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Research Question: To what extent does the type and concentration of protein and the type of culture medium affect the sensitivity of the mouse embryo assay (MEA) to detect Triton X-100 (TX-100) in culture media?, Design: The effect of the concentration of bovine serum albumin (BSA) and human serum albumin (HSA) was assessed by supplementing media with 0.5 or 5 mg/ml. Potassium-supplemented simplex optimized medium (KSOM) and human tubal fluid (HTF) were used as complex and simple formulation media, respectively. Variables were combined, forming study groups where embryos were cultured in test media spiked with a sublethal TX-100 concentration. The conditions of greatest sensitivity were determined by statistical comparison of blastocyst formation rates and total cell counts between groups., Results: Although all of the study groups showed equal capacity for sustaining proper embryo development, the reported sensitivity of the MEA differed between groups when subjected to TX-100. HTF conferred significantly greater sensitivity than KSOM regardless of the type and concentration of protein used, and medium supplementation with 5 mg/ml BSA rather than 0.5 mg/ml BSA resulted in significantly higher sensitivity regardless of the type of medium used. This increase in concentration also resulted in higher sensitivity when supplementing HTF with HSA. The BSA groups provided more sensitivity than their HSA counterparts, except for the KSOM + 0.5 mg/ml BSA group. Cell count analysis did not provide further significant conclusions., Conclusions: For TX-100 detection within culture medium, the type and concentration of protein and the type of culture medium have a direct effect on MEA sensitivity. These results could help to standardize the MEA protocol, and increase its ability to detect sublethal concentrations of embryotoxic substances, especially TX-100, thus avoiding possible clinical harmful effects., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology., Competing Interests: Competing interests statement:The lead laboratory in this work has received private funding from the Venture Capital (VC) Scranton Enterprises. The authors declare that this funder has no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.

Research Question: Can an automated sperm injection robot perform Automated Intracytoplasmic Sperm Injection (ICSIA) for use in human IVF?, Design: The ICSIA robot automated the sperm injection procedure, including injection pipette advancement, zona pellucida and oolemma penetration with piezo pulses, and pipette removal after sperm release. The robot was first tested in mouse, hamster and rabbit oocytes, and subsequently using discarded human oocytes injected with microbeads. A small clinical pilot trial was conducted with donor oocytes to study the feasibility of the robot in a clinical setting. The ICSIA robot was controlled by engineers with no micromanipulation experience. Results were compared with those obtained with manual ICSI conducted by experienced embryologists., Results: The ICSIA robot demonstrated similar results to the manual procedure in the different animal models tested as well as in the pre-clinical validations conducted in discarded human oocytes. In the clinical validation, 13 out of 14 oocytes injected with ICSIA fertilized correctly versus 16 out of 18 in the manual control; eight developed into good-quality blastocysts versus 12 in the manual control; and four were diagnosed as chromosomally normal versus 10 euploid in the manual control. Three euploid blastocysts from the ICSIA robot group have been transferred into two recipients, which resulted in two singleton pregnancies and two babies born., Conclusions: The ICSIA robot showed high proficiency in injecting animal and human oocytes when operated by inexperienced personnel. The preliminary results obtained in this first clinical pilot trial are within key performance indicators., (Copyright © 2023. Published by Elsevier Ltd.)

Objectives: To gain insights into the technical feasibility of maternal spindle transfer (MST) applied in the context of repeated in vitro fertilization (IVF) failures for the treatment of idiopathic infertility., Design: A prospective pilot study., Setting: IVF center., Patient(s): Twenty-five infertile couples with multiple previous unsuccessful IVF cycles (range, 3-11), no previous pregnancy, and no history of mitochondrial DNA (mtDNA) disease participated. The study focused on women <40 years, with previous IVF attempts characterized by a pattern of low fertilization rates and/or impaired embryo development. Couples with severe male-factor infertility were not eligible. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice., Intervention(s): We performed MST by transferring metaphase II spindles from the patients' oocytes into the previously enucleated donor oocytes, followed by intracytoplasmic sperm injection, in vitro embryo culture, blastocyst biopsy, and vitrification. Only euploid blastocysts were considered for embryo transfer., Main Outcome Measure(s): Outcome measures included oocyte fertilization, blastocyst development, clinical pregnancy and live birth, incidence of mitochondrial carryover and potential mtDNA reversal, as well as general health of the children born., Result(s): Twenty-eight MST cycles produced 6 children (19 embryo transfers, 7 clinical pregnancies). Pediatric follow-up of the children, performed at intervals from birth to 12-24 months of age, revealed their development to be unremarkable. DNA fingerprinting confirmed that the nuclear DNA of MST children was inherited from both parents, without any contribution from the oocyte donor. For 5 of the children, mtDNA was derived almost exclusively (>99%) from the donor. However, 1 child, who had similarly low mtDNA carryover (0.8%) at the blastocyst stage, showed an increase in the maternal mtDNA haplotype, accounting for 30% to 60% of the total at birth., Conclusion(s): This pilot study provides the first insights into the feasibility of applying MST for patients with idiopathic infertility and repeated IVF failures. Reconstructed oocytes produced embryos capable of implanting, developing to term and producing apparently healthy newborns/children. However, claims concerning the efficacy of MST with respect to infertility treatment would be premature considering the limitations of this study. Importantly, mtDNA reversal was detected in one child born after MST, a finding with possible implications for mitochondrial replacement therapies., Clinical Trial Registration Number: Pilot trial registry number, ISRCTN11455145. The date of registration: 20/02/2018. The date of enrolment of the first patients: 18/03/2018., (Copyright © 2023. Published by Elsevier Inc.)

Study Question: Are there significant differences between the available commercial oil brands used for human IVF?, Summary Answer: Important differences have been detected among the tested oil brands in their potential to stabilize culture conditions and, more importantly, in their direct effect on embryo development and viability., What Is Known Already: Mineral oil is a critical component of the human culture system due to its protective and stabilizing roles during in vitro embryo development. Many different oils are available on the market, with differences in their viscosity, density and overall quality., Study Design, Size, Duration: Thirteen different commercial oil brands were compared., Participants/materials, Setting, Methods: Each oil was firstly analyzed to assess its viscosity, density, peroxide value and potential oxidation. Secondly, the capacity of each oil to reduce pH, osmolality and temperature fluctuations during embryo culture and manipulation was compared. Lastly, a sensitive mouse embryo assay (MEA) protocol, previously optimized to detect toxicity in oils samples, was used to compare the overall quality of the different brands in terms of embryo developmental rates up to the blastocyst stage. At the end of the MEAs, a triple labeling protocol was applied to analyze Oct4+ cells, apoptotic cells and total cell counts in the blastocysts obtained by fluorescence microscopy., Main Results and the Role of Chance: Significant divergences were detected in the rise of osmolality and the equilibration and stability of pH between different oils, which could be correlated to their physico-chemical characteristics. In particular, oil samples with a higher viscosity tended to offer an additional protection against fluctuations in the culture conditions, however, the differences in temperature stability between oils were minor. Two out of the 13 oil samples, which were commercially available, were identified as embryo-toxic by applying the MEA protocol with increased sensitivity for toxicity detection. Additionally, substantial differences in the total number of cells and the number of cells in the inner cell mass of the obtained blastocysts were also detected between oil groups., Limitations, Reasons for Caution: A single lot of oil was used for each brand and, thus, lot-to-lot variations in oil quality could not be determined. However, several bottles from the same oil were included to account for potential intra-lot variability., Wider Implications of the Findings: Commercial oils differ in both their physical characteristics and their performance in maintaining the stability of the culture conditions during in vitro embryo culture. Oil selection is important for embryo culture success. Additionally, the detection of embryo-toxic oils which had already been released to the human IVF market showcases the importance of applying sensitive MEA protocols for a better detection of toxicity in this type of samples., Study Funding/competing Interest(s): This study was privately funded., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Study Question: Which lab-related factors impact the culture system's capacity to maintain a stable osmolality during human embryo culture?, Summary Answer: Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture., What Is Known Already: Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes., Study Design, Size, Duration: The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation., Participants/materials, Setting, Methods: Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates., Main Results and the Role of Chance: Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator's characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture., Limitations, Reasons for Caution: While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories., Wider Implications of the Findings: Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables., Study Funding/competing Interest(s): This study was privately funded., Trial Registration Number: N/A., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To study the effect of zona opening (ZO) and 2 zona thinning (ZT) techniques on the hatching process of mouse embryos using a last-generation laser system and time-lapse microscopy (TLM)., Design: Prospective randomized study., Setting: Private research center., Animals: A total of 267 F1 hybrid (B6/CBA) mice embryos were included., Intervention(s): Morulae were randomly selected and the zona pellucida (ZP) manipulated using a laser system according to 4 experimental groups: control (ZP intact, n = 59), ZO (25 μm hole, n = 70), ZT25 (25% perimeter thinned, n = 71), and ZT35 (35% perimeter thinned, n = 67). Embryo development was monitored by TLM until day 6., Main Outcome Measure(s): Time to first breach the ZP, hatching time, time to complete hatching, multiple breaching, multiple hatching, loss of cells, hole size, and embryo quality were analyzed., Result(s): No significant differences in the proportion of completely hatched embryos were found among groups. However, the time (average hours ± SD) to complete hatching was significantly delayed in the control group compared with all laser-treated groups: 118.3 ± 9.5 hours in the ZT25 group, 116.6 ± 8.7 hours in the ZT35 group, and 120.4 ± 9.9 hours in the ZO group. The applied laser techniques did not interfere with the quality of the blastocysts at day 5/6 of culture., Conclusion(s): ZO, ZT25, and ZT35 embryos hatched significantly earlier than the zona intact group without increasing the multiple hatching rates, suggesting an improvement of the hatching process. This study found that the pattern of the hatching process after ZT and ZO differs., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies., Competing Interests: NC, KS, IM, EM, RB, AS, MG, IV, JG, KR, DW, GC No competing interests declared, (© 2020, Costa-Borges et al.)

Research Question: Can culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples?, Design: Protein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA] + α/β-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity., Results: High concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSA + alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used., Conclusions: Current quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females., (Copyright © 2018. Published by Elsevier Inc.)