Media temperature control: A potentially important  quality control parameter in human oocyte vitrification

Media temperature control: A potentially important quality control parameter in human oocyte vitrification | 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 Media temperature control: A potentially important quality control parameter in human oocyte vitrification Munevver Serdarogullari, Zafer Atayurt, Georges Raad, Hayriye Karakaya, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7702661/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: The purpose of this study is to evaluate the role of controlling media temperature during human oocyte vitrification. This study aimed to retrospectively compare embryological outcomes following oocyte warming, fertilisation and embryo development to the blastocyst stage for two separate periods during which the vitrification solution temperature was not controlled and controlled. Method: A retrospective observational study was conducted at Ventus IVF Center, Cyprus (March 2024–February 2025), evaluating the effect of vitrification solution temperature during vitrification on oocyte survival and embryological outcomes. All oocytes were sourced from 16 donor cycles, with informed consent obtained. Oocytes were vitrified either under uncontrolled ambient conditions (n = 87 oocytes) or with temperature control (n = 83 oocytes) implemented using a heated stage. Vitrification and equilibration solution temperatures were recorded using a calibrated UNI-T UT322 thermometer. Results: There were no significant differences in baseline characteristics of oocyte donors between the controlled and uncontrolled temperature groups (p > 0.05). During vitrification, vitrification media temperature was significantly higher in the controlled group (23.85 ± 0.43°C) compared to the uncontrolled group (19.65 ± 0.43°C; p < 0.001). No significant differences were observed in oocyte survival post-warming (82/83 vs. 80/87; p = 0.081) or fertilisation rates (69/82 vs. 70/87; p = 0.671) between groups. Compared to the controlled group, the uncontrolled group showed markedly higher developmental arrest at the pronuclear (10/70 vs. 0/69), post-pronuclear (20/70 vs. 0/69), and cleavage (35/70 vs. 0/69) stages, and significantly reduced blastocyst formation (5/70 vs. 56/69; all p < 0.001). Conclusion: Maintaining vitrification solution temperature rather than relying on ambient conditions, significantly improves oocyte vitrification efficiency and pre-implantation embryo development parameters. quality control vitrification solution temperature human oocyte cryopreservation Figures Figure 1 Key Message The temperature of the vitrification media plays an important role in successful oocyte cryopreservation outcomes. Recognising vitrification solution temperature as a critical and novel quality control parameter may facilitate refinements in vitrification protocols and quality control in laboratories, ultimately improving the efficacy of oocyte cryopreservation. Introduction Vitrification has emerged as the leading method for oocyte cryopreservation, marking a major advancement in medically assisted reproduction (MAR), as demonstrated by its clinical effectiveness, safety, and widespread adoption in fertility preservation and elective oocyte freezing worldwide ( 6 , 2 , 3 , 4 , 5 ). Its established role in fertility preservation for medical and non-medical indications as well as oocyte donation programs, particularly through decoupling donor-recipient cycle synchronization, further supports its position as the gold standard ( 6 , 7 ). However, given the widespread use of oocyte vitrification, evidence-based modifications to current protocols are essential to optimise outcomes. Human oocytes are large cells with high water content and a distinct cytoskeleton ( 8 ). During in vitro handling and culture, even a short drop from the standard 37°C culture temperature has been associated with effects on the meiotic spindle ( 9 , 10 ), Spindle disorganisation begins after ~ 10 min at 33–34°C ( 11 ), becomes complete within minutes at 25°C ( 12 ), and exposure to 0°C has been shown to cause loss of spindle integrity and chromosomal mis-alignment ( 13 ). Nonetheless, such effects have been observed to be transient, with the spindle being a dynamic structure capable of depolymerization and repolymerization ( 14 ). The process of vitrification entails exposure of oocyte/s in cryoprotectant (CPA) solutions followed by rapid cooling, turning water into a glass-like structure and avoiding hazardous intracellular ice formation ( 7 ). The method of vitrification works effectively only if all factors stay in balance ( 7 , 15 ). If CPA concentration or cooling speed is too low, ice forms; if CPA exposure or concentration is too high, chemical toxicity and sudden water movement can create cryo-injury to the oocyte ( 7 , 15 ). Temperature has a pivotal role in retaining the balance ( 16 ). Handling at warmer conditions accelerates CPA diffusion into and out of the oocyte, so the oocyte spends less time in potentially toxic solutions ( 16 ). Temperature loss is the first link in the chain of events leading to oocyte damage with this damage possibly remaining unnoticed until pre-implantation embryo development ( 16 ). Vitrification commercial kit manufacturers suggest working at a stable room temperature. In practice however, several parameters can affect the temperature at which oocytes are exposed while handling within the CPA solutions. These can include amongst others open dishes (without lid) on an unheated laminar flow hood as well as laminar flow hood air flow speed that can cause cooling effects, dish material and air-conditioning ( 16 , 17 ). These conditions can create substantial differences even in laboratories that apply the same vitrification protocols and in accordance with vitrification media manufacturers’ instructions ( 18). Among these conditions, temperature control during vitrification has traditionally focused on cooling and warming rates, with less attention given to the temperature of the vitrification solutions during handling and processing ( 19 ). This retrospective observational study was conducted in a single centre, and covered two separate periods. During the first period, oocyte vitrification was performed according to the manufacturer’s instructions, with active monitoring of vitrification drop temperature but no control of the drop temperature and maintenance within a designated temperature range( 18 ). This practice coincided with a noticeable decline in embryo developmental outcomes post-warming, raising concerns about the potential impact of vitrification solution temperature, an aspect that had not previously been addressed as a critical quality control parameter. As part of troubleshooting, corrective measures were implemented to ensure strict temperature control of the vitrification solutions in subsequent procedures which consisted of the second period. This study aimed to summarise results of these two separate periods comparing embryological outcomes following oocyte warming, fertilisation and embryo development to the blastocyst stage. By analysing survival rates and developmental competence, the aim was to identify if vitrification solution drop temperature is an important quality control parameter that requires control. Materials and Methods 2.1 Study design and ethical considerations This is a retrospective observational study conducted at Ventus IVF Center, a newly established clinic in Cyprus, between March 2024 and February 2025. The clinic implemented a broad internal KPI monitoring system to build a reference dataset and ensure quality, with temperature routinely measured as an environmental indicator. Temperature measurements included monitoring of ambient room temperature, monitoring of all surface temperatures during handling, continuous monitoring of culture temperatures as well as monitoring of solution temperatures including vitrification solution drop temperature. Surface, solution and room temperatures were monitored using the UNI-T UT322 thermometer (Uni-Trend Technology Co., Ltd, China), which was validated against an NIST-traceable mercury-calibrated thermometer. The Vermox thermometer was equipped with a T-type thermocouple as the drop sensor and a PT100 RTD as the surface sensor. The UNI-T thermometer used a K-type thermocouple as the drop sensor, and temperature measurements were recorded at the frequency specified by the manufacturer until the temperature stabilised and no longer fluctuated. During all stages of in vitro handling including oocyte and embryo warming, denudation, fertilisation check as well as ICSI, solution temperature was measured and documented using the validated thermometer and sensor. Measurements included both the solution drop temperature and the corresponding surface temperature (± SD) and adjustments to the surface temperature were made where necessary to ensure control of handling temperature at 37.0 ± 0.2°C. During oocyte and embryo culture within the incubators, temperature was monitored continuously using temperature data loggers and sensors to record temperature over time, providing real-time data and historical records for analysis integrated with automated alarms to ensure optimal incubator conditions and signal any deviations for immediate intervention. Prior to allocating donor oocytes to recipients, the efficiency of the oocyte cryopreservation programme for donors for which oocytes were frozen between March 2024- August 2024 was tested. During this period, whilst vitrification solution temperature was monitored, there was no control of the temperature within a designated range, as this was not in the list of recommendations by the vitrification media manufacturer. Donor oocytes were warmed and fertilised with donor sperm. The outcomes in terms of oocyte survival and subsequent embryo development were recorded. Results indicated suboptimal preimplantation embryo development. Troubleshooting was initiated and led to amendment of oocyte vitrification practice for donor oocytes that were vitrified between September 2024 and February 2025, during which a quality control parameter in controlling vitrification solution temperature within a designated range during handling of oocytes was implemented (23 ± 2°C). The outcomes of oocyte survival and preimplantation embryo development were also recorded following the intervention. A retrospective analysis was then performed from the data, comparing outcomes across the two different vitrification strategies (controlled and uncontrolled vitrification solution temperature), to evaluate their impact on oocyte viability and subsequent embryo development. The study received ethical approval from Cyprus International University (EKK24-25/06/10). All participants provided written informed consent for the donation of their oocytes. A total of 170 mature metaphase II (MII) oocytes, donated from 16 consenting donors across 16 donation cycles, were included. These consisted of a controlled solution temperature group (6 donors, n = 83 oocytes) and uncontrolled temperature group (10 donors, n = 87 oocytes). In both groups, solution and room temperatures were monitored using the specified and validated UNI-T UT322 thermometer with the appropriate sensors as described above. In the controlled group, although ambient room conditions were within the recommended range of 23 ± 2°C ( 17 , 20 ), the heated stage of the laminar flow hood for the purpose of oocyte vitrification was increased to 29.5°C to establish vitrification solution drop measurement within room temperature range (23 ± 2°C). The temperature of the vitrification solution was measured immediately before the addition of the oocyte into a mock drop next to the drop used for vitrification. All other parameters, including the type of dish, media volume, and dish placement, were kept consistent throughout the procedures during the study period to ensure stable and representative temperature conditions at the time of oocyte exposure. Nonetheless, most media including vitrification, warming and culture media had different lot numbers between the two study periods. Throughout the duration of the study only one senior embryologist was responsible for oocyte vitrification and warming. The senior embryologist has had extensive experience in both protocols with staff competency in oocyte vitrification and warming assessed regularly, to ensure consistent quality and adherence to best practices. 2.2 Oocyte Donors All participants in the study were anonymous individuals, with ages ranging from 20 to 30 years old at the time of oocyte donation. The candidates underwent thorough screening, including serological testing for Hepatitis B surface antigen, Hepatitis C virus, and HIV antibodies, as well as genetic testing for thalassemia, cystic fibrosis, and chromosomal abnormalities via karyotype analysis. Furthermore, comprehensive evaluations of familial histories concerning genetically inherited diseases were conducted. The controlled ovarian stimulation protocol was performed as previously described by Khurana et al., ( 21 ). Following a baseline transvaginal ultrasound assessment on the second day of menstruation, ovarian stimulation was initiated using recombinant follicle-stimulating hormone (rFSH) (Gonal-F, Merck). The initial dosage was individualised based on the patient's age, antral follicle count (AFC), body mass index (BMI), and, if applicable, previous responses to ovarian stimulation. Concurrently, a fixed dose of medroxyprogesterone acetate (MPA) (5 mg tablet, Deva, Turkey) was administered at 10 mg once daily, beginning on the second day of menstruation and continuing until the day of ovulation trigger. Upon the achievement of at least three follicles measuring 18 mm in diameter, patients were administered 0.2 mg of triptorelin (Gonapeptyl, Ferring), with oocyte retrieval scheduled to occur 35 hours subsequent to the injection. 2.3 Oocyte pick-up, denudation, semen preparation, ICSI and embryo culture Oocyte aspiration was performed 35 hours after HCG administration under ultrasound guidance. Oocytes were washed with handling medium-complete (MHM-C; FuJIFILM Irvine Scientific, CA, USA) containing Gentamicin and Human Serum Albumin (HSA), and collected into pre-equilibrated 750-µl drops of continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA, which was kept under humidified and heated conditions (at 37°C, 6% CO2 and 5% O2) in a benchtop incubator (G210 InviCell From K-Systems™) for 2 h until denudation. Enzymatic removal of cumulus cells was performed using 80 IU/mL hyaluronidase (FUJIFILM Irvine Scientific, CA, USA). Following denudation, intracytoplasmic sperm injection (ICSI) was performed in multipurpose handling medium-complete (MHM-C; FUJIFILM Irvine Scientific, CA, USA) containing Gentamicin and HSA and microinjected oocytes were cultured individually in a special pre-equilibrated culture dish (EmbryoSlide, Vitrolife) for embryo development. In this study, only continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA was used for embryo culture. EmbryoSlide wells were filled with 25–30 µl CSCM-NXM and covered with 1.4 ml Heavy oil (Kitazato) to prevent evaporation and equilibrated overnight before use. All oocytes/embryos were cultured in a time-lapse incubator (EmbryoScope, Vitrolife) at 37°C, 6% CO2 and 5% O2. The sperm used for fertilisation derived from donors and was obtained from a commercial sperm bank (Cryos International), with each sample being thawed and subsequently subjected to two washing cycles utilising a sperm washing medium, specifically a modified HTF medium supplemented with HSA at a concentration of 5.0 mg/mL (FUJIFILM Irvine Scientific, CA, USA). 2.4 Oocyte Vitrification and Warming Procedure The study was conducted in a controlled laboratory environment. The room temperature and vitrification solution temperatures were recorded using the UNI-T UT322 thermometer, validated with an NIST-traceable mercury-calibrated thermometer as described above. The Vitrification Freeze/Warm Kit (Vit Kit-Freeze NX/Vit Kit-Warm NX; FUJIFILM Irvine Scientific, CA, USA) was employed throughout the course of this investigation. The procedures for the vitrification and warming of oocytes were executed in accordance with the specifications provided by the manufacturers. The Cryotop® carrier device (Kitazato, Japan) and Nunc Ivf dish 90mm, non-treated (Thermo Scientific, USA) were utilised for the vitrification processes. In brief, Equilibration NX - ES constitutes a dual buffered solution (HEPES & MOPS) derived from Continuous Single Culture medium (CSCM), which incorporates Gentamicin Sulfate, 7.5% (v/v) of both DMSO and ethylene glycol, alongside 20% (v/v) of Dextran Serum Supplement (DSS). Vitrification NX - VS is characterised as a dual buffered solution (HEPES & MOPS) of CSCM that includes Gentamicin Sulfate, 15% (v/v) of each DMSO and ethylene glycol, 20% (v/v) DSS, in addition to 0.5 M Trehalose. DSS represents a protein supplement composed of 50 mg/mL therapeutic grade HSA and 20 mg/mL Dextran. DSS is incorporated at a concentration of 20% (v/v) in Vit Kit – Freeze NX, yielding a final concentration of 10 mg/mL HSA and 4 mg/mL Dextran. Also, thawing Kit contains a Thawing Solution (TS) that contains 2 mL in each vial of a 1 M sucrose, 20% DSS, and gentamicin composition in an M-199 HEPES buffered medium. Also, kit contains a Dilution Solution (DS) consisting of 2 mL of a composition of 0.5 M sucrose, 20% DSS, and gentamicin in the same M-199 HEPES buffered medium. Finally, the Washing Solution (WS) containing 2 mL contains 20% DSS and gentamicin in an M-199 HEPES buffered medium. After warming, oocytes were checked for viability (warmed oocytes were considered to not survive if lysed, highly vacuolised or otherwise impaired in cytoplasmic or extracytoplasmic structures) and incubated in standard conditions at 37°C (6% CO2, 5%O2) until the ICSI procedure. Fertilisation and Embryo culture: Upon completion of warming of oocytes, ICSI was conducted in multipurpose handling medium-complete (MHM-C; FUJIFILM Irvine Scientific, CA, USA) containing Gentamicin and HSA, with microinjected oocytes cultured individually within a specialized pre-equilibrated culture dish (EmbryoSlide, Vitrolife) to facilitate embryonic development. This investigation exclusively employed continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA for the purpose of embryo culture. The wells of the EmbryoSlide were filled with 25–30 µl CSCM-NXM and subsequently covered with 1.4 ml of Heavy oil (Kitazato) to mitigate evaporation, allowing for overnight equilibration prior to application. All embryos were incubated within a time-lapse incubator (EmbryoScope™+ ,Vitrolife) at 37°C, 6% CO2, and 5% O2. Fertilisation assessment was conducted 16–18 hours post insemination. Embryos were cultured until day 6 of embryo development and Gardner and Schoolcraft's criteria for blastocyst grading additional to morphokinetic data were used to evaluate embryo quality ( 22 ). Statistical Analysis: Data were analysed using both parametric and non-parametric statistical methods as appropriate. For comparison of continuous variables between controlled and uncontrolled temperature groups, including stimulation days, daily usage, BMI, total usage, age, collected oocytes, and temperature measurements, the Mann-Whitney U test was employed due to the small sample sizes and without assuming normal distribution of the data. Results are presented as mean ± standard deviation (M ± SD) along with 95% confidence intervals (CI). For categorical variables related to oocyte and embryo quality outcomes, chi-square tests (χ²) were used to compare proportions between groups, including oocyte survival percentage, fertilisation rate, and blastulation percentage. Fisher's exact test was applied for comparing pronuclear arrest, Post-Pronuclear fading zygote arrest, and cleavage arrest percentages between groups, as this test is more appropriate when expected cell frequencies are small. For these arrest parameters, one-sided 97.5% confidence intervals were calculated to reflect the directionality of the observed differences. Statistical significance was set at p < 0.05 for all analyses. Results Baseline donor characteristics are comparable between controlled and uncontrolled temperature groups Baseline characteristics of oocyte donors did not differ significantly between the controlled (6 donors) and uncontrolled (10 donors) temperature groups across all assessed parameters ( Table 1 ). The mean number of stimulation days was comparable between the uncontrolled (10.36 ± 1.36) and controlled (10.67 ± 1.51) groups (p = 0.76). Daily gonadotropin doses were similar in the uncontrolled (209.09 ± 16.85 IU) and controlled (208.33 ± 12.91 IU) groups (p = 0.88). BMI values were also comparable between groups (22.00 ± 1.00 vs 21.83 ± 0.98; p = 0.80). Total gonadotropin consumption during stimulation did not differ between the uncontrolled (2170.45 ± 224.67 IU) and controlled (2220.83 ± 334.07 IU) groups (p = 0.80). Mean age was comparable (24.64 ± 2.25 vs 25.00 ± 3.03 years; p = 0.80), and the number of oocytes retrieved was similar (22.55 ± 1.92 vs 22.67 ± 2.07; p = 0.96). Table 1 Comparison of patient characteristics between controlled and uncontrolled groups Parameter Uncontrolled group (M ± SD) Controlled group (M ± SD) Test p-value 95% CI Uncontrolled 95% CI Controlled Stimulation Days 10.36 ± 1.36 10.67 ± 1.51 Mann-Whitney U = 29.5 0.76 [9.45, 11.27] [9.09, 12.25] Daily Usage (IU) 209.09 ± 16.85 208.33 ± 12.91 Mann-Whitney U = 31 0.88 [197.77, 220.41] [194.78, 221.88] BMI (Kg/m 2 ) 22 ± 1 21.83 ± 0.98 Mann-Whitney U = 30 0.80 [21.33, 22.67] [20.8, 22.86] Total Usage (IU) 2170.45 ± 224.67 2220.83 ± 334.07 Mann-Whitney U = 30 0.80 [2019.52, 2321.38] [1870.19, 2571.47] Age (years) 24.64 ± 2.25 25 ± 3.03 Mann-Whitney U = 30 0.80 [23.13, 26.15] [21.82, 28.18] Number of collected Oocytes 22.55 ± 1.92 22.67 ± 2.07 Mann-Whitney U = 32 0.96 [21.26, 23.84] [20.5, 24.84] M = mean; SD = standard deviation; CI = confidence interval; BMI = body mass index. Significant differences in vitrification medium temperatures between groups Room temperature was significantly lower in the uncontrolled temperature group (22.82 ± 0.55°C) compared to the controlled group (24.77 ± 0.1°C; Mann–Whitney U = 6, p < 0.001). A more pronounced difference was observed in the temperature of the embryo equilibration and vitrification medium ("drop temperature"), which averaged 19.65 ± 0.43°C under uncontrolled conditions versus 23.85 ± 0.43°C in the controlled setting (Mann–Whitney U = 0, p < 0.001) ( Table 2 ) . Graphical presentation of temperature curves over time for room temperature and vitrification solution temperature for the 2 groups is shown in Supplementary Fig. 1. Excluding vitrification, for all other processes of oocyte and embryo handling, heated surface and drop temperature measurements are shown in Supplementary Table 1. Table 2 Comparison of oocyte and embryo quality between Uncontrolled group and Controlled group conditions Parameter Uncontrolled group Controlled group Test p-value 95% CI Uncontrolled 95% CI Controlled Room temperature °C (mean ± SD) 22.85 ± 0.839 24.77 ± 0.09776 Mann-Whitney U = 6 < 0.001 [22.67, 23.03] [24.56, 24.98] Vitrification Medium temperature °C (Drop temp) mean ± SD 19.65 ± 0.839 23.85 ± 0.3780 Mann-Whitney U = 0 < 0.001 [19.47, 19.83] [23.77, 23.93] Total number of MII oocytes (n) 87 83 — — — — Oocyte survival % 80/87 82/83 χ² = 3.0387 0.081 [0.8412, 0.9670] [0.4475, 0.6891] Fertilization rate % 70/87 69/82 χ² = 0.181 0.671 [0.7057, 0.8819] [0.7442, 0.9128] Pronuclear arrest %* 10/70 0/69 Fisher’s exact < 0.001 [0.0707, 0.2471] [0, 0.0521] Post-Pronuclear fading zygote arrest %* 20/70 0/69 Fisher’s exact < 0.001 [0.1840, 0.4062] [0, 0.0521] Cleavage arrest %* (blocked at cleavage stage) 35/70 0/69 Fisher’s exact < 0.001 [0.3780, 0.6220] [0, 0.0521] Cleavage rate %* 40/70 69/69 Fisher’s exact < 0.001 [0.4475, 0.6891] [0.9479, 1] Blastulation % 5/70 56/69 χ² = 90.5211 < 0.001 [0.0236, 0.1589] [0.6994, 0.8957] CI = confidence interval; MII = metaphase II; * One-sided 97.5% CI was used for these parameters. Comparable oocyte survival and fertilisation rates under controlled and uncontrolled conditions A total of 87 metaphase II (MII) oocytes were exposed to uncontrolled conditions and 83 to controlled conditions. Fertilisation rates were comparable under uncontrolled and controlled conditions (70/87 vs. 69/82; χ² = 0.18, p = 0.671) ( Table 2 ) . Oocyte survival following warming did not differ significantly between the two groups (80/87 vs. 82/83; χ² = 3.04, p = 0.081), with overlapping confidence intervals. Pronuclear, Post-Pronuclear fading zygote arrest, and cleavage arrest rates were significantly higher under uncontrolled conditions. Pronuclear arrest was observed in 10/70 zygotes in the uncontrolled group, whereas no arrest occurred in the controlled group (0/69; p < 0.001, Fisher’s exact test). Post-Pronuclear fading zygote arrest followed a similar pattern, affecting 20/70 oocytes in the uncontrolled group and none in the controlled group (0/69; p < 0.001). Cleavage arrest occurred in 35/70 oocytes cultured under uncontrolled conditions, while all zygotes cleaved in the controlled group (69/69; p < 0.001). As a result, the overall cleavage rate was significantly reduced in the uncontrolled group (40/70). Blastulation was also markedly impaired, with only 5/70 oocytes reaching the blastocyst stage in the uncontrolled group compared to 56/69 in the controlled group (χ² = 90.52, p < 0.001) ( Table 2 ) . Morphokinetic data comparisons for the 2 groups are provided in Supplementary Table 2. Comparisons of embryological and clinical outcomes between fresh autologous and donor oocyte cycles from March 2024 to February 2025 are shown in Supplementary Table 3. Discussion This study investigates the impact of vitrification media drop temperature as a potential novel quality control parameter to improve cryopreservation outcomes based on oocyte vitrification practice that was introduced in a newly built for purpose IVF laboratory. While oocyte survival and fertilisation rates remained unaffected, embryo development was significantly compromised under uncontrolled vitrification solution drop temperatures (~ 20°C). Controlled drop temperatures (~ 24°C) yielded higher blastulation rates and reduced developmental arrest. Comparable patient characteristics across groups strengthen the possible attribution of these effects to vitrification media drop temperature rather than baseline variability. In this observational study, cleavage arrest was significantly more frequent in the uncontrolled temperature group (50%) compared to the controlled group (0%), reinforcing the notion that thermal instability can impair embryonic development ( 17 ). Previous studies have shown the importance of precise temperature control during vitrification, as even slight deviations may disrupt oocyte physiology ( 16 ). Oocytes are particularly vulnerable to thermal fluctuations ( 7 , 12 ). Previous studies have reported that cold-induced spindle disassembly can lead to developmental arrest ( 9 , 12 ). Oocytes are extremely thermosensitive and are particularly vulnerable to cryoinjuries ( 11 ). Their vulnerability can be attributed primarily to their considerable large size, high water content (75–85%), and distinctive intracellular architecture, rendering them among the most complex biological entities to successfully undergo freezing procedures ( 11 , 15 ). The predominant physical stressors associated with cryopreservation encompass chilling injuries and the formation of ice crystals. Abrupt reductions in temperature can precipitate cold-shock injuries in temperature-sensitive structures, compromising their functionality by modifying membrane permeability and inflicting damage on intracellular organelles, such as the cytoskeleton and meiotic spindle ( 12 , 15 ). The extreme vulnerability of oocytes to cryoinjury is largely attributed to the sensitivity of the meiotic spindle to temperature fluctuations ( 11 ). During all stages of in vitro handling, not only regarding cryopreservation, variations in temperature that fall beneath the physiological range (37.0 ± 0.2°C) can have a direct impact on the developmental capacity of oocytes, resulting in disturbances in cytoskeletal structures, particularly the depolymerisation of the meiotic spindle ( 17 , 23 ). Even brief exposure to temperatures near 20°C can disrupt spindle integrity, significantly impairing subsequent embryonic development after fertilization ( 11 ). At 0°C, rapid depolymerisation occurs, leading to irreversible spindle disruption ( 11 ). While cryoprotectants help preserve spindle structure during freezing, the warming process often results in severe compromise ( 11 ). However, studies have shown that the spindle can reassemble post-cryopreservation, and acquire correct chromosome alignment and segregation following fertilisation ( 7 , 11 , 13 , 17 , 24 – 27 ) Osmotic shock, arising from abrupt alterations in osmolarity during the addition or removal of cryoprotectant agents (CPAs), poses a risk to cellular membranes and diminishes oocyte viability ( 15 ). The selection, concentration, and potential toxicity of CPAs are paramount, as these substances inhibit ice crystallisation but may exhibit cytotoxicity at elevated concentrations or extended exposure durations ( 7 ). The manipulation of temperature, CPA concentration, and exposure duration must be meticulously calibrated to achieve a harmonious equilibrium between cryoprotection and cellular integrity ( 15 ). Furthermore, the preservation of stable pH levels through robust buffering systems is vital for averting cellular injury ( 15 ). The architecture of cryopreservation devices, including vitrification straws or cooling mechanisms, affects the cooling rate, which must be sufficiently rapid to prevent ice formation ( 15 ). Finally, human elements, particularly the proficiency of laboratory personnel can affect quality control variables that collectively can affect oocyte viability and embryo development following fertilisation ( 15 ). One of the most important parameters during oocyte vitrification is temperature ( 16 ). Elevated temperatures augment the kinetic energy and consequently the motility of the molecules, thereby enhancing the diffusion rate ( 15 ). Given that the processes of loading and unloading permeable CPAs are entirely contingent upon the diffusion rate, an increase in the temperature employed for oocyte vitrification correlates with a reduction in the duration required for the loading and unloading of CPAs( 15 , 19 ). In accordance with the majority of protocols from commercially available kits for oocyte vitrification, it is customary to utilise room temperature (22–25°C) as a reference temperature for the loading and unloading of CPAs across the oocyte membrane due to its procedural simplicity ( 15 , 7 ). Routine quality control procedures in most IVF laboratories include temperature monitoring of the heated stage, ensuring that media remain at 37 ± 0.2°C using validated instruments. However, less attention is typically paid to the temperature of cryopreservation solutions during handling on non-heated stages. It remains to be established whether controlling vitrification drop temperature can improve vitrification protocols in terms of overall cryopreservation outcomes. Current commercially available vitrification kits typically specify room temperature (22–27°C) for CPA loading and unloading; however, these protocols often lack adequate control over the vitrification media temperature ( 16 , 19 ). Every laboratory operates under different conditions. Laboratory factors such as room temperature, air velocity within the laminar flow hood, and dish type may inadvertently lower the temperature of the vitrification solution, potentially affecting the efficiency and consistency of cryopreservation ( 16 , 17 , 26 ). Our findings, upon verification, will highlight the importance of continuously monitoring and regulating vitrification media temperature to ensure optimal cryopreservation outcomes. Published benchmarks and key performance indicators (KPIs) for cryopreservation offer crucial insights for evaluating procedural effectiveness and results ( 18 ). Nevertheless, these benchmarks might not completely reflect the advancements in techniques and the introduction of new quality control measures. The Alpha consensus expert assembly has delineated 14 KPIs for cryopreservation; however, it did not provide specific guidance on particular cryopreservation techniques or equipment ( 18 ). Importantly, the consensus has not taken into consideration the possible impact of solution temperature regulation on vitrification outcomes. Considering that fluctuations in oocyte vitrification solution temperature can significantly affect downstream developmental capacity as shown from the outcomes of this study, this aspect represents a neglected quality control measure. The lack of standardised vitrification solution temperature monitoring in current protocols may lead to inconsistencies in results. By integrating solution temperature as an additional quality control criterion, existing KPIs could be enhanced, potentially increasing the dependability of vitrification success rates. Furthermore, even the vitrification protocol manuals issued by commercial firms, which contain specifications from the manufacturers, very commonly use room temperature with no mention of the working vitrification medium solution temperature ( 18 , 28 ) Despite the fact that our investigation yielded significant insights, some limitations must be acknowledged including the observational nature of the study which inherently limits the ability to draw causal conclusions, as well as the small sample size and limited number of donors, which constrain both the statistical power and the generalisability of the findings. Additionally, since donors were recruited at different time frames there is a possibility that the outcomes reflected biological variability between the donors, even though there were no significant differences between background characteristics and with all recruited donors having proven parity. Furthermore, potential confounding factors should be considered, particularly the fact that the observations were made in a newly established laboratory setting. A prospective investigation using sibling oocytes to control for donor related factors would offer a more robust framework, but raises ethical concerns that preclude its implementation. Further experiments using animal models could help determine whether the findings of this study are specifically attributable to the altered drop temperature of the vitrification solution. Use of apoptotic biomarkers, such as Capase-3, in addition to developmental parameters can be explored. The oocytes in this study were obtained from young, healthy donors, which may not accurately represent the wider patient demographic, including the elderly and those exhibiting diminished ovarian reserve and other infertility causes. Additionally, although our study demonstrates a possibility that temperature stability is important, the exact optimal temperature range for vitrification solutions will need to be determined. In conclusion, this study suggests that temperature may influence oocyte developmental outcomes following fertilisation but this is not conclusively shown here due to the many confounding variables that have not been assessed. Controlled vitrification solution drop temperature shows improved and not compromised embryo development, while uncontrolled temperatures can potentially compromise blastulation rates and increase developmental arrest. Regardless of room temperature, the media temperature potentially plays a more crucial role in achieving successful oocyte cryopreservation outcomes. By identifying the importance of temperature stability as a novel quality control parameter, we can refine vitrification protocols and improve cryopreservation outcomes. Declarations Ethical Approval This study was approved by the Cyprus International University Scientific Research and Publication Ethics Committee. Conflict of Interest Statement All the authors report no conflict of interest directly or indirectly related to this work submitted for publication. Funding This study did not receive any external funding. Author Contribution Conceptualization: MS;ZA;GR;GL;MM. Data acquisition: MS;ZA;HY;YY. Formal analysis: MS, Z Y; GR; OA. Methodology: MS,ZA,GR, MM,GL,GR,OA,BY. Writing review and editing: MS, GL,GR,OA, ZA, ZY. Supervision: MM,GL.All authors reviewed the results and approved the final version of the manuscript. References Cobo A, García-Velasco JA, Remohí J, Pellicer A. Oocyte vitrification for fertility preservation for both medical and nonmedical reasons. Fertil Steril. 2021 May;115(5):1091–101. Ethics Committee of the American Society for Reproductive Medicine. Electronic address: [email protected] , Ethics Committee of the American Society for Reproductive Medicine. Planned oocyte cryopreservation for women seeking to preserve future reproductive potential: an Ethics Committee opinion. Fertil Steril. 2018 Nov;110(6):1022–8. Argyle CE, Harper JC, Davies MC. Oocyte cryopreservation: where are we now? Hum Reprod Update. 2016 Jun;22(4):440–9. Johnston M, Richings NM, Leung A, Sakkas D, Catt S. A major increase in oocyte cryopreservation cycles in the USA, Australia and New Zealand since 2010 is highlighted by younger women but a need for standardized data collection. Hum Reprod Oxf Engl. 2021 Feb 18;36(3):624–35. Cascante SD, Berkeley AS, Licciardi F, McCaffrey C, Grifo JA. Planned oocyte cryopreservation: the state of the ART. Reprod Biomed Online. 2023 Dec;47(6):103367. Cobo A, Remohí J, Chang CC, Nagy ZP. Oocyte cryopreservation for donor egg banking. Reprod Biomed Online. 2011 Sep;23(3):341–6. Martinez-Rodero I, Gallardo M, Pisaturo V, Scarica C, Conaghan J, Liebermann J, et al. The development of shorter protocols for vitrification and post-warming dilution of human oocytes and embryos: a narrative review. Reprod Biomed Online [Internet]. 2025 Feb 6 [cited 2025 May 16];0(0). Available from: https://www.rbmojournal.com/article/S1472-6483(25)00064-1/fulltext Segovia Y, Victory N, Peinado I, García-Valverde LM, García M, Aizpurua J, et al. Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation. J Reprod Dev. 2017 Aug 19;63(4):377–82. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod Oxf Engl. 2001 Nov;16(11):2374–8. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril. 2001 Feb;75(2):348–53. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril. 2001 Apr;75(4):769–77. Larman MG, Minasi MG, Rienzi L, Gardner DK. Maintenance of the meiotic spindle during vitrification in human and mouse oocytes. Reprod Biomed Online. 2007 Dec;15(6):692–700. Koutlaki N, Schoepper B, Maroulis G, Diedrich K, Al-Hasani S. Human oocyte cryopreservation: past, present and future. Reprod Biomed Online. 2006 Sep;13(3):427–36. Sharma RK, Azeem A, Agarwal A. Spindle and Chromosomal Alterations in Metaphase II Oocytes. Reprod Sci. 2013 Nov 1;20(11):1293–301. Chang CC, Shapiro DB, Nagy ZP. Human Oocyte Vitrification. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner’s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 135–41. Available from: https://doi.org/10.1007/978-3-031-58214-1_13 Elder K, Dale B. In-Vitro Fertilization [Internet]. 4th ed. Cambridge: Cambridge University Press; 2020 [cited 2025 May 16]. Available from: https://www.cambridge.org/core/books/invitro-fertilization/141E123DA1050BE98BC00965A436D1E8 Sjoblom C, Liperis G. Control of Variables. In: Nagy ZP, Varghese AC, Agarwal A, editors. In Vitro Fertilization: A Textbook of Current and Emerging Methods and Devices [Internet]. Cham: Springer International Publishing; 2019 [cited 2025 May 16]. p. 57–68. Available from: https://doi.org/10.1007/978-3-319-43011-9_7 Alpha Scientists In Reproductive Medicine. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod Biomed Online. 2012 Aug;25(2):146–67. Stachecki JJ. Cryopreservation Principles. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner’s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 29–37. Available from: https://doi.org/10.1007/978-3-031-58214-1_3 Butler JM, Johnson JE, Boone WR. The heat is on: room temperature affects laboratory equipment--an observational study. J Assist Reprod Genet. 2013 Oct;30(10):1389–93. Khurana RK, Rao V, Nayak C, Pranesh GT, Rao KA. Comparing Progesterone Primed Ovarian Stimulation (PPOS) to GnRH Antagonist Protocol in Oocyte Donation Cycles. J Hum Reprod Sci. 2022;15(3):278–83. Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999 Jun;11(3):307–11. Almeida PA, Bolton VN. The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte. Zygote Camb Engl. 1995 Nov;3(4):357–65. Martínez-Burgos M, Herrero L, Megías D, Salvanes R, Montoya MC, Cobo AC, et al. Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril. 2011 Jan;95(1):374–7. Tamura AN, Huang TTF, Marikawa Y. Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes. Biol Reprod. 2013 Nov;89(5):112. Liperis G, Sjöblom C. Quality Control in the IVF Laboratory: Continuous Improvement. In: Morbeck DE, Montag MHM, editors. Principles of IVF Laboratory Practice: Laboratory Set-Up, Training and Daily Operation [Internet]. 2nd ed. Cambridge: Cambridge University Press; 2023 [cited 2025 May 16]. p. 86–95. Available from: https://www.cambridge.org/core/books/principles-of-ivf-laboratory-practice/quality-control-in-the-ivf-laboratory/175B34A2FEFD844DEB39E198E2200FD9 Pantos K, Maziotis E, Trypidi A, Grigoriadis S, Agapitou K, Pantou A, et al. The Effect of Open and Closed Oocyte Vitrification Systems on Embryo Development: A Systematic Review and Network Meta-Analysis. J Clin Med. 2024 Jan;13(9):2651. Campbell A, Barrie A. Continuous Quality Improvement (CQI) and Key Performance Indicators (KPIs) in Cryopreservation: Aspiring for the Best Results. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner’s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 277–86. Available from: https://doi.org/10.1007/978-3-031-58214-1_28 Additional Declarations No competing interests reported. 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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-7702661","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534105044,"identity":"c9590499-cb68-4e04-b1fb-43b51652764b","order_by":0,"name":"Munevver Serdarogullari","email":"","orcid":"","institution":"Ventus IVF Centre","correspondingAuthor":false,"prefix":"","firstName":"Munevver","middleName":"","lastName":"Serdarogullari","suffix":""},{"id":534105046,"identity":"251747b0-2d65-4e62-b2f2-320bde38861a","order_by":1,"name":"Zafer Atayurt","email":"","orcid":"","institution":"Ventus IVF 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1","display":"","copyAsset":false,"role":"figure","size":629882,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"GATEMPFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7702661/v1/596a9211a8681732269b7ce0.jpg"},{"id":100405712,"identity":"b4ca7980-85aa-4602-971c-3224e55af347","added_by":"auto","created_at":"2026-01-16 12:16:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1528239,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7702661/v1/f2b324e8-a08d-4859-8134-6992ca2a6871.pdf"},{"id":94457056,"identity":"e6990118-32dc-47b7-b463-cc068f6145d7","added_by":"auto","created_at":"2025-10-27 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vitrification media plays an important role in successful oocyte cryopreservation outcomes. Recognising vitrification solution temperature as a critical and novel quality control parameter may facilitate refinements in vitrification protocols and quality control in laboratories, ultimately improving the efficacy of oocyte cryopreservation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eVitrification has emerged as the leading method for oocyte cryopreservation, marking a major advancement in medically assisted reproduction (MAR), as demonstrated by its clinical effectiveness, safety, and widespread adoption in fertility preservation and elective oocyte freezing worldwide (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Its established role in fertility preservation for medical and non-medical indications as well as oocyte donation programs, particularly through decoupling donor-recipient cycle synchronization, further supports its position as the gold standard (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, given the widespread use of oocyte vitrification, evidence-based modifications to current protocols are essential to optimise outcomes.\u003c/p\u003e\u003cp\u003eHuman oocytes are large cells with high water content and a distinct cytoskeleton (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). During \u003cem\u003ein vitro\u003c/em\u003e handling and culture, even a short drop from the standard 37°C culture temperature has been associated with effects on the meiotic spindle (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), Spindle disorganisation begins after ~ 10 min at 33–34°C (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), becomes complete within minutes at 25°C (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and exposure to 0°C has been shown to cause loss of spindle integrity and chromosomal mis-alignment (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Nonetheless, such effects have been observed to be transient, with the spindle being a dynamic structure capable of depolymerization and repolymerization (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe process of vitrification entails exposure of oocyte/s in cryoprotectant (CPA) solutions followed by rapid cooling, turning water into a glass-like structure and avoiding hazardous intracellular ice formation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The method of vitrification works effectively only if all factors stay in balance (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). If CPA concentration or cooling speed is too low, ice forms; if CPA exposure or concentration is too high, chemical toxicity and sudden water movement can create cryo-injury to the oocyte (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTemperature has a pivotal role in retaining the balance (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Handling at warmer conditions accelerates CPA diffusion into and out of the oocyte, so the oocyte spends less time in potentially toxic solutions (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Temperature loss is the first link in the chain of events leading to oocyte damage with this damage possibly remaining unnoticed until pre-implantation embryo development (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Vitrification commercial kit manufacturers suggest working at a stable room temperature. In practice however, several parameters can affect the temperature at which oocytes are exposed while handling within the CPA solutions. These can include amongst others open dishes (without lid) on an unheated laminar flow hood as well as laminar flow hood air flow speed that can cause cooling effects, dish material and air-conditioning (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These conditions can create substantial differences even in laboratories that apply the same vitrification protocols and in accordance with vitrification media manufacturers’ instructions ( 18). Among these conditions, temperature control during vitrification has traditionally focused on cooling and warming rates, with less attention given to the temperature of the vitrification solutions during handling and processing (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis retrospective observational study was conducted in a single centre, and covered two separate periods. During the first period, oocyte vitrification was performed according to the manufacturer’s instructions, with active monitoring of vitrification drop temperature but no control of the drop temperature and maintenance within a designated temperature range(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This practice coincided with a noticeable decline in embryo developmental outcomes post-warming, raising concerns about the potential impact of vitrification solution temperature, an aspect that had not previously been addressed as a critical quality control parameter. As part of troubleshooting, corrective measures were implemented to ensure strict temperature control of the vitrification solutions in subsequent procedures which consisted of the second period. This study aimed to summarise results of these two separate periods comparing embryological outcomes following oocyte warming, fertilisation and embryo development to the blastocyst stage. By analysing survival rates and developmental competence, the aim was to identify if vitrification solution drop temperature is an important quality control parameter that requires control.\u003c/p\u003e\n\n"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003e2.1 Study design and ethical considerations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis is a retrospective observational study conducted at Ventus IVF Center, a newly established clinic in Cyprus, between March 2024 and February 2025. The clinic implemented a broad internal KPI monitoring system to build a reference dataset and ensure quality, with temperature routinely measured as an environmental indicator. Temperature measurements included monitoring of ambient room temperature, monitoring of all surface temperatures during handling, continuous monitoring of culture temperatures as well as monitoring of solution temperatures including vitrification solution drop temperature. Surface, solution and room temperatures were monitored using the UNI-T UT322 thermometer (Uni-Trend Technology Co., Ltd, China), which was validated against an NIST-traceable mercury-calibrated thermometer. The Vermox thermometer was equipped with a T-type thermocouple as the drop sensor and a PT100 RTD as the surface sensor. The UNI-T thermometer used a K-type thermocouple as the drop sensor, and temperature measurements were recorded at the frequency specified by the manufacturer until the temperature stabilised and no longer fluctuated.\u003c/p\u003e\u003cp\u003eDuring all stages of \u003cem\u003ein vitro\u003c/em\u003e handling including oocyte and embryo warming, denudation, fertilisation check as well as ICSI, solution temperature was measured and documented using the validated thermometer and sensor. Measurements included both the solution drop temperature and the corresponding surface temperature (± SD) and adjustments to the surface temperature were made where necessary to ensure control of handling temperature at 37.0 ± 0.2°C. During oocyte and embryo culture within the incubators, temperature was monitored continuously using temperature data loggers and sensors to record temperature over time, providing real-time data and historical records for analysis integrated with automated alarms to ensure optimal incubator conditions and signal any deviations for immediate intervention.\u003c/p\u003e\u003cp\u003ePrior to allocating donor oocytes to recipients, the efficiency of the oocyte cryopreservation programme for donors for which oocytes were frozen between March 2024- August 2024 was tested. During this period, whilst vitrification solution temperature was monitored, there was no control of the temperature within a designated range, as this was not in the list of recommendations by the vitrification media manufacturer. Donor oocytes were warmed and fertilised with donor sperm. The outcomes in terms of oocyte survival and subsequent embryo development were recorded. Results indicated suboptimal preimplantation embryo development. Troubleshooting was initiated and led to amendment of oocyte vitrification practice for donor oocytes that were vitrified between September 2024 and February 2025, during which a quality control parameter in controlling vitrification solution temperature within a designated range during handling of oocytes was implemented (23 ± 2°C). The outcomes of oocyte survival and preimplantation embryo development were also recorded following the intervention. A retrospective analysis was then performed from the data, comparing outcomes across the two different vitrification strategies (controlled and uncontrolled vitrification solution temperature), to evaluate their impact on oocyte viability and subsequent embryo development. The study received ethical approval from Cyprus International University (EKK24-25/06/10). All participants provided written informed consent for the donation of their oocytes. A total of 170 mature metaphase II (MII) oocytes, donated from 16 consenting donors across 16 donation cycles, were included. These consisted of a controlled solution temperature group (6 donors, n = 83 oocytes) and uncontrolled temperature group (10 donors, n = 87 oocytes).\u003c/p\u003e\u003cp\u003eIn both groups, solution and room temperatures were monitored using the specified and validated UNI-T UT322 thermometer with the appropriate sensors as described above. In the controlled group, although ambient room conditions were within the recommended range of 23 ± 2°C (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), the heated stage of the laminar flow hood for the purpose of oocyte vitrification was increased to 29.5°C to establish vitrification solution drop measurement within room temperature range (23 ± 2°C). The temperature of the vitrification solution was measured immediately before the addition of the oocyte into a mock drop next to the drop used for vitrification. All other parameters, including the type of dish, media volume, and dish placement, were kept consistent throughout the procedures during the study period to ensure stable and representative temperature conditions at the time of oocyte exposure. Nonetheless, most media including vitrification, warming and culture media had different lot numbers between the two study periods. Throughout the duration of the study only one senior embryologist was responsible for oocyte vitrification and warming. The senior embryologist has had extensive experience in both protocols with staff competency in oocyte vitrification and warming assessed regularly, to ensure consistent quality and adherence to best practices.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.2 Oocyte Donors\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll participants in the study were anonymous individuals, with ages ranging from 20 to 30 years old at the time of oocyte donation. The candidates underwent thorough screening, including serological testing for Hepatitis B surface antigen, Hepatitis C virus, and HIV antibodies, as well as genetic testing for thalassemia, cystic fibrosis, and chromosomal abnormalities via karyotype analysis. Furthermore, comprehensive evaluations of familial histories concerning genetically inherited diseases were conducted. The controlled ovarian stimulation protocol was performed as previously described by Khurana et al., (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Following a baseline transvaginal ultrasound assessment on the second day of menstruation, ovarian stimulation was initiated using recombinant follicle-stimulating hormone (rFSH) (Gonal-F, Merck). The initial dosage was individualised based on the patient's age, antral follicle count (AFC), body mass index (BMI), and, if applicable, previous responses to ovarian stimulation. Concurrently, a fixed dose of medroxyprogesterone acetate (MPA) (5 mg tablet, Deva, Turkey) was administered at 10 mg once daily, beginning on the second day of menstruation and continuing until the day of ovulation trigger. Upon the achievement of at least three follicles measuring 18 mm in diameter, patients were administered 0.2 mg of triptorelin (Gonapeptyl, Ferring), with oocyte retrieval scheduled to occur 35 hours subsequent to the injection.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.3 Oocyte pick-up, denudation, semen preparation, ICSI and embryo culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOocyte aspiration was performed 35 hours after HCG administration under ultrasound guidance. Oocytes were washed with handling medium-complete (MHM-C; FuJIFILM Irvine Scientific, CA, USA) containing Gentamicin and Human Serum Albumin (HSA), and collected into pre-equilibrated 750-µl drops of continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA, which was kept under humidified and heated conditions (at 37°C, 6% CO2 and 5% O2) in a benchtop incubator (G210 InviCell From K-Systems™) for 2 h until denudation. Enzymatic removal of cumulus cells was performed using 80 IU/mL hyaluronidase (FUJIFILM Irvine Scientific, CA, USA). Following denudation, intracytoplasmic sperm injection (ICSI) was performed in multipurpose handling medium-complete (MHM-C; FUJIFILM Irvine Scientific, CA, USA) containing Gentamicin and HSA and microinjected oocytes were cultured individually in a special pre-equilibrated culture dish (EmbryoSlide, Vitrolife) for embryo development. In this study, only continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA was used for embryo culture. EmbryoSlide wells were filled with 25–30 µl CSCM-NXM and covered with 1.4 ml Heavy oil (Kitazato) to prevent evaporation and equilibrated overnight before use. All oocytes/embryos were cultured in a time-lapse incubator (EmbryoScope, Vitrolife) at 37°C, 6% CO2 and 5% O2. The sperm used for fertilisation derived from donors and was obtained from a commercial sperm bank (Cryos International), with each sample being thawed and subsequently subjected to two washing cycles utilising a sperm washing medium, specifically a modified HTF medium supplemented with HSA at a concentration of 5.0 mg/mL (FUJIFILM Irvine Scientific, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.4 Oocyte Vitrification and Warming Procedure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study was conducted in a controlled laboratory environment. The room temperature and vitrification solution temperatures were recorded using the UNI-T UT322 thermometer, validated with an NIST-traceable mercury-calibrated thermometer as described above. The Vitrification Freeze/Warm Kit (Vit Kit-Freeze NX/Vit Kit-Warm NX; FUJIFILM Irvine Scientific, CA, USA) was employed throughout the course of this investigation. The procedures for the vitrification and warming of oocytes were executed in accordance with the specifications provided by the manufacturers. The Cryotop® carrier device (Kitazato, Japan) and Nunc Ivf dish 90mm, non-treated (Thermo Scientific, USA) were utilised for the vitrification processes. In brief, Equilibration NX - ES constitutes a dual buffered solution (HEPES \u0026amp; MOPS) derived from Continuous Single Culture medium (CSCM), which incorporates Gentamicin Sulfate, 7.5% (v/v) of both DMSO and ethylene glycol, alongside 20% (v/v) of Dextran Serum Supplement (DSS). Vitrification NX - VS is characterised as a dual buffered solution (HEPES \u0026amp; MOPS) of CSCM that includes Gentamicin Sulfate, 15% (v/v) of each DMSO and ethylene glycol, 20% (v/v) DSS, in addition to 0.5 M Trehalose. DSS represents a protein supplement composed of 50 mg/mL therapeutic grade HSA and 20 mg/mL Dextran. DSS is incorporated at a concentration of 20% (v/v) in Vit Kit – Freeze NX, yielding a final concentration of 10 mg/mL HSA and 4 mg/mL Dextran. Also, thawing Kit contains a Thawing Solution (TS) that contains 2 mL in each vial of a 1 M sucrose, 20% DSS, and gentamicin composition in an M-199 HEPES buffered medium. Also, kit contains a Dilution Solution (DS) consisting of 2 mL of a composition of 0.5 M sucrose, 20% DSS, and gentamicin in the same M-199 HEPES buffered medium. Finally, the Washing Solution (WS) containing 2 mL contains 20% DSS and gentamicin in an M-199 HEPES buffered medium. After warming, oocytes were checked for viability (warmed oocytes were considered to not survive if lysed, highly vacuolised or otherwise impaired in cytoplasmic or extracytoplasmic structures) and incubated in standard conditions at 37°C (6% CO2, 5%O2) until the ICSI procedure.\u003c/p\u003e\u003ch3\u003eFertilisation and Embryo culture:\u003c/h3\u003e\u003cp\u003eUpon completion of warming of oocytes, ICSI was conducted in multipurpose handling medium-complete (MHM-C; FUJIFILM Irvine Scientific, CA, USA) containing Gentamicin and HSA, with microinjected oocytes cultured individually within a specialized pre-equilibrated culture dish (EmbryoSlide, Vitrolife) to facilitate embryonic development. This investigation exclusively employed continuous single culture-NX Complete (CSCM-NXM) supplemented with Gentamicin and HSA for the purpose of embryo culture. The wells of the EmbryoSlide were filled with 25–30 µl CSCM-NXM and subsequently covered with 1.4 ml of Heavy oil (Kitazato) to mitigate evaporation, allowing for overnight equilibration prior to application. All embryos were incubated within a time-lapse incubator (EmbryoScope™+ ,Vitrolife) at 37°C, 6% CO2, and 5% O2. Fertilisation assessment was conducted 16–18 hours post insemination. Embryos were cultured until day 6 of embryo development and Gardner and Schoolcraft's criteria for blastocyst grading additional to morphokinetic data were used to evaluate embryo quality (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eStatistical Analysis:\u003c/h2\u003e\u003cp\u003eData were analysed using both parametric and non-parametric statistical methods as appropriate. For comparison of continuous variables between controlled and uncontrolled temperature groups, including stimulation days, daily usage, BMI, total usage, age, collected oocytes, and temperature measurements, the Mann-Whitney U test was employed due to the small sample sizes and without assuming normal distribution of the data. Results are presented as mean ± standard deviation (M ± SD) along with 95% confidence intervals (CI).\u003c/p\u003e\u003cp\u003eFor categorical variables related to oocyte and embryo quality outcomes, chi-square tests (χ²) were used to compare proportions between groups, including oocyte survival percentage, fertilisation rate, and blastulation percentage. Fisher's exact test was applied for comparing pronuclear arrest, Post-Pronuclear fading zygote arrest, and cleavage arrest percentages between groups, as this test is more appropriate when expected cell frequencies are small. For these arrest parameters, one-sided 97.5% confidence intervals were calculated to reflect the directionality of the observed differences. Statistical significance was set at p \u0026lt; 0.05 for all analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eBaseline donor characteristics are comparable between controlled and uncontrolled temperature groups\u003c/h2\u003e\u003cp\u003eBaseline characteristics of oocyte donors did not differ significantly between the controlled (6 donors) and uncontrolled (10 donors) temperature groups across all assessed parameters \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The mean number of stimulation days was comparable between the uncontrolled (10.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36) and controlled (10.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51) groups (p\u0026thinsp;=\u0026thinsp;0.76). Daily gonadotropin doses were similar in the uncontrolled (209.09\u0026thinsp;\u0026plusmn;\u0026thinsp;16.85 IU) and controlled (208.33\u0026thinsp;\u0026plusmn;\u0026thinsp;12.91 IU) groups (p\u0026thinsp;=\u0026thinsp;0.88). BMI values were also comparable between groups (22.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 vs 21.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98; p\u0026thinsp;=\u0026thinsp;0.80). Total gonadotropin consumption during stimulation did not differ between the uncontrolled (2170.45\u0026thinsp;\u0026plusmn;\u0026thinsp;224.67 IU) and controlled (2220.83\u0026thinsp;\u0026plusmn;\u0026thinsp;334.07 IU) groups (p\u0026thinsp;=\u0026thinsp;0.80). Mean age was comparable (24.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 vs 25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03 years; p\u0026thinsp;=\u0026thinsp;0.80), and the number of oocytes retrieved was similar (22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92 vs 22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07; p\u0026thinsp;=\u0026thinsp;0.96).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of patient characteristics between controlled and uncontrolled groups\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUncontrolled group\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControlled group\u003c/p\u003e\u003cp\u003e(M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95% CI Uncontrolled\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e95% CI Controlled\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStimulation Days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e10.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;29.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[9.45, 11.27]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[9.09, 12.25]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDaily Usage (IU)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e209.09\u0026thinsp;\u0026plusmn;\u0026thinsp;16.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e208.33\u0026thinsp;\u0026plusmn;\u0026thinsp;12.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[197.77, 220.41]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[194.78, 221.88]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI (Kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e21.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[21.33, 22.67]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[20.8, 22.86]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal Usage (IU)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2170.45\u0026thinsp;\u0026plusmn;\u0026thinsp;224.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e2220.83\u0026thinsp;\u0026plusmn;\u0026thinsp;334.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[2019.52, 2321.38]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[1870.19, 2571.47]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e24.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[23.13, 26.15]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[21.82, 28.18]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of collected Oocytes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e[21.26, 23.84]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e[20.5, 24.84]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eM\u0026thinsp;=\u0026thinsp;mean; SD\u0026thinsp;=\u0026thinsp;standard deviation; CI\u0026thinsp;=\u0026thinsp;confidence interval; BMI\u0026thinsp;=\u0026thinsp;body mass index.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSignificant differences in vitrification medium temperatures between groups\u003c/h3\u003e\n\u003cp\u003eRoom temperature was significantly lower in the uncontrolled temperature group (22.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u0026deg;C) compared to the controlled group (24.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C; Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A more pronounced difference was observed in the temperature of the embryo equilibration and vitrification medium (\"drop temperature\"), which averaged 19.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u0026deg;C under uncontrolled conditions versus 23.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u0026deg;C in the controlled setting (Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Graphical presentation of temperature curves over time for room temperature and vitrification solution temperature for the 2 groups is shown in Supplementary Fig.\u0026nbsp;1. Excluding vitrification, for all other processes of oocyte and embryo handling, heated surface and drop temperature measurements are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of oocyte and embryo quality between Uncontrolled group and Controlled group conditions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUncontrolled group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControlled group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95% CI Uncontrolled\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e95% CI Controlled\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRoom temperature \u0026deg;C (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09776\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[22.67, 23.03]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[24.56, 24.98]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitrification Medium temperature \u0026deg;C (Drop temp) mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3780\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMann-Whitney U\u0026thinsp;=\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[19.47, 19.83]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[23.77, 23.93]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal number of MII oocytes (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOocyte survival %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80/87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82/83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eχ\u0026sup2; = 3.0387\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.081\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.8412, 0.9670]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0.4475, 0.6891]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFertilization rate %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70/87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69/82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eχ\u0026sup2; = 0.181\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.671\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.7057, 0.8819]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0.7442, 0.9128]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePronuclear arrest %*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10/70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.0707, 0.2471]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0, 0.0521]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePost-Pronuclear fading zygote arrest %*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20/70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.1840, 0.4062]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0, 0.0521]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCleavage arrest %* (blocked at cleavage stage)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35/70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.3780, 0.6220]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0, 0.0521]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCleavage rate %*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40/70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69/69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFisher\u0026rsquo;s exact\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.4475, 0.6891]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0.9479, 1]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlastulation %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5/70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56/69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eχ\u0026sup2; = 90.5211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e[0.0236, 0.1589]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e[0.6994, 0.8957]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eCI\u0026thinsp;=\u0026thinsp;confidence interval; MII\u0026thinsp;=\u0026thinsp;metaphase II;\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e* One-sided 97.5% CI was used for these parameters.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eComparable oocyte survival and fertilisation rates under controlled and uncontrolled conditions\u003c/h3\u003e\n\u003cp\u003eA total of 87 metaphase II (MII) oocytes were exposed to uncontrolled conditions and 83 to controlled conditions. Fertilisation rates were comparable under uncontrolled and controlled conditions (70/87 vs. 69/82; χ\u0026sup2; = 0.18, p\u0026thinsp;=\u0026thinsp;0.671) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Oocyte survival following warming did not differ significantly between the two groups (80/87 vs. 82/83; χ\u0026sup2; = 3.04, p\u0026thinsp;=\u0026thinsp;0.081), with overlapping confidence intervals.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePronuclear, Post-Pronuclear fading zygote arrest, and cleavage arrest rates were significantly higher under uncontrolled conditions.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePronuclear arrest was observed in 10/70 zygotes in the uncontrolled group, whereas no arrest occurred in the controlled group (0/69; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fisher\u0026rsquo;s exact test). Post-Pronuclear fading zygote arrest followed a similar pattern, affecting 20/70 oocytes in the uncontrolled group and none in the controlled group (0/69; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Cleavage arrest occurred in 35/70 oocytes cultured under uncontrolled conditions, while all zygotes cleaved in the controlled group (69/69; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As a result, the overall cleavage rate was significantly reduced in the uncontrolled group (40/70). Blastulation was also markedly impaired, with only 5/70 oocytes reaching the blastocyst stage in the uncontrolled group compared to 56/69 in the controlled group (χ\u0026sup2; = 90.52, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Morphokinetic data comparisons for the 2 groups are provided in Supplementary Table\u0026nbsp;2. Comparisons of embryological and clinical outcomes between fresh autologous and donor oocyte cycles from March 2024 to February 2025 are shown in Supplementary Table\u0026nbsp;3.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigates the impact of vitrification media drop temperature as a potential novel quality control parameter to improve cryopreservation outcomes based on oocyte vitrification practice that was introduced in a newly built for purpose IVF laboratory. While oocyte survival and fertilisation rates remained unaffected, embryo development was significantly compromised under uncontrolled vitrification solution drop temperatures (~\u0026thinsp;20\u0026deg;C). Controlled drop temperatures (~\u0026thinsp;24\u0026deg;C) yielded higher blastulation rates and reduced developmental arrest. Comparable patient characteristics across groups strengthen the possible attribution of these effects to vitrification media drop temperature rather than baseline variability.\u003c/p\u003e\u003cp\u003eIn this observational study, cleavage arrest was significantly more frequent in the uncontrolled temperature group (50%) compared to the controlled group (0%), reinforcing the notion that thermal instability can impair embryonic development (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Previous studies have shown the importance of precise temperature control during vitrification, as even slight deviations may disrupt oocyte physiology (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Oocytes are particularly vulnerable to thermal fluctuations (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Previous studies have reported that cold-induced spindle disassembly can lead to developmental arrest (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOocytes are extremely thermosensitive and are particularly vulnerable to cryoinjuries (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Their vulnerability can be attributed primarily to their considerable large size, high water content (75\u0026ndash;85%), and distinctive intracellular architecture, rendering them among the most complex biological entities to successfully undergo freezing procedures (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The predominant physical stressors associated with cryopreservation encompass chilling injuries and the formation of ice crystals. Abrupt reductions in temperature can precipitate cold-shock injuries in temperature-sensitive structures, compromising their functionality by modifying membrane permeability and inflicting damage on intracellular organelles, such as the cytoskeleton and meiotic spindle (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The extreme vulnerability of oocytes to cryoinjury is largely attributed to the sensitivity of the meiotic spindle to temperature fluctuations (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). During all stages of in vitro handling, not only regarding cryopreservation, variations in temperature that fall beneath the physiological range (37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C) can have a direct impact on the developmental capacity of oocytes, resulting in disturbances in cytoskeletal structures, particularly the depolymerisation of the meiotic spindle (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Even brief exposure to temperatures near 20\u0026deg;C can disrupt spindle integrity, significantly impairing subsequent embryonic development after fertilization (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). At 0\u0026deg;C, rapid depolymerisation occurs, leading to irreversible spindle disruption (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). While cryoprotectants help preserve spindle structure during freezing, the warming process often results in severe compromise (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, studies have shown that the spindle can reassemble post-cryopreservation, and acquire correct chromosome alignment and segregation following fertilisation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eOsmotic shock, arising from abrupt alterations in osmolarity during the addition or removal of cryoprotectant agents (CPAs), poses a risk to cellular membranes and diminishes oocyte viability (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The selection, concentration, and potential toxicity of CPAs are paramount, as these substances inhibit ice crystallisation but may exhibit cytotoxicity at elevated concentrations or extended exposure durations (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The manipulation of temperature, CPA concentration, and exposure duration must be meticulously calibrated to achieve a harmonious equilibrium between cryoprotection and cellular integrity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Furthermore, the preservation of stable pH levels through robust buffering systems is vital for averting cellular injury (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The architecture of cryopreservation devices, including vitrification straws or cooling mechanisms, affects the cooling rate, which must be sufficiently rapid to prevent ice formation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Finally, human elements, particularly the proficiency of laboratory personnel can affect quality control variables that collectively can affect oocyte viability and embryo development following fertilisation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). One of the most important parameters during oocyte vitrification is temperature (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Elevated temperatures augment the kinetic energy and consequently the motility of the molecules, thereby enhancing the diffusion rate (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Given that the processes of loading and unloading permeable CPAs are entirely contingent upon the diffusion rate, an increase in the temperature employed for oocyte vitrification correlates with a reduction in the duration required for the loading and unloading of CPAs(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn accordance with the majority of protocols from commercially available kits for oocyte vitrification, it is customary to utilise room temperature (22\u0026ndash;25\u0026deg;C) as a reference temperature for the loading and unloading of CPAs across the oocyte membrane due to its procedural simplicity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Routine quality control procedures in most IVF laboratories include temperature monitoring of the heated stage, ensuring that media remain at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C using validated instruments. However, less attention is typically paid to the temperature of cryopreservation solutions during handling on non-heated stages. It remains to be established whether controlling vitrification drop temperature can improve vitrification protocols in terms of overall cryopreservation outcomes.\u003c/p\u003e\u003cp\u003eCurrent commercially available vitrification kits typically specify room temperature (22\u0026ndash;27\u0026deg;C) for CPA loading and unloading; however, these protocols often lack adequate control over the vitrification media temperature (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Every laboratory operates under different conditions. Laboratory factors such as room temperature, air velocity within the laminar flow hood, and dish type may inadvertently lower the temperature of the vitrification solution, potentially affecting the efficiency and consistency of cryopreservation (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Our findings, upon verification, will highlight the importance of continuously monitoring and regulating vitrification media temperature to ensure optimal cryopreservation outcomes. Published benchmarks and key performance indicators (KPIs) for cryopreservation offer crucial insights for evaluating procedural effectiveness and results (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Nevertheless, these benchmarks might not completely reflect the advancements in techniques and the introduction of new quality control measures. The Alpha consensus expert assembly has delineated 14 KPIs for cryopreservation; however, it did not provide specific guidance on particular cryopreservation techniques or equipment (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Importantly, the consensus has not taken into consideration the possible impact of solution temperature regulation on vitrification outcomes. Considering that fluctuations in oocyte vitrification solution temperature can significantly affect downstream developmental capacity as shown from the outcomes of this study, this aspect represents a neglected quality control measure. The lack of standardised vitrification solution temperature monitoring in current protocols may lead to inconsistencies in results. By integrating solution temperature as an additional quality control criterion, existing KPIs could be enhanced, potentially increasing the dependability of vitrification success rates. Furthermore, even the vitrification protocol manuals issued by commercial firms, which contain specifications from the manufacturers, very commonly use room temperature with no mention of the working vitrification medium solution temperature (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eDespite the fact that our investigation yielded significant insights, some limitations must be acknowledged including the observational nature of the study which inherently limits the ability to draw causal conclusions, as well as the small sample size and limited number of donors, which constrain both the statistical power and the generalisability of the findings. Additionally, since donors were recruited at different time frames there is a possibility that the outcomes reflected biological variability between the donors, even though there were no significant differences between background characteristics and with all recruited donors having proven parity. Furthermore, potential confounding factors should be considered, particularly the fact that the observations were made in a newly established laboratory setting. A prospective investigation using sibling oocytes to control for donor related factors would offer a more robust framework, but raises ethical concerns that preclude its implementation. Further experiments using animal models could help determine whether the findings of this study are specifically attributable to the altered drop temperature of the vitrification solution. Use of apoptotic biomarkers, such as Capase-3, in addition to developmental parameters can be explored. The oocytes in this study were obtained from young, healthy donors, which may not accurately represent the wider patient demographic, including the elderly and those exhibiting diminished ovarian reserve and other infertility causes. Additionally, although our study demonstrates a possibility that temperature stability is important, the exact optimal temperature range for vitrification solutions will need to be determined. In conclusion, this study suggests that temperature may influence oocyte developmental outcomes following fertilisation but this is not conclusively shown here due to the many confounding variables that have not been assessed. Controlled vitrification solution drop temperature shows improved and not compromised embryo development, while uncontrolled temperatures can potentially compromise blastulation rates and increase developmental arrest. Regardless of room temperature, the media temperature potentially plays a more crucial role in achieving successful oocyte cryopreservation outcomes. By identifying the importance of temperature stability as a novel quality control parameter, we can refine vitrification protocols and improve cryopreservation outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical Approval\u003c/h2\u003e\u003cp\u003e This study was approved by the Cyprus International University Scientific Research and Publication Ethics Committee.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\u003cp\u003eAll the authors report no conflict of interest directly or indirectly related to this work submitted for publication.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study did not receive any external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: MS;ZA;GR;GL;MM. Data acquisition: MS;ZA;HY;YY. Formal analysis: MS, Z Y; GR; OA. Methodology: MS,ZA,GR, MM,GL,GR,OA,BY. Writing review and editing: MS, GL,GR,OA, ZA, ZY. Supervision: MM,GL.All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCobo A, Garc\u0026iacute;a-Velasco JA, Remoh\u0026iacute; J, Pellicer A. Oocyte vitrification for fertility preservation for both medical and nonmedical reasons. Fertil Steril. 2021 May;115(5):1091\u0026ndash;101. \u003c/li\u003e\n\u003cli\u003eEthics Committee of the American Society for Reproductive Medicine. Electronic address: [email protected], Ethics Committee of the American Society for Reproductive Medicine. Planned oocyte cryopreservation for women seeking to preserve future reproductive potential: an Ethics Committee opinion. Fertil Steril. 2018 Nov;110(6):1022\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eArgyle CE, Harper JC, Davies MC. Oocyte cryopreservation: where are we now? Hum Reprod Update. 2016 Jun;22(4):440\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eJohnston M, Richings NM, Leung A, Sakkas D, Catt S. A major increase in oocyte cryopreservation cycles in the USA, Australia and New Zealand since 2010 is highlighted by younger women but a need for standardized data collection. Hum Reprod Oxf Engl. 2021 Feb 18;36(3):624\u0026ndash;35. \u003c/li\u003e\n\u003cli\u003eCascante SD, Berkeley AS, Licciardi F, McCaffrey C, Grifo JA. Planned oocyte cryopreservation: the state of the ART. Reprod Biomed Online. 2023 Dec;47(6):103367. \u003c/li\u003e\n\u003cli\u003eCobo A, Remoh\u0026iacute; J, Chang CC, Nagy ZP. Oocyte cryopreservation for donor egg banking. Reprod Biomed Online. 2011 Sep;23(3):341\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eMartinez-Rodero I, Gallardo M, Pisaturo V, Scarica C, Conaghan J, Liebermann J, et al. The development of shorter protocols for vitrification and post-warming dilution of human oocytes and embryos: a narrative review. Reprod Biomed Online [Internet]. 2025 Feb 6 [cited 2025 May 16];0(0). Available from: https://www.rbmojournal.com/article/S1472-6483(25)00064-1/fulltext \u003c/li\u003e\n\u003cli\u003eSegovia Y, Victory N, Peinado I, Garc\u0026iacute;a-Valverde LM, Garc\u0026iacute;a M, Aizpurua J, et al. Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation. J Reprod Dev. 2017 Aug 19;63(4):377\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eWang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod Oxf Engl. 2001 Nov;16(11):2374\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eWang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril. 2001 Feb;75(2):348\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eZenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril. 2001 Apr;75(4):769\u0026ndash;77. \u003c/li\u003e\n\u003cli\u003eLarman MG, Minasi MG, Rienzi L, Gardner DK. Maintenance of the meiotic spindle during vitrification in human and mouse oocytes. Reprod Biomed Online. 2007 Dec;15(6):692\u0026ndash;700. \u003c/li\u003e\n\u003cli\u003eKoutlaki N, Schoepper B, Maroulis G, Diedrich K, Al-Hasani S. Human oocyte cryopreservation: past, present and future. Reprod Biomed Online. 2006 Sep;13(3):427\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eSharma RK, Azeem A, Agarwal A. Spindle and Chromosomal Alterations in Metaphase II Oocytes. Reprod Sci. 2013 Nov 1;20(11):1293\u0026ndash;301. \u003c/li\u003e\n\u003cli\u003eChang CC, Shapiro DB, Nagy ZP. Human Oocyte Vitrification. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner\u0026rsquo;s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 135\u0026ndash;41. Available from: https://doi.org/10.1007/978-3-031-58214-1_13 \u003c/li\u003e\n\u003cli\u003eElder K, Dale B. In-Vitro Fertilization [Internet]. 4th ed. Cambridge: Cambridge University Press; 2020 [cited 2025 May 16]. Available from: https://www.cambridge.org/core/books/invitro-fertilization/141E123DA1050BE98BC00965A436D1E8 \u003c/li\u003e\n\u003cli\u003eSjoblom C, Liperis G. Control of Variables. In: Nagy ZP, Varghese AC, Agarwal A, editors. In Vitro Fertilization: A Textbook of Current and Emerging Methods and Devices [Internet]. Cham: Springer International Publishing; 2019 [cited 2025 May 16]. p. 57\u0026ndash;68. Available from: https://doi.org/10.1007/978-3-319-43011-9_7 \u003c/li\u003e\n\u003cli\u003eAlpha Scientists In Reproductive Medicine. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting. Reprod Biomed Online. 2012 Aug;25(2):146\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eStachecki JJ. Cryopreservation Principles. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner\u0026rsquo;s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 29\u0026ndash;37. Available from: https://doi.org/10.1007/978-3-031-58214-1_3 \u003c/li\u003e\n\u003cli\u003eButler JM, Johnson JE, Boone WR. The heat is on: room temperature affects laboratory equipment--an observational study. J Assist Reprod Genet. 2013 Oct;30(10):1389\u0026ndash;93. \u003c/li\u003e\n\u003cli\u003eKhurana RK, Rao V, Nayak C, Pranesh GT, Rao KA. Comparing Progesterone Primed Ovarian Stimulation (PPOS) to GnRH Antagonist Protocol in Oocyte Donation Cycles. J Hum Reprod Sci. 2022;15(3):278\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eGardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999 Jun;11(3):307\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eAlmeida PA, Bolton VN. The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte. Zygote Camb Engl. 1995 Nov;3(4):357\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Burgos M, Herrero L, Meg\u0026iacute;as D, Salvanes R, Montoya MC, Cobo AC, et al. Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertil Steril. 2011 Jan;95(1):374\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eTamura AN, Huang TTF, Marikawa Y. Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes. Biol Reprod. 2013 Nov;89(5):112. \u003c/li\u003e\n\u003cli\u003eLiperis G, Sj\u0026ouml;blom C. Quality Control in the IVF Laboratory: Continuous Improvement. In: Morbeck DE, Montag MHM, editors. Principles of IVF Laboratory Practice: Laboratory Set-Up, Training and Daily Operation [Internet]. 2nd ed. Cambridge: Cambridge University Press; 2023 [cited 2025 May 16]. p. 86\u0026ndash;95. Available from: https://www.cambridge.org/core/books/principles-of-ivf-laboratory-practice/quality-control-in-the-ivf-laboratory/175B34A2FEFD844DEB39E198E2200FD9 \u003c/li\u003e\n\u003cli\u003ePantos K, Maziotis E, Trypidi A, Grigoriadis S, Agapitou K, Pantou A, et al. The Effect of Open and Closed Oocyte Vitrification Systems on Embryo Development: A Systematic Review and Network Meta-Analysis. J Clin Med. 2024 Jan;13(9):2651. \u003c/li\u003e\n\u003cli\u003eCampbell A, Barrie A. Continuous Quality Improvement (CQI) and Key Performance Indicators (KPIs) in Cryopreservation: Aspiring for the Best Results. In: Nagy ZP, Varghese AC, Agarwal A, editors. Cryopreservation in Assisted Reproduction: A Practitioner\u0026rsquo;s Guide to Methods, Management and Organization [Internet]. Cham: Springer International Publishing; 2024 [cited 2025 May 16]. p. 277\u0026ndash;86. Available from: https://doi.org/10.1007/978-3-031-58214-1_28 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"quality control, vitrification solution temperature, human oocyte, cryopreservation","lastPublishedDoi":"10.21203/rs.3.rs-7702661/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7702661/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose:\u003c/h2\u003e\u003cp\u003eThe purpose of this study is to evaluate the role of controlling media temperature during human oocyte vitrification. This study aimed to retrospectively compare embryological outcomes following oocyte warming, fertilisation and embryo development to the blastocyst stage for two separate periods during which the vitrification solution temperature was not controlled and controlled.\u003c/p\u003e\u003ch2\u003eMethod:\u003c/h2\u003e\u003cp\u003eA retrospective observational study was conducted at Ventus IVF Center, Cyprus (March 2024\u0026ndash;February 2025), evaluating the effect of vitrification solution temperature during vitrification on oocyte survival and embryological outcomes. All oocytes were sourced from 16 donor cycles, with informed consent obtained. Oocytes were vitrified either under uncontrolled ambient conditions (n\u0026thinsp;=\u0026thinsp;87 oocytes) or with temperature control (n\u0026thinsp;=\u0026thinsp;83 oocytes) implemented using a heated stage. Vitrification and equilibration solution temperatures were recorded using a calibrated UNI-T UT322 thermometer.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eThere were no significant differences in baseline characteristics of oocyte donors between the controlled and uncontrolled temperature groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). During vitrification, vitrification media temperature was significantly higher in the controlled group (23.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u0026deg;C) compared to the uncontrolled group (19.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u0026deg;C; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant differences were observed in oocyte survival post-warming (82/83 vs. 80/87; p\u0026thinsp;=\u0026thinsp;0.081) or fertilisation rates (69/82 vs. 70/87; p\u0026thinsp;=\u0026thinsp;0.671) between groups. Compared to the controlled group, the uncontrolled group showed markedly higher developmental arrest at the pronuclear (10/70 vs. 0/69), post-pronuclear (20/70 vs. 0/69), and cleavage (35/70 vs. 0/69) stages, and significantly reduced blastocyst formation (5/70 vs. 56/69; all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eMaintaining vitrification solution temperature rather than relying on ambient conditions, significantly improves oocyte vitrification efficiency and pre-implantation embryo development parameters.\u003c/p\u003e","manuscriptTitle":"Media temperature control: A potentially important quality control parameter in human oocyte vitrification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 11:41:41","doi":"10.21203/rs.3.rs-7702661/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bae1d2e7-738e-458f-bc74-df20c8b51f04","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-16T01:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 11:41:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7702661","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7702661","identity":"rs-7702661","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}