Redefining safety standards: A large-scale comparative analysis of bovine versus murine models for medical device embryotoxicity testing.

Redefining safety standards: A large-scale comparative analysis of bovine versus murine models for medical device embryotoxicity testing. | 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 Redefining safety standards: A large-scale comparative analysis of bovine versus murine models for medical device embryotoxicity testing. Raquel Romar, Jon Romero, María Maroto, Alfonso Gutiérrez-Adán, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8967516/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 Structured Abstract Purpose To overcome the limited sensitivity of the standard Mouse Embryo Assay (MEA) for embryotoxicity screening of assisted reproduction devices and to assess the Bovine Embryo Assay (BEA) as a more sensitive alternative. Methods : In a large comparative laboratory study, bovine cumulus–oocyte complexes (from slaughterhouse ovaries) were fertilized with frozen semen from the same bull, and mouse cumulus–oocyte complexes, epididymal sperm, and one-cell embryos were used for MEA. Sperm selection, fertilization, and embryo culture media from two suppliers (A: Vitrolife; B: Genea Biomedx) were tested in parallel using BEA, standard MEA, and an extended MEA including fertilization. BEA assessed cleavage, blastocyst development and kinetics, post-warming re-expansion/hatching, total cell number, ICM and TE allocation, and ICM/TE ratio. Obstetrics & Gynecology embryotoxicity human culture media assisted reproduction mouse embryo assay bovine embryo assay Figures Figure 1 Figure 2 Figure 3 Capsule The bovine model detects toxicities in commercial media that the standard mouse assay fails to identify, improving the safety of human embryos. INTRODUCTION Human assisted reproduction relies on culture media and a range of embryo-contact devices whose safety is typically assured through premarket and lot-release quality control [ 1 , 2 ]. For decades, the standard mouse embryo assay has been the principal biological test used to screen assisted reproduction devices for embryotoxicity [ 3 – 5 ]. However, routine mouse embryo testing is widely criticized for limited discriminatory power under current acceptance thresholds [ 6 ], frequently classifying tested lots as non-toxic while failing to reveal sublethal effects that may impair embryo competence. Under commonly applied guidance from FDA [ 7 ], lots are typically accepted when at least 80% of embryos reach the expanded blastocyst stage, a criterion that is often met and may mask meaningful between-lot or between-product differences. This concern is amplified by the lack of standardization of key parameters (culture duration, embryo numbers and endpoints) across laboratories and by known physiological differences between mouse and human early development [ 8 – 10 ]. In parallel, societal and regulatory pressure to reduce animal use in testing has intensified the need for more informative approaches that can be implemented in quality control settings [ 11 , 12 ]. A practical alternative should preserve the conceptual strengths of embryo-based testing (integrating complex cell-microenvironment interactions) while improving sensitivity and reducing reliance on animals bred and sacrificed for this purpose [ 13 , 14 ]. Bovine in vitro embryo production offers abundant material sourced as slaughterhouse by-products, enabling large sample sizes and a broader set of developmental and quality endpoints than typically used in the mouse embryo assay [ 13 ]. Here, we performed a large-scale comparison of a bovine embryo assay with mouse embryo assays currently used for assisted reproduction device testing. Using identical batches of sperm selection, fertilization and embryo culture media from two commercial suppliers, we quantified developmental success, kinetics, cell allocation and cryotolerance in bovine embryos, and compared the ability of mouse embryo assays to detect differences across the same test articles. MATERIALS AND METHODS Study overview Four experiments were performed to compare bovine and mouse embryo-based embryotoxicity testing across three categories of assisted reproduction culture media: sperm selection media, fertilization media and embryo culture media. Experiments 1–3 evaluated each category separately using a bovine embryo assay (BEA) in parallel with the standard mouse embryo assay (Std-MEA) performed by an independent certified laboratory. Experiment 4 evaluated the complete, sequential use of each supplier's media line in a mouse assay that included in vitro fertilization (IVF-MEA). Test articles The same production batches of culture media from two commercial suppliers were used throughout. Supplier A was Vitrolife AB (Sweden) and supplier B was Genea Biomedx (Sydney, Australia). Each supplier contributed one medium for sperm selection (SpermRinse™ and Sperm Buffer medium, respectively), one for fertilization (Gx-IVF™ and Fertilisation Medium) and one for embryo culture (Gx-TL™ and Geri® Medium). Species-appropriate control media were used for bovine procedures (EmbryoCloud, Murcia, Spain) and for mouse procedures, as described below. Bovine embryo assay (BEA) Bovine ovaries were obtained from a commercial abattoir and transported to the laboratory in tempered saline within two hours. Cumulus-oocyte complexes were aspirated from 2–8 mm antral follicles, selected by morphology, rinsed in COW-WASH medium (EmbryoCloud, Murcia, Spain) and matured in vitro in COW-IVM medium (EmbryoCloud) for 22–24 hours at 38.5º C in a humidified atmosphere containing 5% CO 2 . In vitro fertilization was performed using frozen-thawed semen from a single bull of known fertility after sperm selection by swim-up in COW-SUM medium (EmbryoCloud). Gametes were co-incubated in COW-IVF medium (EmbryoCloud) for 24 hours at 38.5 ºC under 5% CO 2 . Putative zygotes were denuded and cultured under oil in COW-IVC medium (EmbryoCloud) at 38.5 ºC in a humidified atmosphere with 5% CO 2 and 5% O 2 for up to eight days. A full description of BEA is provided in Supplemental Materials. Endpoints included cleavage rate at day 2, cumulative blastocyst yield at days 7 and 8, and developmental stage distribution at days 7 and 8. Blastocyst quality was assessed by differential cell staining [ 15 ] to quantify total cell number, inner cell mass cell number (ICM), trophectoderm cell number (TE) and the inner cell mass to trophectoderm ratio (ICM/TE). Cryotolerance was evaluated by vitrifying day 7 expanded blastocysts (quality grades 1–2) and recording re-expansion and survival at 3 h and 24 h after warming [ 16 ]. Detailed protocols for differential staining and vitrification-warming followed previously published methods with minor modifications[ 17 , 18 ]; only modifications relevant to the present experiments are described in the Supplemental Materials. Mouse embryo assays (MEA) 1. Standard mouse embryo assay (Std-MEA): The Std-MEA was conducted by Embryotools S.L. (Barcelona, Spain), an ISO 17025-certified independent laboratory, following current regulatory guidance [ 7 , 19 ]. In brief, in vivo-derived one-cell mouse embryos were cultured in test media for durations aligned with each medium's intended use and then continued in control conditions. Acceptance criteria and scoring followed the certified laboratory workflow, including a requirement that at least 80% of embryos develop to the expanded blastocyst stage within the specified culture window. Cleavage and blastocyst development and morphology were assessed using the laboratory's certified workflow and acceptance criteria. 2. Mouse assay including in vitro fertilization (IVF-MEA): A research laboratory (INIA-CSIC, Madrid, Spain) performed a mouse assay that included sperm preparation, in vitro fertilization and embryo culture using each supplier's media line sequentially. Control sperm preparation and fertilization were conducted in standard mouse fertilization media, HTF-HSA and KSOM respectively. A full description of IVF-MEA is provided in Supplemental Materials to enable reproducibility. Ethics and oversight Institutional Review Board status: not applicable; no human participants, clinical specimens or identifiable patient data were used. Bovine material consisted of slaughterhouse by-products obtained from routine commercial operations; no live animals were handled or subjected to experimental procedures for this study. Institutional Animal Care and Use Committee approval: all procedures for IVF-MEA were performed in accordance with European and national legislation for animal research and were approved by the institutional ethics committee and competent authority under protocol PROEX 137.2/21. Statistical analysis Developmental rates were analyzed as binomial outcomes. Group comparisons were performed using analysis of variance after appropriate transformation when required, followed by post hoc multiple comparisons. Statistical significance was set at P < 0.05. Detailed statistics procedure is provided in Supplemental Materials. RESULTS Bovine dataset and replicate structure Across experiments 1–3, a total of 4,118 bovine oocytes were used, with at least four independent in vitro fertilization cycles per medium category and supplier and at least 50 oocytes per group per replicate. The distribution by experiment was 1,249 oocytes for sperm selection media, 1,392 oocytes for fertilization media and 1,477 oocytes for embryo culture media. Mouse embryo assays were performed on the same batches of media in parallel (51 oocytes in sperm selection testing, 75 one-cell embryos for fertilization media testing and 80 one-cell embryos for embryo culture media testing in Std-MEA). As for IVF-MEA, a total of 1,028 oocytes were used to test each supplier's media line sequentially. Experiment 1: Sperm selection media When bull sperm were selected using sperm selection media from supplier A (Vitrolife; SpermRinse™) or supplier B (Genea Biomedx; Sperm Buffer medium) and then used to fertilize bovine oocytes under identical bovine fertilization and embryo culture conditions, no differences were observed relative to the bovine control medium for cleavage, blastocyst yield at days 7 and 8 or developmental kinetics (Figs. 1 A-C-D). Blastocyst quality metrics, including total cell number, inner cell mass cell number, trophectoderm cell number (Fig. 1 B) and inner cell mass to trophectoderm ratio ( 0.70 ± 0.05, 0.75 ± 0.05 and 0.75 ± 0.05, respectively, for control, supplier A and supplier B groups) were comparable across groups Cryotolerance, assessed by re-expansion and survival at 3 hours and 24 hours after warming, also did not differ among groups. (Figs. 1 E-F). The Std-MEA results for the same sperm selection media batches likewise showed no differences relative to control (Table 1 ). Table 1 Embryo yield a n d ki n etics obtai n ed in the standard Mouse Embryo Assay (std-MEA) with sperm selection media from two different suppliers (SpermRinse™ from Vitrolife and Sperm Buffer from Genea Biomedx). Control n Day 2 Two-cell stage n (%) Day 5 Expanded blastocyst* n (%) Good Quality (morphology) Blastocysts n (%) Result 16 15 (93.75) 15 (93.75) 11 (73.33) Passed SpermRinse™ 15 15 (100) 15 (100) 8 (53.33) Passed Sperm Buffer 20 20 (100) 18 (90) 15 (83.33) Passed P-value 0.342 0.476 0.411 *Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P < 0.05). NS: not significant. HTF-BSA and KSOM media were used in control group. Experiment 2: Fertilization media Human fertilization media did not support sperm penetration into bovine oocytes under the conditions tested; therefore, embryotoxicity was assessed in a manner analogous to Std-MEA by exposing bovine zygotes to test fertilization media for 24 h and then continuing culture in bovine embryo culture medium. Under this design, the day 8 blastocyst yield was lower in supplier A's fertilization medium (Vitrolife, Gx-IVF™), compared with the bovine control ( P < 0.005), while the supplier B (Genea; Sperm Buffer medium) showed intermediate yield (Fig. 2 A). Beyond yield, the bovine embryo assay identified differences in embryo quality: total cell number, inner cell mass cell number (Fig. 2 B) and the inner cell mass to trophectoderm ratio (0.78 ± 0.08, 0.60 ± 0.05 and 0.92 ± 0.07, respectively for control, supplier A and supplier B groups, respectively) were lower after exposure to supplier A fertilization medium compared with supplier B ( P < 0.05) Developmental stage distribution also differed, with a higher proportion of hatched blastocysts in the better-performing fertilization medium (supplier B) at day 8 ( P < 0.005) (Fig. 2 D). Post-warming survival did not differ significantly among groups (Figs. 2 E-F). In contrast, Std-MEA did not detect significant differences between fertilization media and control under its routine readouts, and both media met acceptance criteria (Table 2 ). Table 2 Embryo yield a n d ki n etics obtai n ed in the standard Mouse Embryo Assay (std-MEA) with in vitro fertilization media from two different suppliers (Gx IVF™ from Vitrolife and Fertilisation Medium from Genea Biomedx). Control n Day 2 Two-cell stage n (%) Day 5 Expanded blastocyst* n (%) Good Quality (morphology) Blastocysts n (%) Result 15 15 (100) 15 (100) 14 (93.33) Passed Gx IVF™ 30 30 (100) 30 (100) 26 (86.67) Passed Fertilisation Medium 30 30 (100) 30 (100) 24 (80.00) Passed P-value - - 0.485 *Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P < 0.05). NS: not significant. HTF-BSA and KSOM media were used in control group. Experiment 3: Embryo culture media When bovine zygotes were cultured for up to eight days in embryo culture media from the two suppliers, the bovine embryo assay detected differences consistent with impaired developmental competence and reduced embryo robustness in supplier B’s medium (Genea Biomedx; Geri® Medium). Specifically, day 8 blastocyst yield was lower in supplier B compared with the bovine control ( P < 0.05), and supplier A (Vitrolife, Gx-TL™) showed intermediate performance (Fig. 3 A). As for blastocyst quality endpoints, total cell number, trophectoderm cell number (Fig. 3 B) and the inner cell mass to trophectoderm ratio (0.75 ± 0.04, 0.67 ± 0.03 and 0.86 ± 0.07 respectively for control, supplier A and supplier B) were reduced in supplier B medium ( P < 0.05). Developmental kinetics were slower in the supplier B medium, with fewer hatched blastocysts at days 7 and 8 ( P < 0.05) (Figs. 3 C-D). These sublethal effects translated into reduced cryotolerance, with survival at 24 h after warming approximately 40% in the supplier B medium versus more than 70% in the supplier A medium and the bovine control ( P < 0.05) (Figs. 3 E-F). As in Experiment 2, Std-MEA did not detect differences between embryo culture media and control, and both media met acceptance criteria (Table 3 ). Total cell number per blastocyst was similar between groups (206.9 ± 19.2, 174.8 ± 22.9, and 180.2 ± 41.2 respectively for Control, supplier A and supplier B). Table 3 Embryo yield a n d ki n etics obtai n ed in the standard Mouse Embryo Assay (std-MEA) with embryo culture media from two different suppliers (GX-TL ™ from Vitrolife and Geri ® Medium from Genea Biomedx). Control n Day 2 Two-cell stage n (%) Day 5 Expanded blastocyst* n (%) Good Quality (morphology) Blastocysts n (%) Result 16 16 (100) 16 (100) 15 (93.75) Passed Gx-TL™ 32 32 (100) 31 (96.88) 26 (83.87) Passed Geri® Medium 32 31 (96.88) 31 (96.88) 27 (87.10) Passed P-value 0.478 0.781 0.526 *Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P < 0.05). NS: not significant. HTF-BSA and KSOM media were used in control group. Experiment 4: Mouse embryo assay including in vitro fertilization (IVF-MEA) In a mouse assay that included sperm preparation, fertilization and embryo culture performed sequentially using each supplier's media line (1,028 oocytes across four replicates), overall developmental rates to blastocyst were similar between suppliers (Table 4 ). A small shift toward earlier blastocyst formation was observed for supplier B, but the assay did not reproduce the separation between embryo culture media that was evident in the bovine embryo assay. Table 4 Embryo yield and kinetics obtained in the in vitro fertilization Mouse Embryo Assay (IVF-MEA) performed with the sequential use of two supplier's (Vitrolife and Genea Biomedx) media lines. Group Embryo yield Embryo kinetics n Day 2 Two-cell stage n (%) Day 6 Expanded blastocyst stage** n (%) Day 4 Blastocyst stage n (%) Day 5 Blastocyst stage n (%) Day 6 Blastocyst stage n (%) Control* 190 91 (47.89 ± 3.63) 32 (35.16 ± 5.03) a 4 (4.11 ± 2.16) a 27 (26.97 ± 4.85) 1 (1.11 ± 1.09) Vitrolife 389 179 (46.02 ± 2.53) 84 (46.93 ± 3.74) ab 41 (24.58 ± 3.22) c 31 (20.11 ± 3.00) 2 (1.68 ± 0.96) Genea Biomedx 449 212 (47.22 ± 2.35) 122 (57.55 ± 3.40) b 75 (38.21 ± 3.34) b 31 (18.04 ± 2.66) 2 (0.94 ± 0.66) P-value NS 0.001 < 0.001 NS NS *Control embryos were produced by using HTF-BSA and KSOM media. ** Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P < 0.05). NS: not significant. DISCUSSION This comparative analysis shows that, under routine designs and acceptance thresholds, mouse embryo assays may have limited sensitivity to discriminate between assisted reproduction culture media that differ in their effects on embryo competence. Across fertilization and embryo culture media, neither the Std-MEA nor the MEA including IVF identified relevant differences between suppliers despite bovine embryo assay findings that were consistent, statistically supported and biologically meaningful, including altered developmental kinetics, reduced lineage allocation and diminished cryotolerance. Several features of current mouse embryo testing likely contribute to low discriminatory power. The standard assay typically relies on a small number of embryos and a restricted set of endpoints dominated by morphology-based blastocyst scoring [ 7 , 8 ]. In addition, the use of in vivo-derived embryos bypasses potential sensitivity at earlier stages, including sperm exposure and fertilization, and short exposure windows can further reduce the likelihood of detecting sublethal toxicity[ 9 ]. Finally, the std-MEA relies on the use of specific mouse strains that are crossed to maintain a specific genetic background, despite different studies which propose that only a pooled sample of different mouse strains can be used for comprehensive media MEA testing and that embryos from outbred mice may be more sensitive [ 20 , 21 ] These limitations are compounded by acceptance criteria that can be met by most lots [ 7 ], which supports lot release but does not necessarily maximize the ability to detect performance differences among devices [ 14 ]. By contrast, the bovine embryo assay leverages a large number of oocytes that are readily available as slaughterhouse by-products, enabling higher statistical power without breeding and sacrificing animals for testing[ 13 , 14 ] [ 22 ] Importantly, these high sample size allows the bovine workflow incorporate, without ethical concerns, endpoints that are linked to embryo robustness, such as lineage allocation and post-warming survival, which are not captured by routine mouse embryo testing. Also, the bovine assay is not restricted to a single inbred strain with homogeneous genetics, and bovine in vitro embryo production outcomes are consistently reported within ~ 30–40% expanded blastocysts on Day 8 across laboratories and countries [ 23 – 26 ]. In order to identify plausible explanations for the observed variations in performance between the media evaluated in the bovine assay in this study, it is essential to consider both qualitative and quantitative composition. Analytical profiling studies have shown that commercially available embryo culture media differ markedly in concentrations of energy substrates, amino acids and inorganic ions[ 27 – 29 ], and that such compositional differences can measurably influence blastocyst development in mouse models[ 28 , 30 ]. In the present study, it is hard to pinpoint which specific component(s) in Supplier A versus Supplier B IVF media drive any Bovine embryo assay-related differences in blastocyst quality, because publicly available formulation details are limited. Supplier B media trace back to Mortimer’s STF/Sydney IVF lineage[ 31 ], whereas Supplier A media stem from Gardner & Lane’s sequential “back-to-nature” approach[ 32 ]. Both appear to be low-glucose formulations, but the most consistent compositional contrast is the lactate:pyruvate (L:P) ratio: Supplier A is markedly lactate-rich (high L:P), while Supplier B is relatively more pyruvate-rich with a more balanced L:P[ 28 , 29 ]. Also, supplier B reports 5 mg/mL HSA, while Supplier A has been indicated at ~ 10 mg/mL in earlier manuals[ 33 ]. Finally, Supplier A includes a triple antioxidant system (acetyl-L-carnitine, alpha-lipoic acid, N-acetyl-L-cysteine), which could mitigate redox effects associated with high lactate. Therefore, L:P ratio, HSA and antioxidants (among other factors) are plausible drivers of the differences found, but targeted experiments are needed to confirm causality. For embryo culture (IVC) media, outcomes clearly favored Supplier A under a continuous, no-renewal protocol (up to 8 days): Its IVC medium yielded more blastocysts, faster Day 7–8 kinetics, > 3× more hatched blastocysts, higher cell numbers (especially TE), a higher ICM/TE ratio, and > 2× better survival/re-expansion after vitrification than Supplier B. As with IVF media, published comparisons suggest two distinct formulation strategies: Supplier A-type systems are typically lactate-dominant, whereas Supplier B–derived media show lower lactate and relatively higher pyruvate[ 28 ]. Marked differences are also reported in amino acids (sequential essential AA introduction in Supplier A vs earlier inclusion and much higher glycine/taurine in Supplier B, largely linked to HSA) and smaller but consistent differences in salts (Na/K/Mg slightly higher in Supplier A-type media)[ 29 ]. Again, these compositional contrasts may or may not underlie the BEA performance differences, so targeted component-by-component studies are needed. The last experiment, where IVF-MEA was performed with large numbers of mouse gametes (> 1000) in several replicates, only detected minor differences. A limitation of the IVF-MEA experiment is the absence of a direct comparison with the bovine embryo assay due to the fact that IVF-MEA used all three media together, whereas the bovine assay involved testing them separately. IVF-MEA exhibited a higher proportion of Day-4 blastocysts with the Supplier B media, whereas the bovine test indicated that Supplier B IVF medium primarily enhanced blastocyst quality in terms of total cell number and the proportion of hatched blastocysts. The potential for these overlapping advantages to persist during culture in IVF-MEA may have obscured the superior performance of the Supplier A embryo culture medium, as observed in the bovine test. Collectively, these characteristics position the bovine embryo assay as a pragmatic candidate for enhancing the sensitivity of embryotoxicity assessment, thereby aligning with the principles of replacement, reduction and refinement in animal research[ 34 ]. This study also highlights considerations for implementation. Human fertilization media were not suitable for bovine fertilization in our hands, requiring an exposure design focused on zygote culture, analogous to current mouse embryo testing. We evaluated two suppliers and specific production batches; broader testing, including ring trials across laboratories, will be required to define robust performance benchmarks, acceptance criteria and reference materials. Finally, the present work was not designed to link assay outcomes with clinical outcomes; future studies could explore how sensitive embryo quality endpoints relate to clinically relevant performance measures. Overall, these data support the use of a bovine embryo assay as a more sensitive embryo-based approach to detect embryotoxicity and functional impairment in assisted reproduction culture media, and potentially other embryo-contact devices. Conclusions The standard mouse embryo assay, as routinely implemented, showed limited ability to discriminate between assisted reproduction culture media batches that differed in their effects on embryo development and robustness. A bovine embryo assay using slaughterhouse-derived oocytes detected differences in developmental kinetics, lineage allocation and cryotolerance that were not revealed by mouse assays. These findings support the bovine model as a practical, more sensitive option to redefine embryotoxicity safety standards for assisted reproduction devices, pending broader inter-laboratory validation and consensus acceptance criteria. Authorship Contribution Statemen: Conceptualization and experimental design: P.C., R.R; Data curation: P.C., R.R., M.M; Formal analysis: P.C., M.M.; Methodology: J.R-A, M.M, A.G-A; Validation: P.C., R.R.; Writing of original draft: P.C., R.R.; Writing: review and editing: P.C., R.R., J.R-A., M.M., A.G-A.; Funding acquisition: P.C. All authors have read and approved the final version of this manuscript. Declarations Authorship Contribution Statemen: Conceptualization and experimental design: P.C., R.R; Data curation: P.C., R.R., M.M; Formal analysis: P.C., M.M.; Methodology: J.R-A, M.M, A.G-A; Validation: P.C., R.R.; Writing of original draft: P.C., R.R.; Writing: review and editing: P.C., R.R., J.R-A., M.M., A.G-A.; Funding acquisition: P.C. All authors have read and approved the final version of this manuscript. Acknowledgments The authors thank Alicia Masegosa, M.Sc., Ángela Juan, M.Sc., and María López M.Sc., from the embryology laboratory of EmbryoCloud SL for their assistance in performing standard IVF procedures. The authors are grateful to Gabriel Pastor for technical support during development of experiments and Matadero Orihuela S.A, for providing bovine ovaries. 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G-Series Manual. Vitrolife Sweden AB, Göteborg, Sweden, Göteborg, p 56 Russell W, Burch R (1959) The Principles of Humane Experimental Technique. London: Methuen & Co. Limited, London Additional Declarations The authors declare potential competing interests as follows: Supplementary Files JARGSupplMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8967516","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596914396,"identity":"d3c85f5d-5692-4b72-8fb9-eddd031dea0e","order_by":0,"name":"Raquel Romar","email":"","orcid":"https://orcid.org/0000-0003-0492-289X","institution":"University of Murcia","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Romar","suffix":""},{"id":596914397,"identity":"d19c9819-d69c-4c9b-911a-bdf8b1cb0ca8","order_by":1,"name":"Jon Romero","email":"","orcid":"https://orcid.org/0000-0001-7779-6869","institution":"University of Murcia","correspondingAuthor":false,"prefix":"","firstName":"Jon","middleName":"","lastName":"Romero","suffix":""},{"id":596914398,"identity":"031d3678-4a1e-40e8-9057-6b1c87eed2b3","order_by":2,"name":"María Maroto","email":"","orcid":"https://orcid.org/0000-0001-8738-5932","institution":"INIA-CSIC","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Maroto","suffix":""},{"id":596914399,"identity":"35f210dc-f3da-46ef-8a01-e212c67e1cd4","order_by":3,"name":"Alfonso Gutiérrez-Adán","email":"","orcid":"https://orcid.org/0000-0001-9893-9179","institution":"INIA-CSIC","correspondingAuthor":false,"prefix":"","firstName":"Alfonso","middleName":"","lastName":"Gutiérrez-Adán","suffix":""},{"id":596914400,"identity":"95ed7ee4-6f7e-4a2c-b8c9-cb69b10ffdb5","order_by":4,"name":"Pilar Coy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAiElEQVRIiWNgGAWjYLCCDyTrYJxBshZmHpKUm7MfPiZt27aNgZ//AJFaLHvS0qRz224zSM5IIFKLwQ0eM7AWgxvEOgysxRKoxf48sQ4Da2EE2cJArMOAfkm27Dl3m0fiBrFagCF28MaPstty/P1EO4yBgUUCSJMQNUAtzKSnl1EwCkbBKBhZAADVSiPu4bDW3QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3943-1890","institution":"University of Murcia","correspondingAuthor":true,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Coy","suffix":""}],"badges":[],"createdAt":"2026-02-25 12:05:04","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8967516/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8967516/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103552201,"identity":"69e64235-4e38-4503-9550-f221edadd70e","added_by":"auto","created_at":"2026-02-27 02:51:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":368965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbryotoxicity and functionality through the BEA of two human sperm selection media\u003c/strong\u003e. SpermRinse™ (Vitrolife, Supplier A) and Sperm Buffer (Genea Biomedx) media are compared with a bovine sperm selection medium (control group, COW-SUM, EmbryoCloud). (\u003cstrong\u003eA\u003c/strong\u003e) Embryo yield with cleavage rate at Day 2 and blastocysts rates at Days 7 and 8, (\u003cstrong\u003eB\u003c/strong\u003e) Total number of cells per blastocyst (TCN), number of cells in the inner cell mass (ICM), number of cells in the trophectoderm (TE) for bovine blastocysts on Day 7 (expanded category) or Day 8 of culture (blastocyst, expanded, hatching and hatched categories), (\u003cstrong\u003eC\u003c/strong\u003e) Blastocysts developmental kinetics at Day 7 (\u003cstrong\u003eD\u003c/strong\u003e) Blastocysts developmental kinetics at Day 8, (\u003cstrong\u003eE\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 3 h, (\u003cstrong\u003eF\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 24 h. Blast: blastocyst.\u003c/p\u003e","description":"","filename":"JARGFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8967516/v1/098df7a3880a89ca693aabfb.png"},{"id":104398485,"identity":"03764459-f07d-47bb-9164-2d660e6465dc","added_by":"auto","created_at":"2026-03-11 12:02:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":385000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbryotoxicity and functionality through the BEA of two human in vitro fertilization media\u003c/strong\u003e. Gx-IVF™ (Vitrolife) and Fertilisation Medium (Genea Biomedx) media are compared with a bovine sperm selection medium (control group, COW-IVF, EmbryoCloud). (\u003cstrong\u003eA\u003c/strong\u003e) Embryo yield with cleavage rate at Day 2 and blastocysts rates at Days 7 and 8, (\u003cstrong\u003eB\u003c/strong\u003e) Total number of cells per blastocyst (TCN), number of cells in the inner cell mass (ICM), number of cells in the trophectoderm (TE) for bovine blastocysts on Day 7 (expanded category) or Day 8 of culture (blastocyst, expanded, hatching and hatched categories), (\u003cstrong\u003eC\u003c/strong\u003e) Blastocysts developmental kinetics at Day 7 (\u003cstrong\u003eD\u003c/strong\u003e) Blastocysts developmental kinetics at Day 8, (\u003cstrong\u003eE\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 3 h, (\u003cstrong\u003eF\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 24 h. Blast: blastocyst.\u003c/p\u003e","description":"","filename":"JARGFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8967516/v1/c0a20be66a0172126f8f5941.png"},{"id":103552202,"identity":"9cbb5a60-f4fd-4ed8-84ee-9af6ab840861","added_by":"auto","created_at":"2026-02-27 02:51:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":377775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbryotoxicity and functionality through the BEA of two human embryo culture media\u003c/strong\u003e. Gx-TL™ (Vitrolife) and Geri® Medium (Genea Biomedx) media are compared with a bovine sperm selection medium (control group, COW-IVC, EmbryoCloud). (\u003cstrong\u003eA\u003c/strong\u003e) Embryo yield with cleavage rate at Day 2 and blastocysts rates at Days 7 and 8, (\u003cstrong\u003eB\u003c/strong\u003e) Total number of cells per blastocyst (TCN), number of cells in the inner cell mass (ICM), number of cells in the trophectoderm (TE) for bovine blastocysts on Day 7 (expanded category) or Day 8 of culture (blastocyst, expanded, hatching and hatched categories), (\u003cstrong\u003eC\u003c/strong\u003e) Blastocysts developmental kinetics at Day 7 (\u003cstrong\u003eD\u003c/strong\u003e) Blastocysts developmental kinetics at Day 8, (\u003cstrong\u003eE\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 3 h, (\u003cstrong\u003eF\u003c/strong\u003e) Post-vitrification survival rate and blastocyst re-expansion at 24 h. Blast: blastocyst.\u003c/p\u003e","description":"","filename":"JARGFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8967516/v1/7fc9fd8e1a65a2c033aec6e9.png"},{"id":104407495,"identity":"9798f8fc-37f9-45b6-b188-bc0441da4d9d","added_by":"auto","created_at":"2026-03-11 12:38:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2184950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8967516/v1/11a14488-7bcf-4b3c-9d52-35bc03684ece.pdf"},{"id":104398219,"identity":"416a8c9f-f226-4131-afd8-91190ab97769","added_by":"auto","created_at":"2026-03-11 12:00:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31507,"visible":true,"origin":"","legend":"","description":"","filename":"JARGSupplMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8967516/v1/6d0662b96e45a4f7c52dd5bf.docx"}],"financialInterests":"The authors declare potential competing interests as follows: ","formattedTitle":"\u003cp\u003eRedefining safety standards: A large-scale comparative analysis of bovine versus murine models for medical device embryotoxicity testing.\u003c/p\u003e","fulltext":[{"header":"Capsule","content":"\u003cp\u003eThe bovine model detects toxicities in commercial media that the standard mouse assay fails to identify, improving the safety of human embryos.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eHuman assisted reproduction relies on culture media and a range of embryo-contact devices whose safety is typically assured through premarket and lot-release quality control [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For decades, the standard mouse embryo assay has been the principal biological test used to screen assisted reproduction devices for embryotoxicity [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, routine mouse embryo testing is widely criticized for limited discriminatory power under current acceptance thresholds [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], frequently classifying tested lots as non-toxic while failing to reveal sublethal effects that may impair embryo competence. Under commonly applied guidance from FDA [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], lots are typically accepted when at least 80% of embryos reach the expanded blastocyst stage, a criterion that is often met and may mask meaningful between-lot or between-product differences. This concern is amplified by the lack of standardization of key parameters (culture duration, embryo numbers and endpoints) across laboratories and by known physiological differences between mouse and human early development [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn parallel, societal and regulatory pressure to reduce animal use in testing has intensified the need for more informative approaches that can be implemented in quality control settings [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. A practical alternative should preserve the conceptual strengths of embryo-based testing (integrating complex cell-microenvironment interactions) while improving sensitivity and reducing reliance on animals bred and sacrificed for this purpose [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBovine in vitro embryo production offers abundant material sourced as slaughterhouse by-products, enabling large sample sizes and a broader set of developmental and quality endpoints than typically used in the mouse embryo assay [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Here, we performed a large-scale comparison of a bovine embryo assay with mouse embryo assays currently used for assisted reproduction device testing. Using identical batches of sperm selection, fertilization and embryo culture media from two commercial suppliers, we quantified developmental success, kinetics, cell allocation and cryotolerance in bovine embryos, and compared the ability of mouse embryo assays to detect differences across the same test articles.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy overview\u003c/h2\u003e \u003cp\u003eFour experiments were performed to compare bovine and mouse embryo-based embryotoxicity testing across three categories of assisted reproduction culture media: sperm selection media, fertilization media and embryo culture media. Experiments 1\u0026ndash;3 evaluated each category separately using a bovine embryo assay (BEA) in parallel with the standard mouse embryo assay (Std-MEA) performed by an independent certified laboratory. Experiment 4 evaluated the complete, sequential use of each supplier's media line in a mouse assay that included in vitro fertilization (IVF-MEA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTest articles\u003c/h3\u003e\n\u003cp\u003eThe same production batches of culture media from two commercial suppliers were used throughout. Supplier A was Vitrolife AB (Sweden) and supplier B was Genea Biomedx (Sydney, Australia). Each supplier contributed one medium for sperm selection (SpermRinse\u0026trade; and Sperm Buffer medium, respectively), one for fertilization (Gx-IVF\u0026trade; and Fertilisation Medium) and one for embryo culture (Gx-TL\u0026trade; and Geri\u0026reg; Medium). Species-appropriate control media were used for bovine procedures (EmbryoCloud, Murcia, Spain) and for mouse procedures, as described below.\u003c/p\u003e\n\u003ch3\u003eBovine embryo assay (BEA)\u003c/h3\u003e\n\u003cp\u003eBovine ovaries were obtained from a commercial abattoir and transported to the laboratory in tempered saline within two hours. Cumulus-oocyte complexes were aspirated from 2\u0026ndash;8 mm antral follicles, selected by morphology, rinsed in COW-WASH medium (EmbryoCloud, Murcia, Spain) and matured in vitro in COW-IVM medium (EmbryoCloud) for 22\u0026ndash;24 hours at 38.5\u0026ordm; C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. In vitro fertilization was performed using frozen-thawed semen from a single bull of known fertility after sperm selection by swim-up in COW-SUM medium (EmbryoCloud). Gametes were co-incubated in COW-IVF medium (EmbryoCloud) for 24 hours at 38.5 \u0026ordm;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. Putative zygotes were denuded and cultured under oil in COW-IVC medium (EmbryoCloud) at 38.5 \u0026ordm;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e for up to eight days. A full description of BEA is provided in Supplemental Materials.\u003c/p\u003e \u003cp\u003eEndpoints included cleavage rate at day 2, cumulative blastocyst yield at days 7 and 8, and developmental stage distribution at days 7 and 8. Blastocyst quality was assessed by differential cell staining [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] to quantify total cell number, inner cell mass cell number (ICM), trophectoderm cell number (TE) and the inner cell mass to trophectoderm ratio (ICM/TE). Cryotolerance was evaluated by vitrifying day 7 expanded blastocysts (quality grades 1\u0026ndash;2) and recording re-expansion and survival at 3 h and 24 h after warming [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Detailed protocols for differential staining and vitrification-warming followed previously published methods with minor modifications[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; only modifications relevant to the present experiments are described in the Supplemental Materials.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMouse embryo assays (MEA)\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e1. Standard mouse embryo assay (Std-MEA): The Std-MEA was conducted by Embryotools S.L. (Barcelona, Spain), an ISO 17025-certified independent laboratory, following current regulatory guidance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In brief, in vivo-derived one-cell mouse embryos were cultured in test media for durations aligned with each medium's intended use and then continued in control conditions. Acceptance criteria and scoring followed the certified laboratory workflow, including a requirement that at least 80% of embryos develop to the expanded blastocyst stage within the specified culture window. Cleavage and blastocyst development and morphology were assessed using the laboratory's certified workflow and acceptance criteria.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e2. Mouse assay including in vitro fertilization (IVF-MEA): A research laboratory (INIA-CSIC, Madrid, Spain) performed a mouse assay that included sperm preparation, in vitro fertilization and embryo culture using each supplier's media line sequentially. Control sperm preparation and fertilization were conducted in standard mouse fertilization media, HTF-HSA and KSOM respectively. A full description of IVF-MEA is provided in Supplemental Materials to enable reproducibility.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e\n\u003ch3\u003eEthics and oversight\u003c/h3\u003e\n\u003cp\u003eInstitutional Review Board status: not applicable; no human participants, clinical specimens or identifiable patient data were used.\u003c/p\u003e \u003cp\u003eBovine material consisted of slaughterhouse by-products obtained from routine commercial operations; no live animals were handled or subjected to experimental procedures for this study.\u003c/p\u003e \u003cp\u003e Institutional Animal Care and Use Committee approval: all procedures for IVF-MEA were performed in accordance with European and national legislation for animal research and were approved by the institutional ethics committee and competent authority under protocol PROEX 137.2/21.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eDevelopmental rates were analyzed as binomial outcomes. Group comparisons were performed using analysis of variance after appropriate transformation when required, followed by post hoc multiple comparisons. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Detailed statistics procedure is provided in Supplemental Materials.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBovine dataset and replicate structure\u003c/h2\u003e \u003cp\u003eAcross experiments 1\u0026ndash;3, a total of 4,118 bovine oocytes were used, with at least four independent in vitro fertilization cycles per medium category and supplier and at least 50 oocytes per group per replicate. The distribution by experiment was 1,249 oocytes for sperm selection media, 1,392 oocytes for fertilization media and 1,477 oocytes for embryo culture media. Mouse embryo assays were performed on the same batches of media in parallel (51 oocytes in sperm selection testing, 75 one-cell embryos for fertilization media testing and 80 one-cell embryos for embryo culture media testing in Std-MEA). As for IVF-MEA, a total of 1,028 oocytes were used to test each supplier's media line sequentially.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperiment 1: Sperm selection media\u003c/h3\u003e\n\u003cp\u003eWhen bull sperm were selected using sperm selection media from supplier A (Vitrolife; SpermRinse\u0026trade;) or supplier B (Genea Biomedx; Sperm Buffer medium) and then used to fertilize bovine oocytes under identical bovine fertilization and embryo culture conditions, no differences were observed relative to the bovine control medium for cleavage, blastocyst yield at days 7 and 8 or developmental kinetics (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBlastocyst quality metrics, including total cell number, inner cell mass cell number, trophectoderm cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and inner cell mass to trophectoderm ratio ( 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, respectively, for control, supplier A and supplier B groups) were comparable across groups Cryotolerance, assessed by re-expansion and survival at 3 hours and 24 hours after warming, also did not differ among groups. (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eThe Std-MEA results for the same sperm selection media batches likewise showed no differences relative to control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eEmbryo yield a\u003cem\u003en\u003c/em\u003ed ki\u003cem\u003en\u003c/em\u003eetics obtai\u003cem\u003en\u003c/em\u003eed in the standard Mouse Embryo Assay (std-MEA) with sperm selection media from two different suppliers (SpermRinse\u0026trade; from Vitrolife and Sperm Buffer from Genea Biomedx).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003cp\u003eTwo-cell stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003cp\u003eExpanded blastocyst*\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGood Quality (morphology)\u003c/p\u003e \u003cp\u003eBlastocysts n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (93.75)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (93.75)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (73.33)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpermRinse\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8 (53.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSperm Buffer\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 (90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15 (83.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NS: not significant. HTF-BSA and KSOM media were used in control group.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 2: Fertilization media\u003c/h2\u003e \u003cp\u003eHuman fertilization media did not support sperm penetration into bovine oocytes under the conditions tested; therefore, embryotoxicity was assessed in a manner analogous to Std-MEA by exposing bovine zygotes to test fertilization media for 24 h and then continuing culture in bovine embryo culture medium.\u003c/p\u003e \u003cp\u003eUnder this design, the day 8 blastocyst yield was lower in supplier A's fertilization medium (Vitrolife, Gx-IVF\u0026trade;), compared with the bovine control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005), while the supplier B (Genea; Sperm Buffer medium) showed intermediate yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBeyond yield, the bovine embryo assay identified differences in embryo quality: total cell number, inner cell mass cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the inner cell mass to trophectoderm ratio (0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, respectively for control, supplier A and supplier B groups, respectively) were lower after exposure to supplier A fertilization medium compared with supplier B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003cp\u003eDevelopmental stage distribution also differed, with a higher proportion of hatched blastocysts in the better-performing fertilization medium (supplier B) at day 8 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Post-warming survival did not differ significantly among groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eIn contrast, Std-MEA did not detect significant differences between fertilization media and control under its routine readouts, and both media met acceptance criteria (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\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\u003eEmbryo yield a\u003cem\u003en\u003c/em\u003ed ki\u003cem\u003en\u003c/em\u003eetics obtai\u003cem\u003en\u003c/em\u003eed in the standard Mouse Embryo Assay (std-MEA) with in vitro fertilization media from two different suppliers (Gx IVF\u0026trade; from Vitrolife and Fertilisation Medium from Genea Biomedx).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003cp\u003eTwo-cell stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003cp\u003eExpanded blastocyst*\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGood Quality (morphology)\u003c/p\u003e \u003cp\u003eBlastocysts n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 (100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 (93.33)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGx IVF\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26 (86.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFertilisation Medium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24 (80.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NS: not significant. HTF-BSA and KSOM media were used in control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 3: Embryo culture media\u003c/h2\u003e \u003cp\u003eWhen bovine zygotes were cultured for up to eight days in embryo culture media from the two suppliers, the bovine embryo assay detected differences consistent with impaired developmental competence and reduced embryo robustness in supplier B\u0026rsquo;s medium (Genea Biomedx; Geri\u0026reg; Medium). Specifically, day 8 blastocyst yield was lower in supplier B compared with the bovine control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and supplier A (Vitrolife, Gx-TL\u0026trade;) showed intermediate performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs for blastocyst quality endpoints, total cell number, trophectoderm cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and the inner cell mass to trophectoderm ratio (0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, 0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 respectively for control, supplier A and supplier B) were reduced in supplier B medium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Developmental kinetics were slower in the supplier B medium, with fewer hatched blastocysts at days 7 and 8 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). These sublethal effects translated into reduced cryotolerance, with survival at 24 h after warming approximately 40% in the supplier B medium versus more than 70% in the supplier A medium and the bovine control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003eAs in Experiment 2, Std-MEA did not detect differences between embryo culture media and control, and both media met acceptance criteria (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Total cell number per blastocyst was similar between groups (206.9\u0026thinsp;\u0026plusmn;\u0026thinsp;19.2, 174.8\u0026thinsp;\u0026plusmn;\u0026thinsp;22.9, and 180.2\u0026thinsp;\u0026plusmn;\u0026thinsp;41.2 respectively for Control, supplier A and supplier B).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEmbryo yield a\u003cem\u003en\u003c/em\u003ed ki\u003cem\u003en\u003c/em\u003eetics obtai\u003cem\u003en\u003c/em\u003eed in the standard Mouse Embryo Assay (std-MEA) with embryo culture media from two different suppliers (GX-TL\u003cb\u003e\u0026trade;\u003c/b\u003e from Vitrolife and Geri \u003cb\u003e\u0026reg;\u003c/b\u003eMedium from Genea Biomedx).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003cp\u003eTwo-cell stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003cp\u003eExpanded blastocyst*\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGood Quality (morphology)\u003c/p\u003e \u003cp\u003eBlastocysts n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15 (93.75)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGx-TL\u0026trade;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31 (96.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26 (83.87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGeri\u0026reg; Medium\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31 (96.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31 (96.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27 (87.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePassed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NS: not significant. HTF-BSA and KSOM media were used in control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 4: Mouse embryo assay including in vitro fertilization (IVF-MEA)\u003c/h2\u003e \u003cp\u003eIn a mouse assay that included sperm preparation, fertilization and embryo culture performed sequentially using each supplier's media line (1,028 oocytes across four replicates), overall developmental rates to blastocyst were similar between suppliers (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A small shift toward earlier blastocyst formation was observed for supplier B, but the assay did not reproduce the separation between embryo culture media that was evident in the bovine embryo assay.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEmbryo yield and kinetics obtained in the in vitro fertilization Mouse Embryo Assay (IVF-MEA) performed with the sequential use of two supplier's (Vitrolife and Genea Biomedx) media lines.\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=\".\" 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\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eEmbryo yield\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eEmbryo kinetics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003cp\u003eTwo-cell stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 6\u003c/p\u003e \u003cp\u003eExpanded blastocyst stage**\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDay 4\u003c/p\u003e \u003cp\u003eBlastocyst stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003cp\u003eBlastocyst stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDay 6\u003c/p\u003e \u003cp\u003eBlastocyst stage\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91\u003c/p\u003e \u003cp\u003e(47.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003cp\u003e(35.16\u0026thinsp;\u0026plusmn;\u0026thinsp;5.03) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e(4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16) a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003cp\u003e(26.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVitrolife\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e179\u003c/p\u003e \u003cp\u003e(46.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003cp\u003e(46.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74) ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41\u003c/p\u003e \u003cp\u003e(24.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22) c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003cp\u003e(20.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e(1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenea Biomedx\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e212\u003c/p\u003e \u003cp\u003e(47.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122\u003c/p\u003e \u003cp\u003e(57.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003cp\u003e(38.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34) b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003cp\u003e(18.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e(0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\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\u003eNS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Control embryos were produced by using HTF-BSA and KSOM media.\u003c/p\u003e \u003cp\u003e** Blastocyst rates calculated from cleaved zygotes. Different letters denote significant differences between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NS: not significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis comparative analysis shows that, under routine designs and acceptance thresholds, mouse embryo assays may have limited sensitivity to discriminate between assisted reproduction culture media that differ in their effects on embryo competence. Across fertilization and embryo culture media, neither the Std-MEA nor the MEA including IVF identified relevant differences between suppliers despite bovine embryo assay findings that were consistent, statistically supported and biologically meaningful, including altered developmental kinetics, reduced lineage allocation and diminished cryotolerance.\u003c/p\u003e \u003cp\u003eSeveral features of current mouse embryo testing likely contribute to low discriminatory power. The standard assay typically relies on a small number of embryos and a restricted set of endpoints dominated by morphology-based blastocyst scoring [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, the use of in vivo-derived embryos bypasses potential sensitivity at earlier stages, including sperm exposure and fertilization, and short exposure windows can further reduce the likelihood of detecting sublethal toxicity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Finally, the std-MEA relies on the use of specific mouse strains that are crossed to maintain a specific genetic background, despite different studies which propose that only a pooled sample of different mouse strains can be used for comprehensive media MEA testing and that embryos from outbred mice may be more sensitive [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThese limitations are compounded by acceptance criteria that can be met by most lots [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which supports lot release but does not necessarily maximize the ability to detect performance differences among devices [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBy contrast, the bovine embryo assay leverages a large number of oocytes that are readily available as slaughterhouse by-products, enabling higher statistical power without breeding and sacrificing animals for testing[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e Importantly, these high sample size allows the bovine workflow incorporate, without ethical concerns, endpoints that are linked to embryo robustness, such as lineage allocation and post-warming survival, which are not captured by routine mouse embryo testing. Also, the bovine assay is not restricted to a single inbred strain with homogeneous genetics, and bovine in vitro embryo production outcomes are consistently reported within ~\u0026thinsp;30\u0026ndash;40% expanded blastocysts on Day 8 across laboratories and countries [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to identify plausible explanations for the observed variations in performance between the media evaluated in the bovine assay in this study, it is essential to consider both qualitative and quantitative composition. Analytical profiling studies have shown that commercially available embryo culture media differ markedly in concentrations of energy substrates, amino acids and inorganic ions[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and that such compositional differences can measurably influence blastocyst development in mouse models[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the present study, it is hard to pinpoint which specific component(s) in Supplier A versus Supplier B IVF media drive any Bovine embryo assay-related differences in blastocyst quality, because publicly available formulation details are limited. Supplier B media trace back to Mortimer\u0026rsquo;s STF/Sydney IVF lineage[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], whereas Supplier A media stem from Gardner \u0026amp; Lane\u0026rsquo;s sequential \u0026ldquo;back-to-nature\u0026rdquo; approach[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Both appear to be low-glucose formulations, but the most consistent compositional contrast is the lactate:pyruvate (L:P) ratio: Supplier A is markedly lactate-rich (high L:P), while Supplier B is relatively more pyruvate-rich with a more balanced L:P[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Also, supplier B reports 5 mg/mL HSA, while Supplier A has been indicated at ~\u0026thinsp;10 mg/mL in earlier manuals[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Finally, Supplier A includes a triple antioxidant system (acetyl-L-carnitine, alpha-lipoic acid, N-acetyl-L-cysteine), which could mitigate redox effects associated with high lactate. Therefore, L:P ratio, HSA and antioxidants (among other factors) are plausible drivers of the differences found, but targeted experiments are needed to confirm causality.\u003c/p\u003e \u003cp\u003eFor embryo culture (IVC) media, outcomes clearly favored Supplier A under a continuous, no-renewal protocol (up to 8 days): Its IVC medium yielded more blastocysts, faster Day 7\u0026ndash;8 kinetics, \u0026gt;\u0026thinsp;3\u0026times; more hatched blastocysts, higher cell numbers (especially TE), a higher ICM/TE ratio, and \u0026gt;\u0026thinsp;2\u0026times; better survival/re-expansion after vitrification than Supplier B. As with IVF media, published comparisons suggest two distinct formulation strategies: Supplier A-type systems are typically lactate-dominant, whereas Supplier B\u0026ndash;derived media show lower lactate and relatively higher pyruvate[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Marked differences are also reported in amino acids (sequential essential AA introduction in Supplier A vs earlier inclusion and much higher glycine/taurine in Supplier B, largely linked to HSA) and smaller but consistent differences in salts (Na/K/Mg slightly higher in Supplier A-type media)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Again, these compositional contrasts may or may not underlie the BEA performance differences, so targeted component-by-component studies are needed.\u003c/p\u003e \u003cp\u003eThe last experiment, where IVF-MEA was performed with large numbers of mouse gametes (\u0026gt;\u0026thinsp;1000) in several replicates, only detected minor differences. A limitation of the IVF-MEA experiment is the absence of a direct comparison with the bovine embryo assay due to the fact that IVF-MEA used all three media together, whereas the bovine assay involved testing them separately. IVF-MEA exhibited a higher proportion of Day-4 blastocysts with the Supplier B media, whereas the bovine test indicated that Supplier B IVF medium primarily enhanced blastocyst quality in terms of total cell number and the proportion of hatched blastocysts. The potential for these overlapping advantages to persist during culture in IVF-MEA may have obscured the superior performance of the Supplier A embryo culture medium, as observed in the bovine test.\u003c/p\u003e \u003cp\u003eCollectively, these characteristics position the bovine embryo assay as a pragmatic candidate for enhancing the sensitivity of embryotoxicity assessment, thereby aligning with the principles of replacement, reduction and refinement in animal research[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study also highlights considerations for implementation. Human fertilization media were not suitable for bovine fertilization in our hands, requiring an exposure design focused on zygote culture, analogous to current mouse embryo testing. We evaluated two suppliers and specific production batches; broader testing, including ring trials across laboratories, will be required to define robust performance benchmarks, acceptance criteria and reference materials. Finally, the present work was not designed to link assay outcomes with clinical outcomes; future studies could explore how sensitive embryo quality endpoints relate to clinically relevant performance measures.\u003c/p\u003e \u003cp\u003eOverall, these data support the use of a bovine embryo assay as a more sensitive embryo-based approach to detect embryotoxicity and functional impairment in assisted reproduction culture media, and potentially other embryo-contact devices.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe standard mouse embryo assay, as routinely implemented, showed limited ability to discriminate between assisted reproduction culture media batches that differed in their effects on embryo development and robustness. A bovine embryo assay using slaughterhouse-derived oocytes detected differences in developmental kinetics, lineage allocation and cryotolerance that were not revealed by mouse assays. These findings support the bovine model as a practical, more sensitive option to redefine embryotoxicity safety standards for assisted reproduction devices, pending broader inter-laboratory validation and consensus acceptance criteria.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAuthorship Contribution Statemen:\u003c/h2\u003e \u003cp\u003eConceptualization and experimental design: P.C., R.R; Data curation: P.C., R.R., M.M; Formal analysis: P.C., M.M.; Methodology: J.R-A, M.M, A.G-A; Validation: P.C., R.R.; Writing of original draft: P.C., R.R.; Writing: review and editing: P.C., R.R., J.R-A., M.M., A.G-A.; Funding acquisition: P.C. All authors have read and approved the final version of this manuscript.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statemen:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and experimental design: P.C., R.R; Data curation: P.C., R.R., M.M; Formal analysis: P.C., M.M.; Methodology: J.R-A, M.M, A.G-A; Validation: P.C., R.R.; Writing of original draft: P.C., R.R.; Writing: review and editing: P.C., R.R., J.R-A., M.M., A.G-A.; Funding acquisition: P.C. All authors have read and approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Alicia Masegosa, M.Sc., Ángela Juan, M.Sc., and María López M.Sc., from the embryology laboratory of EmbryoCloud SL for their assistance in performing standard IVF procedures. The authors are grateful to Gabriel Pastor for technical support during development of experiments and Matadero Orihuela S.A, for providing bovine ovaries.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDumoulin JC, Land JA, Van Montfoort AP, Nelissen EC, Coonen E, Derhaag JG et al (2010) Effect of in vitro culture of human embryos on birthweight of newborns. Hum Reprod 25(3):605\u0026ndash;612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKleijkers SHM, van Montfoort APA, Smits LJM, Viechtbauer W, Roseboom TJ, Nelissen ECM et al (2014) IVF culture medium affects post-natal weight in humans during the first 2 years of life. Hum Reprod 29(4):661\u0026ndash;669\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChronopoulou E, Harper JC (2015) IVF culture media: Past, present and future. 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Limited, London\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"b12fc2de-8d8d-4369-b61e-17e0cc54775b","identifier":"10.13039/501100004837","name":"Ministerio de Ciencia e Innovación","awardNumber":"MCIN/AEI10.13039/501100011033 /AEI/","order_by":0},{"identity":"d539018e-be8f-4117-838f-da2a95e9d228","identifier":"10.13039/501100000780","name":"European Commission","awardNumber":"NextGeneration EU/PRTR, PLEC2022-009246","order_by":1},{"identity":"b635556c-ef04-444c-89a4-0309de466154","identifier":"10.13039/100007801","name":"Fundación Séneca","awardNumber":"22253/PDC/23.","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Murcia","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":"embryotoxicity, human culture media, assisted reproduction, mouse embryo assay, bovine embryo assay","lastPublishedDoi":"10.21203/rs.3.rs-8967516/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8967516/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eStructured Abstract\u003c/b\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cdiv class=\"Heading\"\u003ePurpose\u003c/div\u003e \u003cp\u003eTo overcome the limited sensitivity of the standard Mouse Embryo Assay (MEA) for embryotoxicity screening of assisted reproduction devices and to assess the Bovine Embryo Assay (BEA) as a more sensitive alternative.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e: In a large comparative laboratory study, bovine cumulus\u0026ndash;oocyte complexes (from slaughterhouse ovaries) were fertilized with frozen semen from the same bull, and mouse cumulus\u0026ndash;oocyte complexes, epididymal sperm, and one-cell embryos were used for MEA. Sperm selection, fertilization, and embryo culture media from two suppliers (A: Vitrolife; B: Genea Biomedx) were tested in parallel using BEA, standard MEA, and an extended MEA including fertilization. BEA assessed cleavage, blastocyst development and kinetics, post-warming re-expansion/hatching, total cell number, ICM and TE allocation, and ICM/TE ratio.\u003c/p\u003e","manuscriptTitle":"Redefining safety standards: A large-scale comparative analysis of bovine versus murine models for medical device embryotoxicity testing.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 02:51:11","doi":"10.21203/rs.3.rs-8967516/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":"add78b70-071b-4c96-be82-c9403b3d5f7a","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63579796,"name":"Obstetrics \u0026 Gynecology"}],"tags":[],"updatedAt":"2026-02-27T02:51:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 02:51:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8967516","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8967516","identity":"rs-8967516","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}