impact of shortened equilibration time on subsequent embryonic development following fertilization. In this study, therefore, we compared SV and FV procedures in terms of oocyte survival and subsequent early embryonic development following the OW procedure and insemination. Additionally, molecular assessments were performed to compare these two procedures, providing evidence of both efficiency and safety. We believe this information is crucial for evaluating the efficacy of the FV procedure for oocyte vitrification. Materials and Methods Animals and oocyte collection Eight-week-old female C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under controlled environmental conditions (20–22°C, 50–70% relative humidity, 12 h light/dark cycle) with ad libitum access to food and water. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Review Committee of Zhejiang University (ZJU20250897) and were performed in accordance with institutional and national animal welfare guidelines. Female mice were super ovulated by intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG), followed by 10 IU human chorionic gonadotropin (HCG) 46–48 h later. Cumulus-oocyte complexes (COCs) were collected from the oviductal ampulla 14 h post-HCG administration and treated with hyaluronidase solution (80 units/mL) to remove cumulus and granulosa cells, yielding metaphase II (MII) oocytes for the experiments. The method of euthanasia for experimental mice was cervical dislocation [ 18 – 19 ]. Experimental design Two vitrification procedures were compared: 1) The established SV procedure, the oocytes were equilibrated in 200 µL of V1 solution (7.5% DMSO + 7.5% EG, Vitrification Kit, ARSCI Biomedical Inc, Jiaxing, China) for 5 minutes at the room temperature, and then transferred the oocytes to 200 µL of V2 solution (15.0% DMSO + 15.0% EG) for 1 minute and immediately loaded onto RC Straw (ARSCI Biomedical Inc, Jiaxing, China) and then rapidly plunged into liquid nitrogen for storage (Fig. 1 SV); 2) The shortened equilibration time of FV procedure, the oocytes were equilibrated in 200 µL of V1 solution for 1 minute at the room temperature, and then transferred the oocytes to 200 µL of V2 solution for 1 minute and loaded immediately onto RC Straw and then rapidly plunged into liquid nitrogen for storage (Fig. 1 , FV). For warming of the vitrified oocytes, One-Step Warming Solution (OSWS, ARSCI Biomedical Inc, Jiaxing, China) was used (Fig. 1 , OW). Briefly, RC Straw was directly inserted into pre-warmed (37°C) OSWS solution (1.0 M Sucrose) for 1 minute, and then the oocytes were transferred to pre-warmed (37°C) Embryo Development Medium (EDM, ARSCI Biomedical Inc, Jiaxing, China) for washing 1 minute and then cultured in EDM until insemination in 5.0% CO 2 incubator (37°C, 100% humidity). After warming, the survival of the oocytes was assessed by morphological observation, and the survived oocytes were subjected to insemination by intracytoplasmic sperm injection (ICSI) method or other experimental assessments [ 13 ]. Insemination and embryonic development culture ICSI was performed using a PIEZO-driven microinjection system (Eppendorf) [ 20 ]. Briefly mouse sperm heads were separated from tails by pulse vibration in PVP solution (ARSCI Inc, RC-1120) and then injected into oocyte cytoplasm. Injected oocytes were washed three times with Gamete Buffer Medium (ARSCI Inc., RC-1020) and cultured in EDM droplets (50 µL) for fertilization assessment after 10 hours of ICSI procedures. Embryonic development potential was observed at 24 hours (2-cell stage), 48 hours (4-cell stage), 60 hours (8-cell stage), 3 days (compaction), and 4 days (blastocyst formation) post-ICSI, respectively [ 21 ]. Blastocyst development rates were evaluated on the day of end culture [ 22 ] [ 23 ], and the formed blastocysts were performed by apoptosis detection using TUNEL assay. Oocyte immunofluorescence The spindle of oocytes as a crucial structure in oocyte meiosis and fertilization serves as an important indicator for assessing oocyte developmental potential [ 24 ]. The warmed oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes, and then permeabilized and blocked in PBS containing 0.1% Triton X-100 (Sigma) and 10% fetal bovine serum (FBS, Gibco) for 1 hour at room temperature. After three times of washing with PBS, the oocytes were incubated with 1:1000 diluted FITC-conjugated mouse anti-α-Tubulin antibody (Sigma) for 2 hours at room temperature. Finally, DNA was stained with Hoechst 33342 (Sigma) for 10 minutes and images were observed and analyzed by using confocal microscopy. Oocyte mitochondrial Mitotracker live-cell staining The warmed oocytes were incubated in 1:1000 diluted Mitotracker dye (in EDM) at 37°C for 30 minutes, followed by Hoechst 33342 DNA staining for 10 minutes. After three washes with Gamete Buffer Medium (ARSCI Inc., RC-1020), images were observed and analyzed using Evident (Olympus) SpinSR [ 25 ]. Oocyte mitochondrial membrane potential JC-1 detection JC-1 mitochondrial membrane potential detection kit (Beyotime) was used. Briefly, the oocytes were incubated in JC-1 staining working solution at 37°C for 20 minutes and then washed three times with Gamete Buffer Medium (ARSCI Inc., RC-1020). Obtained images were observed using Evident (Olympus) SpinSR to assess membrane potential through green/red fluorescence ratio. Blastocyst apoptosis detection (TUNEL assay) Apoptosis is a normal phenomenon during embryonic development, but excessive apoptosis can affect embryo quality and developmental potential [ 26 ]. TUNEL apoptosis detection kit (Beyotime) was used and combined with confocal microscopic observation [ 27 ]. Briefly, the blastocysts were fixed in 4% PFA at room temperature for 30 minutes, permeabilized with 0.3% Triton X-100 for 5 minutes, washed once with PBS, and then incubated with TUNEL reaction solution at 37°C in the dark for 1 hour. Nuclei were counterstained with Hoechst 33342 for 10 minutes and observed by using confocal microscopy. Statistical analysis Statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., USA). All data are expressed as the mean ± SEM from at least three independent experiments. For comparisons between two groups, Student’s t-test was applied, while one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) test was used for comparisons among multiple groups. A p-value of less than 0.05 was considered statistically significant. Results The FV procedure yielded a comparable oocyte survival rate As shown in Fig. 2 , FV procedure achieved 97.22 ± 1.10% (a total of 141 oocytes with 9 replicates) survival rate, which was comparable to that of SV procedure (98.61 ± 0.92%, a total of 139 oocytes with 9 replicates). Also, the morphological observation of the warmed oocytes was similar among groups (Fig. 2 ). The FV procedure showed no significant impact on spindle morphology Neither FV nor SV procedures affected oocyte spindle morphology (Fig. 3 ). The rates of normal spindle morphology in oocytes were 95.83 ± 2.08% in Fresh (47 oocytes), 95.69 ± 2.16% in SV (46 oocytes), and 95.56 ± 2.22% in FV (45 oocytes), respectively, with no significant differences among groups. The FV procedure showed no significant impact on fertilization and early embryonic development As shown in Table 1, there were no differences in both groups of fertilization and early embryonic development as well blastocyst formation rates, but they were significantly lower (P < 0.01) than Fresh oocyte group. There were also no differences in morphological observation during early embryonic development among groups (Fig. 4 , a total of 133 zygotes 3 replicates). The FV procedure did not affect mitochondrial aggregation As shown in Fig. 5 , Mitotracker staining revealed that the fluorescence intensity of mitochondria in the oocytes was comparable among Fresh, SV, and FV groups. However, mitochondrial staining revealed that Fresh oocytes showed minimal aggregation (15.14 ± 1.81%, a total of 46 oocytes with 3 replicates) and significantly alleviated aggregation compared to SV oocytes (43.47 ± 1.93%, a total of 46 oocytes with 3 replicates, P < 0.001) and FV oocytes (43.61 ± 3.06%, a total of 46 oocytes with 3 replicates, P < 0.01) groups. Mitochondrial membrane potential was well maintained by the FV procedure As shown in Fig. 6 , JC-1 staining indicated that there were no differences in the ratio of mitochondrial membrane potential in the oocytes subjected to the FV procedure (a total of 15 oocytes) compared to SV procedure (a total of 15 oocytes). Also, there were no differences in Fresh (A total of 15 oocytes) compared to two groups. Blastocyst apoptosis was unaffected by FV procedure As shown in Fig. 7 , there were no significant differences in the numbers and percentages of apoptotic cells between blastocysts derived from FV (10.12 ± 0.60%, a total of 15 blastocysts) and SV (9.71 ± 0.62%, a total of 15 blastocysts) oocytes, and both were comparable to Fresh oocyte-derived blastocysts (10.12 ± 0.60%, a total of 15 blastocysts). Discussion The results of this study demonstrated that the FV procedure, employing a shortened equilibration time for oocyte vitrification, maintains oocyte survival, fertilization rates, and early embryonic development in vitro comparable to those achieved with the SV procedure. It further indicated that, compared to SV, the FV procedure does not compromise oocyte spindle integrity, mitochondrial distribution and aggregation, or mitochondrial damage, thereby providing an important theoretical foundation for optimizing clinical oocyte cryopreservation protocols. Initial attempts to cryopreserve oocytes utilized the same slow-freezing methods that were considered the gold standard for embryo cryopreservation. However, slow-freezing of oocytes resulted in very low survival and pregnancy rates, leading to its classification as an experimental procedure [ 28 – 31 ]. Over time, the efficacy of slow-freezing improved with increased sucrose concentrations in the freezing medium [ 32 – 34 ]. At the beginning of the 21st century, the introduction and advancement of vitrification technology marked a significant improvement in the efficacy of oocyte and embryo cryopreservation [ 35 ]. To date, vitrification-based cryopreservation of oocytes and embryos has made substantial progress, holds great promise, and has been widely adopted in clinical practice worldwide using established protocols, namely standard or conventional vitrification procedures [ 36 – 38 ]. Although vitrification and warming procedures are significantly shorter than slow-freezing methods, standard vitrification and warming protocols remain time-consuming, particularly with manual handling. The SV procedure requires a minimum duration of 5 + 1 minutes, involving direct exposure of oocytes or embryos to a low-concentration cryoprotectant solution followed by exposure to a more concentrated vitrification solution. Shortening the duration of vitrification protocols is desirable to improve workflow in busy IVF settings and to reduce exposure time to suboptimal temperatures, non-physiological osmolarity, and potentially toxic cryoprotectants. This is critical because vitrification may induce cryoinjury at multiple levels, including premature intracellular Ca²⁺ release, microtubule disruption, chromosomal abnormalities, actin microfilament damage, and oxidative stress, all of which can lead to mitochondrial and endoplasmic reticulum damage [ 39 ]. Concerns persist regarding oocyte vitrification technology due to the use of relatively high concentrations of cryoprotectants such as ethylene glycol (EG) and dimethyl sulfoxide (DMSO). Exposure to high cryoprotectant concentrations is known to damage oocytes through both cytotoxic and osmotic effects. As the results of the present study indicate, embryonic developmental potential was significantly reduced following vitrification and warming procedures (Table 1). Numerous approaches have been explored to overcome or mitigate cryoinjury, including optimizing cryoprotectant solutions to reduce toxicity, using microtubule stabilizers to prevent depolymerization, and applying antioxidant treatments to alleviate oxidative stress during vitrification [ 40 – 44 ]. Therefore, new strategies capable of acting at multiple levels are needed to improve the survival and developmental potential of vitrified-warmed oocytes and embryos. It is widely accepted that the addition and removal of cryoprotectants from oocytes create osmotic imbalances across the oocyte membrane, potentially inducing large volumetric changes and causing damage to oocyte morphology, cytoskeletal structures, and function. Thus, determining oocyte tolerance to osmotic stress is essential for understanding the functional role of cryoprotectants during vitrification. The key to successful oocyte vitrification lies in balancing the use of minimal cryoprotectant concentrations without compromising their cryoprotective efficacy. Interestingly, it has been reported that oocyte vitrification may not require high cryoprotectant concentrations in the vitrification solution, suggesting that current protocols may need refinement toward less cytotoxic solutions [ 45 ]. It is well established that, despite their protective functions, cryoprotectants can cause damage through osmotically driven cell volume changes and inherent chemical toxicity. In particular, it has been calculated that toxicity is minimized during cryoprotectant addition by allowing the cell to swell to its maximum tolerable volume before transferring it to a higher concentration of cryoprotectants, thereby avoiding excessive shrinkage. This approach may significantly reduce damage due to cryoprotectant toxicity [ 46 ]. Another mathematically optimized cryoprotectant equilibration procedure for human oocyte cryopreservation has been reported, suggesting that piecewise-constant procedures are less toxic [ 47 ]. A theoretical model has been established predicting that exposure causes oocytes to initially shrink and then swell to the maximum cell volume limit. To reach the target intracellular cryoprotectant concentration, oocytes are subsequently induced to shrink to the minimum cell volume limit by exposure to a high-concentration cryoprotectant solution, thereby significantly reducing toxicity [ 4 , 46 , 47 ]. Interestingly, Gallardo et al. reported that human oocyte dehydration occurs very rapidly upon exposure to standard cryoprotectant solutions, with the minimum volume of the shrink-swell curve reached within 60 seconds. This indicates that intracellular water ejection is completed concurrently with the permeation of low-molecular-weight cryoprotectants, resulting in similar intracellular and extracellular solute concentrations [ 14 ]. Thus, prolonging exposure to cryoprotectant solutions does not enhance the cytosolic glass-forming tendency and may be unnecessary. Recently, it has been reported that for vitrification of human immature oocytes, the FV procedure achieves results comparable to the SV procedure [ 16 , 17 ]. García-Martínez et al. reported that limiting exposure time to the equilibration solution in bovine oocytes is a more efficient approach for vitrification preparation, improving post-warming oocyte quality by protecting spindle integrity and reducing DNA fragmentation, thereby enhancing blastocyst rates and embryo quality [ 40 ]. Additionally, using mouse oocytes, Cho et al. demonstrated that shortened equilibration time in FV appears more effective for oocyte vitrification due to efficient cytoplasmic water extraction, reduced osmotic stress, and minimized cell contraction and expansion amplitude, thus compensating for the drawbacks of SV [ 15 ]. Conclusion The results of the present study demonstrate that the FV procedure with shortened equilibration time for oocyte vitrification maintains oocyte survival (Fig. 2 ), fertilization rates, and early embryonic development in vitro (Table 1, Figs. 4 and 7 ) comparable to the SV procedure. Furthermore, the FV procedure does not adversely affect oocyte spindle integrity (Fig. 3 ), mitochondrial distribution and aggregation (Fig. 5 ), or mitochondrial damage (Fig. 6 ) compared to SV. These findings provide a critical theoretical basis for the application of the FV procedure in oocyte cryopreservation for fertility preservation in ovarian cancer patients. Declarations Author Contribution J.X., X.C., R.C. designed research, H.W., Q.J, Q.L., X.S. performed research, H.W., Q.W. analyzed data, H.W., J.X., R.C. wrote the paper. All authors reviewed the manuscript. References Santos ML, Pais AS, Almeida Santos T. Fertility preservation in ovarian cancer patients. Gynecol Endocrinol. 2021;37(6):483–9. Stewart C, Ralyea C, Lockwood S. Ovarian Cancer: An Integrated Review. Semin Oncol Nurs. 2019;35(2):151–6. Alvarez M, Solé M, Devesa M, Fábregas R, Boada M, Tur R, Coroleu B, Veiga A, Barri PN. 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Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Ruvalcaba Castellón LA, García Amador MI, Montoya Sarmiento JE. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online. 2008;16(5):608–10. Chian RC, Gilbert L, Huang JY, Demirtas E, Holzer H, Benjamin A, Buckett WM, Tulandi T, Tan SL. Live birth after vitrification of in vitro matured human oocytes. Fertil Steril. 2009;91(2):372–6. Chian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cui SJ, Buckett WM, Tulandi T, Tan SL. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril. 2009;91(6):2391–8. Mogas T, García-Martínez T, Martínez-Rodero I. Methodological approaches in vitrification: enhancing viability of bovine oocytes and in vitro‐produced embryos. Reprod Domest Anim. 2024 59, e14623. García-Martínez T, Martínez-Rodero I, Roncero-Carol J, Yánez-Ortiz I, Higgins AZ, Mogas T. Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification. Theriogenology. 2022;184:110–23. Morató R, Izquierdo D, Albarracín JL, Anguita B, Palomo MJ, Jiménez-Macedo AR, Paramio MT, Mogas T. Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification. Mol Reprod Dev. 2008;75(1):191–201. Girka E, Gatenby L, Gutierrez EJ, Bondioli KR. The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes. Theriogenology. 2022;182:9–16. García-Martínez T, Vendrell-Flotats M, Martínez-Rodero I, Ordóñez-León EA, Álvarez-Rodríguez M, López-Béjar M, Yeste M, Mogas T. Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming. Int J Mol Sci. 2020;21(20):7547. Cao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. Oxidative Stress and Oocyte Cryopreservation: Recent Advances in Mitigation Strategies Involving Antioxidants. Cells. 2022;11(22):3573. Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution. PLoS ONE. 2012;7(4):e36058. Benson JD, Kearsley AJ, Higgins AZ. Mathematical optimization of procedures for cryoprotectant equilibration using a toxicity cost function. Cryobiology. 2012;64(3):144–51. Davidson AF, Benson JD, Higgins AZ. Mathematically optimized cryoprotectant equilibration procedures for cryopreservation of human oocytes. Theor Biol Med Model. 2014;11:13. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 22 Mar, 2026 First submitted to journal 13 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8975751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625813903,"identity":"e992cb42-5ac0-469c-a445-c1183d3d69e7","order_by":0,"name":"Hao Wang","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wang","suffix":""},{"id":625813905,"identity":"a46e0a88-25eb-4a1d-ad97-88ce75c75913","order_by":1,"name":"Qi Jiang","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Jiang","suffix":""},{"id":625813907,"identity":"ece99899-6e1f-4070-862b-56fb7ce025c8","order_by":2,"name":"Qiaoyun Wu","email":"","orcid":"","institution":"Yangzhou Oriental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiaoyun","middleName":"","lastName":"Wu","suffix":""},{"id":625813932,"identity":"8dbcb31d-a03d-410e-81f5-352d39fed4b6","order_by":3,"name":"Qiaodan Li","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Qiaodan","middleName":"","lastName":"Li","suffix":""},{"id":625813939,"identity":"6d2fca25-2815-400e-8b26-85263f97bf74","order_by":4,"name":"Xue Sun","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Sun","suffix":""},{"id":625813949,"identity":"6b37004b-889c-4960-a7e9-65bda362f704","order_by":5,"name":"Xiao Chen","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Chen","suffix":""},{"id":625813950,"identity":"de219641-a08b-4309-915b-6e11dac16987","order_by":6,"name":"Jian Xu","email":"","orcid":"","institution":"The Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Xu","suffix":""},{"id":625813951,"identity":"7e1519eb-747d-4291-ab7f-8296558ff4cd","order_by":7,"name":"Ri-Cheng Chian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYFACHoYDDAw2PPzsDaRpSZOR7DlAghYgOGxjcMOBSA3m7WcPHvi54zwPww0Gxg8fc4jQInMmL+Fg75nbPIyzG5glZ24jQouEBI/BAd622zzMMgfYmHmJ1XLwb9s5HjaJBBK0HOZtO8DDQ7wWnhyDw7JtyTwSPAebifQL+xnjj2/b7Oztjzcf/PCRGC1IgLGBNPWjYBSMglEwCnADAKVGMna+xQHbAAAAAElFTkSuQmCC","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Ri-Cheng","middleName":"","lastName":"Chian","suffix":""}],"badges":[],"createdAt":"2026-02-26 09:09:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8975751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8975751/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107896299,"identity":"0f98cfde-e2a2-4e9b-99f1-0ee6936be0a7","added_by":"auto","created_at":"2026-04-27 10:53:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110165,"visible":true,"origin":"","legend":"\u003cp\u003eStandard Vitrification (SV) and Fast Vitrification (FV) as well as One-step Warming (OW) procedures for oocyte vitrification and warming.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/2a52731fddcba5d438eb40a1.png"},{"id":107896307,"identity":"88485e89-9e82-4c05-990d-c06c258b36f4","added_by":"auto","created_at":"2026-04-27 10:53:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1549886,"visible":true,"origin":"","legend":"\u003cp\u003eThe morphology and survival rates of Fresh, SV and FV oocytes. No morphological differences were observed and no differences for the survival rates among groups. Scale bars=100 μm. Data are presented as mean ± SEM (a total of 419 oocytes with 9 replicates).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/ceb3f407ed7828be74ec2d16.png"},{"id":107896300,"identity":"79c2ddd4-7013-4ba0-b2a6-08051097372c","added_by":"auto","created_at":"2026-04-27 10:53:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":758068,"visible":true,"origin":"","legend":"\u003cp\u003eThe assessment of spindle and chromosome alignment morphologies in Fresh, SV and FV oocytes. Spindle integrity and stability showed no significant differences among the groups. Scale bars=10 μm. Data are presented as mean ± SEM (A total of 138 oocytes with 3 replicates).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/bf0a22eed9b771822a0b842a.png"},{"id":108006538,"identity":"76f97308-8cea-44b8-8c6d-7b4d1e428979","added_by":"auto","created_at":"2026-04-28 12:55:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4864667,"visible":true,"origin":"","legend":"\u003cp\u003eThe early embryonic development potential of the oocytes following ICSI. (A) Representative micrographs depicting embryonic development from the zygote to the blastocyst stage on Fresh, SV and FV oocytes following ICSI. (B) Early embryonic development showed no differences in SV and FV groups, but from 8-cell stage to morula and blastocyst stages in both groups were significantly lower (P\u0026lt;0.05) than Fresh group. Scale bars=100 μm. Data are presented as mean ± SEM (A total of 133 zygotes with 3 replicates).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/6f3761fdacf7e6def3bcda48.png"},{"id":108006458,"identity":"d7f774ab-f437-4dbc-96ee-2095886894ce","added_by":"auto","created_at":"2026-04-28 12:55:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1728680,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution of mitochondria within the oocyte. (A) Representative micrographs showing the distribution of mitochondria in Fresh, SV and FV oocytes. (B) The fluorescence intensity of mitochondria was comparable among Fresh, SV, and FV oocytes. (C) Mitochondrial staining revealed that Fresh group was significantly alleviated by cryopreservation-induced mitochondrial aggregation compared to SV group (***P\u0026lt;0.001) and FV group (**P\u0026lt;0.01). Scale bars=50 μm. Data are presented as mean ± SEM (A total of 138 oocytes with 3 replicates).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/ad1d159ab7f84b1d9ce01d2e.png"},{"id":108006540,"identity":"7df15dde-cf8a-4a3b-9c22-c2f538410d8a","added_by":"auto","created_at":"2026-04-28 12:55:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1621505,"visible":true,"origin":"","legend":"\u003cp\u003eOocytes labeled with JC-1. (A) Representative micrographs of JC-1 staining in Fresh, SV and FV oocytes. (B) JC-1 staining revealed that ratio of mitochondrial membrane potential in oocytes was no differences when the oocytes subjected to SV procedure (A total of 15 oocytes) compared to FV procedure (A total of 15 oocytes). Also, there were no differences between Fresh (A total of 15 oocytes) and two groups. Scale bars=100 μm. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/3a4a030972a45da4a4dcaae3.png"},{"id":107896303,"identity":"0b925417-1396-4e25-9de5-be58416f60ea","added_by":"auto","created_at":"2026-04-27 10:53:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1083443,"visible":true,"origin":"","legend":"\u003cp\u003eThe TUNEL assay of blastocysts. (A) Representative micrographs of TUNEL staining in blastocysts produced from Fresh, SV and FV. (B-C) The TUNEL assay and confocal microscopy revealed no significant differences in the number and percentage of positive cells in blastocysts between the two groups (SV 9.71±0.62% versus FV 10.12±0.60%). These results were comparable to those observed in the Fresh group (10.51±0.80%). Scale bars=10 μm. Data are presented as Mean ± SEM (Each group with 10 blastocysts).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/ee1a4d283f0736981421092e.png"},{"id":108008571,"identity":"815cbafa-d351-42f6-a9fc-0c0a196434ee","added_by":"auto","created_at":"2026-04-28 13:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11898794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/cd850d2e-76fb-40e4-af3d-0bf932c8f325.pdf"},{"id":108006677,"identity":"cc2919d0-1809-4c3c-a519-a19a8db774c0","added_by":"auto","created_at":"2026-04-28 12:56:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":97432,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8975751/v1/47a1c9c5095b51be248d9ded.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing Vitrification Equilibration Methods: Toward a Better Protocol for the Recovery and Subsequent Development of Mature Oocytes from Ovarian Cancer Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor young women diagnosed with ovarian cancer, fertility preservation has become an integral component of comprehensive oncological care [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although fertility-sparing surgery may be feasible for select patients with early-stage disease, adjuvant chemotherapy is often required and poses a significant threat to future fertility due to the well-established gonadotoxic effects of platinum-based agents and alkylating agents [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Oocyte cryopreservation following controlled ovarian stimulation represents the standard approach for fertility preservation in post-pubertal women facing urgent cancer treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the introduction of vitrification, there has been a significant improvement in the efficacy of oocyte cryopreservation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Vitrification achieves rapid cooling, transforming intracellular water into a glass-like state, thereby preventing ice crystal formation and reducing oocyte damage [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The standard vitrification (SV) procedure, which has been widely established, relies on the use of high concentrations of cryoprotectants during an equilibration phase that allows cryoprotectants to penetrate the cell membrane and replace intracellular water over a few minutes (with longer durations potentially required for blastocysts) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This is followed by rapid cooling, which facilitates the transition of intracellular water into a glass-like state and avoids structural cellular damage. The SV procedure has significantly improved oocyte survival, fertilization, and pregnancy rates following embryo transfer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, novel alternatives to the SV procedure, such as ultra-fast vitrification (UFV) and one-step warming (OW), have been reported to achieve high survival rates for both oocytes and embryos. Most clinical reports have demonstrated that OW for embryos following the SV procedure, particularly for blastocysts, achieves comparable survival and higher pregnancy rates [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, reports on the application of UFV and OW specifically for oocytes remain limited [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGallardo et al. reported that oocyte dehydration upon exposure to standard cryoprotectant solutions occurs very rapidly, with the minimum volume of the shrink-swell curve reached within 60 seconds [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. At this point, the ejection of intracellular water is complete, and coupled with the permeation of low molecular weight cryoprotectants, results in similar intracellular and extracellular solute concentrations. This finding suggests that prolonging exposure to cryoprotectant solutions does not enhance the cytosolic glass-forming tendency and may be unnecessary. It implies that short exposures to increasingly hypertonic solutions could represent a more time-efficient strategy for preparing human oocytes and embryos for vitrification. Indeed, in clinical practice, the SV procedure is time-consuming, with most of the duration dedicated to a long equilibration phase (minimum of 5\u0026ndash;8 minutes) in a lower concentration of cryoprotectants, followed by exposure to a higher concentration vitrification solution (minimum of 1 minute). Reducing the duration of vitrification and warming procedures is desirable to improve workflow in a busy IVF setting and to minimize exposure to suboptimal temperatures and osmolarity, as well as the potential toxicity of cryoprotectants.\u003c/p\u003e \u003cp\u003eAlthough it has been reported that ultra-fast vitrification (FV) appears more effective for oocyte cryopreservation due to efficient cytoplasmic water extraction, reduced osmotic stress, and minimization of cell contraction and expansion amplitude [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the mechanistic functions of the FV procedure remain unclear. This is particularly true concerning the impact of shortened equilibration time on subsequent embryonic development following fertilization. In this study, therefore, we compared SV and FV procedures in terms of oocyte survival and subsequent early embryonic development following the OW procedure and insemination. Additionally, molecular assessments were performed to compare these two procedures, providing evidence of both efficiency and safety. We believe this information is crucial for evaluating the efficacy of the FV procedure for oocyte vitrification.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eAnimals and oocyte collection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eEight-week-old female C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under controlled environmental conditions (20\u0026ndash;22\u0026deg;C, 50\u0026ndash;70% relative humidity, 12 h light/dark cycle) with ad libitum access to food and water. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Review Committee of Zhejiang University (ZJU20250897) and were performed in accordance with institutional and national animal welfare guidelines.\u003c/p\u003e \u003cp\u003eFemale mice were super ovulated by intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG), followed by 10 IU human chorionic gonadotropin (HCG) 46\u0026ndash;48 h later. Cumulus-oocyte complexes (COCs) were collected from the oviductal ampulla 14 h post-HCG administration and treated with hyaluronidase solution (80 units/mL) to remove cumulus and granulosa cells, yielding metaphase II (MII) oocytes for the experiments. The method of euthanasia for experimental mice was cervical dislocation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eTwo vitrification procedures were compared: 1) The established SV procedure, the oocytes were equilibrated in 200 \u0026micro;L of V1 solution (7.5% DMSO\u0026thinsp;+\u0026thinsp;7.5% EG, Vitrification Kit, ARSCI Biomedical Inc, Jiaxing, China) for 5 minutes at the room temperature, and then transferred the oocytes to 200 \u0026micro;L of V2 solution (15.0% DMSO\u0026thinsp;+\u0026thinsp;15.0% EG) for 1 minute and immediately loaded onto RC Straw (ARSCI Biomedical Inc, Jiaxing, China) and then rapidly plunged into liquid nitrogen for storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e SV); 2) The shortened equilibration time of FV procedure, the oocytes were equilibrated in 200 \u0026micro;L of V1 solution for 1 minute at the room temperature, and then transferred the oocytes to 200 \u0026micro;L of V2 solution for 1 minute and loaded immediately onto RC Straw and then rapidly plunged into liquid nitrogen for storage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, FV).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor warming of the vitrified oocytes, One-Step Warming Solution (OSWS, ARSCI Biomedical Inc, Jiaxing, China) was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, OW). Briefly, RC Straw was directly inserted into pre-warmed (37\u0026deg;C) OSWS solution (1.0 M Sucrose) for 1 minute, and then the oocytes were transferred to pre-warmed (37\u0026deg;C) Embryo Development Medium (EDM, ARSCI Biomedical Inc, Jiaxing, China) for washing 1 minute and then cultured in EDM until insemination in 5.0% CO\u003csub\u003e2\u003c/sub\u003e incubator (37\u0026deg;C, 100% humidity). After warming, the survival of the oocytes was assessed by morphological observation, and the survived oocytes were subjected to insemination by intracytoplasmic sperm injection (ICSI) method or other experimental assessments [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eInsemination and embryonic development culture\u003c/h3\u003e\n\u003cp\u003eICSI was performed using a PIEZO-driven microinjection system (Eppendorf) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly mouse sperm heads were separated from tails by pulse vibration in PVP solution (ARSCI Inc, RC-1120) and then injected into oocyte cytoplasm. Injected oocytes were washed three times with Gamete Buffer Medium (ARSCI Inc., RC-1020) and cultured in EDM droplets (50 \u0026micro;L) for fertilization assessment after 10 hours of ICSI procedures. Embryonic development potential was observed at 24 hours (2-cell stage), 48 hours (4-cell stage), 60 hours (8-cell stage), 3 days (compaction), and 4 days (blastocyst formation) post-ICSI, respectively [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Blastocyst development rates were evaluated on the day of end culture [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and the formed blastocysts were performed by apoptosis detection using TUNEL assay.\u003c/p\u003e\n\u003ch3\u003eOocyte immunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe spindle of oocytes as a crucial structure in oocyte meiosis and fertilization serves as an important indicator for assessing oocyte developmental potential [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The warmed oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes, and then permeabilized and blocked in PBS containing 0.1% Triton X-100 (Sigma) and 10% fetal bovine serum (FBS, Gibco) for 1 hour at room temperature. After three times of washing with PBS, the oocytes were incubated with 1:1000 diluted FITC-conjugated mouse anti-α-Tubulin antibody (Sigma) for 2 hours at room temperature. Finally, DNA was stained with Hoechst 33342 (Sigma) for 10 minutes and images were observed and analyzed by using confocal microscopy.\u003c/p\u003e\n\u003ch3\u003eOocyte mitochondrial Mitotracker live-cell staining\u003c/h3\u003e\n\u003cp\u003eThe warmed oocytes were incubated in 1:1000 diluted Mitotracker dye (in EDM) at 37\u0026deg;C for 30 minutes, followed by Hoechst 33342 DNA staining for 10 minutes. After three washes with Gamete Buffer Medium (ARSCI Inc., RC-1020), images were observed and analyzed using Evident (Olympus) SpinSR [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOocyte mitochondrial membrane potential JC-1 detection\u003c/h2\u003e \u003cp\u003eJC-1 mitochondrial membrane potential detection kit (Beyotime) was used. Briefly, the oocytes were incubated in JC-1 staining working solution at 37\u0026deg;C for 20 minutes and then washed three times with Gamete Buffer Medium (ARSCI Inc., RC-1020). Obtained images were observed using Evident (Olympus) SpinSR to assess membrane potential through green/red fluorescence ratio.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlastocyst apoptosis detection (TUNEL assay)\u003c/h3\u003e\n\u003cp\u003eApoptosis is a normal phenomenon during embryonic development, but excessive apoptosis can affect embryo quality and developmental potential [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. TUNEL apoptosis detection kit (Beyotime) was used and combined with confocal microscopic observation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, the blastocysts were fixed in 4% PFA at room temperature for 30 minutes, permeabilized with 0.3% Triton X-100 for 5 minutes, washed once with PBS, and then incubated with TUNEL reaction solution at 37\u0026deg;C in the dark for 1 hour. Nuclei were counterstained with Hoechst 33342 for 10 minutes and observed by using confocal microscopy.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., USA). All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments. For comparisons between two groups, Student\u0026rsquo;s t-test was applied, while one-way ANOVA followed by Tukey\u0026rsquo;s Honestly Significant Difference (HSD) test was used for comparisons among multiple groups. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe FV procedure yielded a comparable oocyte survival rate\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, FV procedure achieved 97.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10% (a total of 141 oocytes with 9 replicates) survival rate, which was comparable to that of SV procedure (98.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92%, a total of 139 oocytes with 9 replicates). Also, the morphological observation of the warmed oocytes was similar among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe FV procedure showed no significant impact on spindle morphology\u003c/h2\u003e \u003cp\u003eNeither FV nor SV procedures affected oocyte spindle morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The rates of normal spindle morphology in oocytes were 95.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08% in Fresh (47 oocytes), 95.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16% in SV (46 oocytes), and 95.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22% in FV (45 oocytes), respectively, with no significant differences among groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe FV procedure showed no significant impact on fertilization and early embryonic development\u003c/h2\u003e \u003cp\u003eAs shown in Table\u0026nbsp;1, there were no differences in both groups of fertilization and early embryonic development as well blastocyst formation rates, but they were significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) than Fresh oocyte group. There were also no differences in morphological observation during early embryonic development among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a total of 133 zygotes 3 replicates).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe FV procedure did not affect mitochondrial aggregation\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Mitotracker staining revealed that the fluorescence intensity of mitochondria in the oocytes was comparable among Fresh, SV, and FV groups. However, mitochondrial staining revealed that Fresh oocytes showed minimal aggregation (15.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81%, a total of 46 oocytes with 3 replicates) and significantly alleviated aggregation compared to SV oocytes (43.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93%, a total of 46 oocytes with 3 replicates, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and FV oocytes (43.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06%, a total of 46 oocytes with 3 replicates, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential was well maintained by the FV procedure\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, JC-1 staining indicated that there were no differences in the ratio of mitochondrial membrane potential in the oocytes subjected to the FV procedure (a total of 15 oocytes) compared to SV procedure (a total of 15 oocytes). Also, there were no differences in Fresh (A total of 15 oocytes) compared to two groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBlastocyst apoptosis was unaffected by FV procedure\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, there were no significant differences in the numbers and percentages of apoptotic cells between blastocysts derived from FV (10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60%, a total of 15 blastocysts) and SV (9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62%, a total of 15 blastocysts) oocytes, and both were comparable to Fresh oocyte-derived blastocysts (10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60%, a total of 15 blastocysts).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study demonstrated that the FV procedure, employing a shortened equilibration time for oocyte vitrification, maintains oocyte survival, fertilization rates, and early embryonic development in vitro comparable to those achieved with the SV procedure. It further indicated that, compared to SV, the FV procedure does not compromise oocyte spindle integrity, mitochondrial distribution and aggregation, or mitochondrial damage, thereby providing an important theoretical foundation for optimizing clinical oocyte cryopreservation protocols.\u003c/p\u003e \u003cp\u003eInitial attempts to cryopreserve oocytes utilized the same slow-freezing methods that were considered the gold standard for embryo cryopreservation. However, slow-freezing of oocytes resulted in very low survival and pregnancy rates, leading to its classification as an experimental procedure [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Over time, the efficacy of slow-freezing improved with increased sucrose concentrations in the freezing medium [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At the beginning of the 21st century, the introduction and advancement of vitrification technology marked a significant improvement in the efficacy of oocyte and embryo cryopreservation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To date, vitrification-based cryopreservation of oocytes and embryos has made substantial progress, holds great promise, and has been widely adopted in clinical practice worldwide using established protocols, namely standard or conventional vitrification procedures [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough vitrification and warming procedures are significantly shorter than slow-freezing methods, standard vitrification and warming protocols remain time-consuming, particularly with manual handling. The SV procedure requires a minimum duration of 5\u0026thinsp;+\u0026thinsp;1 minutes, involving direct exposure of oocytes or embryos to a low-concentration cryoprotectant solution followed by exposure to a more concentrated vitrification solution. Shortening the duration of vitrification protocols is desirable to improve workflow in busy IVF settings and to reduce exposure time to suboptimal temperatures, non-physiological osmolarity, and potentially toxic cryoprotectants. This is critical because vitrification may induce cryoinjury at multiple levels, including premature intracellular Ca\u0026sup2;⁺ release, microtubule disruption, chromosomal abnormalities, actin microfilament damage, and oxidative stress, all of which can lead to mitochondrial and endoplasmic reticulum damage [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcerns persist regarding oocyte vitrification technology due to the use of relatively high concentrations of cryoprotectants such as ethylene glycol (EG) and dimethyl sulfoxide (DMSO). Exposure to high cryoprotectant concentrations is known to damage oocytes through both cytotoxic and osmotic effects. As the results of the present study indicate, embryonic developmental potential was significantly reduced following vitrification and warming procedures (Table\u0026nbsp;1). Numerous approaches have been explored to overcome or mitigate cryoinjury, including optimizing cryoprotectant solutions to reduce toxicity, using microtubule stabilizers to prevent depolymerization, and applying antioxidant treatments to alleviate oxidative stress during vitrification [\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, new strategies capable of acting at multiple levels are needed to improve the survival and developmental potential of vitrified-warmed oocytes and embryos.\u003c/p\u003e \u003cp\u003eIt is widely accepted that the addition and removal of cryoprotectants from oocytes create osmotic imbalances across the oocyte membrane, potentially inducing large volumetric changes and causing damage to oocyte morphology, cytoskeletal structures, and function. Thus, determining oocyte tolerance to osmotic stress is essential for understanding the functional role of cryoprotectants during vitrification. The key to successful oocyte vitrification lies in balancing the use of minimal cryoprotectant concentrations without compromising their cryoprotective efficacy. Interestingly, it has been reported that oocyte vitrification may not require high cryoprotectant concentrations in the vitrification solution, suggesting that current protocols may need refinement toward less cytotoxic solutions [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is well established that, despite their protective functions, cryoprotectants can cause damage through osmotically driven cell volume changes and inherent chemical toxicity. In particular, it has been calculated that toxicity is minimized during cryoprotectant addition by allowing the cell to swell to its maximum tolerable volume before transferring it to a higher concentration of cryoprotectants, thereby avoiding excessive shrinkage. This approach may significantly reduce damage due to cryoprotectant toxicity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Another mathematically optimized cryoprotectant equilibration procedure for human oocyte cryopreservation has been reported, suggesting that piecewise-constant procedures are less toxic [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. A theoretical model has been established predicting that exposure causes oocytes to initially shrink and then swell to the maximum cell volume limit. To reach the target intracellular cryoprotectant concentration, oocytes are subsequently induced to shrink to the minimum cell volume limit by exposure to a high-concentration cryoprotectant solution, thereby significantly reducing toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, Gallardo et al. reported that human oocyte dehydration occurs very rapidly upon exposure to standard cryoprotectant solutions, with the minimum volume of the shrink-swell curve reached within 60 seconds. This indicates that intracellular water ejection is completed concurrently with the permeation of low-molecular-weight cryoprotectants, resulting in similar intracellular and extracellular solute concentrations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, prolonging exposure to cryoprotectant solutions does not enhance the cytosolic glass-forming tendency and may be unnecessary. Recently, it has been reported that for vitrification of human immature oocytes, the FV procedure achieves results comparable to the SV procedure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Garc\u0026iacute;a-Mart\u0026iacute;nez et al. reported that limiting exposure time to the equilibration solution in bovine oocytes is a more efficient approach for vitrification preparation, improving post-warming oocyte quality by protecting spindle integrity and reducing DNA fragmentation, thereby enhancing blastocyst rates and embryo quality [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, using mouse oocytes, Cho et al. demonstrated that shortened equilibration time in FV appears more effective for oocyte vitrification due to efficient cytoplasmic water extraction, reduced osmotic stress, and minimized cell contraction and expansion amplitude, thus compensating for the drawbacks of SV [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of the present study demonstrate that the FV procedure with shortened equilibration time for oocyte vitrification maintains oocyte survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), fertilization rates, and early embryonic development in vitro (Table\u0026nbsp;1, Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) comparable to the SV procedure. Furthermore, the FV procedure does not adversely affect oocyte spindle integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), mitochondrial distribution and aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), or mitochondrial damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) compared to SV. These findings provide a critical theoretical basis for the application of the FV procedure in oocyte cryopreservation for fertility preservation in ovarian cancer patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.X., X.C., R.C. designed research, H.W., Q.J, Q.L., X.S. performed research, H.W., Q.W. analyzed data, H.W., J.X., R.C. wrote the paper. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSantos ML, Pais AS, Almeida Santos T. Fertility preservation in ovarian cancer patients. Gynecol Endocrinol. 2021;37(6):483\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart C, Ralyea C, Lockwood S. Ovarian Cancer: An Integrated Review. Semin Oncol Nurs. 2019;35(2):151\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarez M, Sol\u0026eacute; M, Devesa M, F\u0026aacute;bregas R, Boada M, Tur R, Coroleu B, Veiga A, Barri PN. Live birth using vitrified\u0026ndash;warmed oocytes in invasive ovarian cancer: case report and literature review. Reprod Biomed Online. 2014;28(6):663\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChian RC, Wang Y, Li YR. Oocyte vitrification: advances, progress and future goals. J Assist Reprod Genet. 2014;31(4):411\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai T. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev. 2004;50(6):685\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagli MC, Lappi M, Ferraretti AP, Capoti A, Ruberti A, Gianaroli L. Impact of oocyte cryopreservation on embryo development. Fertil Steril. 2010;93(2):510\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRienzi L, Gracia C, Maggiulli R, et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23(2):139\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasciani V, Monseur B, Cimadomo D, Alvero R, Rienzi L. Oocyte and embryo cryopreservation in assisted reproductive technology: past achievements and current challenges. Fertil Steril. 2023;120(3 Pt 1):506\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWozniak K, Reichelderfer R, Ghaemi S, Hupp D, Fuzesi P, Ringler G, Marrs RP, Schiewe MC. Ultra-fast vitrification and rapid elution of human oocytes: Part II - verification of blastocyst development from mature oocytes. Reprod Biomed Online. 2024;49(6):104690.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiebermann J, Brohammer R, Wagner Y, Smith R, Even K, Hirshfeld-Cytron J, Uhler ML. Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol. Reprod Biomed Online. 2024;49(1):103976.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang VS, Cherouveim P, Naert MN, Dimitriadis I, Souter I, Bormann CL. 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Proc Natl Acad Sci U S A. 2018;115(23):E5326\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin H, Zhang T, Wang H, Hu X, Hou X, Fang X, Yin Y, Li H, Shi L, Su YQ. Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice. Front Cell Dev Biol. 2021;9:687522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Okitsu O, Zhao XM, Sun Y, Di W, Chian RC. The effect of minimal concentration of ethylene glycol (EG) combined with polyvinylpyrrolidone (PVP) on mouse oocyte survival and subsequent embryonic development following vitrification. J Assist Reprod Genet. 2014;31(1):55\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamjoo M, Brison DR, Kimber SJ. Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture. Mol Reprod Dev. 2002;61(1):67\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKyrylkova K, Kyryachenko S, Leid M, Kioussi C. Detection of apoptosis by TUNEL assay. Methods Mol Biol. 2012;887:41\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum Reprod. 1996;11(7):1513\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod. 1998;13(11):3156\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril. 1997;68(4):724\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGook DA, Edgar DH. Human oocyte cryopreservation. Hum Reprod Update. 2007;13:591\u0026ndash;605.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril. 2004;82(3):601\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorini A, Sciajno R, Bianchi V, Sereni E, Flamigni C, Coticchio G. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod. 2006;21(2):512\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBianchi V, Lappi M, Bonu MA, Borini A. Oocyte slow freezing using a 0.2\u0026ndash;0.3 M sucrose concentration protocol: is it really the time to trash the cryopreservation machine? Fertil Steril. 2012;97(5):1101\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapis K. A brief overview of the development of oocyte and embryo cryopreservation strategies with a focus on the roles of sugars. Cryobiology. 2025;122:105577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Ruvalcaba Castell\u0026oacute;n LA, Garc\u0026iacute;a Amador MI, Montoya Sarmiento JE. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed Online. 2008;16(5):608\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChian RC, Gilbert L, Huang JY, Demirtas E, Holzer H, Benjamin A, Buckett WM, Tulandi T, Tan SL. Live birth after vitrification of in vitro matured human oocytes. Fertil Steril. 2009;91(2):372\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChian RC, Huang JY, Gilbert L, Son WY, Holzer H, Cui SJ, Buckett WM, Tulandi T, Tan SL. Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes. Fertil Steril. 2009;91(6):2391\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMogas T, Garc\u0026iacute;a-Mart\u0026iacute;nez T, Mart\u0026iacute;nez-Rodero I. Methodological approaches in vitrification: enhancing viability of bovine oocytes and in vitro‐produced embryos. Reprod Domest Anim. 2024 59, e14623.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Mart\u0026iacute;nez T, Mart\u0026iacute;nez-Rodero I, Roncero-Carol J, Y\u0026aacute;nez-Ortiz I, Higgins AZ, Mogas T. Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification. Theriogenology. 2022;184:110\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorat\u0026oacute; R, Izquierdo D, Albarrac\u0026iacute;n JL, Anguita B, Palomo MJ, Jim\u0026eacute;nez-Macedo AR, Paramio MT, Mogas T. Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification. Mol Reprod Dev. 2008;75(1):191\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGirka E, Gatenby L, Gutierrez EJ, Bondioli KR. The effects of microtubule stabilizing and recovery agents on vitrified bovine oocytes. Theriogenology. 2022;182:9\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Mart\u0026iacute;nez T, Vendrell-Flotats M, Mart\u0026iacute;nez-Rodero I, Ord\u0026oacute;\u0026ntilde;ez-Le\u0026oacute;n EA, \u0026Aacute;lvarez-Rodr\u0026iacute;guez M, L\u0026oacute;pez-B\u0026eacute;jar M, Yeste M, Mogas T. Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming. Int J Mol Sci. 2020;21(20):7547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. Oxidative Stress and Oocyte Cryopreservation: Recent Advances in Mitigation Strategies Involving Antioxidants. Cells. 2022;11(22):3573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to -196\u0026deg;c in dilutions of a standard vitrification solution. PLoS ONE. 2012;7(4):e36058.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenson JD, Kearsley AJ, Higgins AZ. Mathematical optimization of procedures for cryoprotectant equilibration using a toxicity cost function. Cryobiology. 2012;64(3):144\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavidson AF, Benson JD, Higgins AZ. Mathematically optimized cryoprotectant equilibration procedures for cryopreservation of human oocytes. Theor Biol Med Model. 2014;11:13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ovarian Cancer, Fertility preservation, Oocyte, Fast vitrification, One-step warming","lastPublishedDoi":"10.21203/rs.3.rs-8975751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8975751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOvarian cancer affects a significant number of women of reproductive age, and while advances in treatment have dramatically improved survival rates, the gonadotoxic effects of chemotherapy and radiotherapy pose a major threat to future fertility. For these patients facing an urgent need to begin cancer therapy, oocyte cryopreservation via vitrification is the standard method for fertility preservation. The success of vitrification hinges on the equilibration step, where oocytes are exposed to cryoprotectants. While standard protocols use a gradual approach, a fast vitrification (FV) method with a shortened equilibration time has been developed.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eTo evaluate whether the shortened equilibration time in fast vitrification (FV) achieves high oocyte survival and maintains functional integrity post-fertilization, thereby supporting its application for fertility preservation in ovarian cancer patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo compare standard vitrification (SV) and fast vitrification (FV) procedures in mouse oocyte survival after vitrification-warming (VW) and subsequent early embryonic development following fertilization. For SV procedure was as that the oocytes were equilibrated in V1 (7.5% dimethyl sulphoxide, DMSO and 7.5% ethylene glycol, EG) solution for 5 minutes and then transferred the oocytes to V2 (15% DMSO and 15% EG) for 1 minute, and the oocytes were immediately loaded onto a carrier for cooling directly plunging into LN\u003csub\u003e2\u003c/sub\u003e; For FV procedure was as that oocytes were equilibrated in V1 solution for 1 minute and then transferred to V2 solution for 1 minute, and the oocytes immediately loaded onto a carrier for cooling by directly plunging into LN\u003csub\u003e2\u003c/sub\u003e. For warming, one-step warming (OW) procedure was applied in both groups. The survival rates and early embryonic development potentials of the warmed oocytes were compared following insemination by piezo-ICSI. Spindle integrity of the warmed oocytes was measured by α-tubulin/Hoechst staining, and mitochondrial status was assessed by Mitotracker/JC-1 staining and the mitochondrial damages were analyzed by confocal microscope. Additionally, the apoptosis status of the formed blastocysts in each group was compared via TUNEL assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWith shortened equilibration time, the FV procedure maintained comparable oocyte survival and fertilization as well as early embryonic development rates to SV procedure. Mechanistic measures revealed that there were no differences between the oocytes vitrified by SV and FV procedures in terms of spindle integrity, mitochondrial status and damages as well as apoptosis status in the formed blastocysts.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eBy reducing exposure time to cryoprotectants and limiting time under suboptimal temperature and osmotic conditions, fast vitrification (FV) offers an efficient oocyte cryopreservation method. The improved workflow afforded by FV supports its application in fertility preservation for ovarian cancer patients.\u003c/p\u003e","manuscriptTitle":"Optimizing Vitrification Equilibration Methods: Toward a Better Protocol for the Recovery and Subsequent Development of Mature Oocytes from Ovarian Cancer Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 10:52:41","doi":"10.21203/rs.3.rs-8975751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"289526461579420350406626687145786071776","date":"2026-04-28T12:08:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-19T14:21:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T10:08:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-23T03:47:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2026-03-13T22:40:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"444c837b-8c34-4849-bf4c-6e1e72a82729","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T10:52:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 10:52:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8975751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8975751","identity":"rs-8975751","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}