Influence of the storage conditions of embryo culture media on mouse development.

The culture of preimplantation embryos in vitro is an important method for human and mouse reproductive technology. This study aims to investigate the influence of different conditions of culture media on the preimplantation stage of mouse embryos cultured in vitro, and monitor the post-implantation development of new mice after embryo transfer to surrogate females. We demonstrated here that mouse embryos cultured in vitro in fresh M16, KSOM, Global, and HTF embryo culture media from one cell to the blastocyst stage and the subsequent embryo transfer to surrogate females are able to proceed through post-implantation development and, after birth, develop into healthy mice. However, culture of embryos in differently aged media shows various (often unpredictable) results. To find the optimal storage conditions of culture media, we suggest that the freezing and long-term storage of these media at - 80°C will not influence the quality of the media. To test this hypothesis, we grew embryos from one cell to blastocysts in vitro in the selected media after thawing and subsequently transferring them to surrogate females. Embryo culture in these four media after thawing does not affect preimplantation and postnatal mouse development. Thus, we have shown that storage of embryo culture media at low temperature (- 80°C) does not impact the quality of the media, and subsequently, it can be used for the culture of embryos for the full preimplantation period, the same as in fresh media.

A novel machine-learning framework based on early embryo morphokinetics identifies a feature signature associated with blastocyst development.

BACKGROUND: Artificial Intelligence entails the application of computer algorithms to the huge and heterogeneous amount of morphodynamic data produced by Time-Lapse Technology. In this context, Machine Learning (ML) methods were developed in order to assist embryologists with automatized and objective predictive models able to standardize human embryo assessment. In this study, we aimed at developing a novel ML-based strategy to identify relevant patterns associated with the prediction of blastocyst development stage on day 5. METHODS: We retrospectively analysed the morphokinetics of 575 embryos obtained from 80 women who underwent IVF at our Unit. Embryo morphokinetics was registered using the Geri plus® time-lapse system. Overall, 30 clinical, morphological and morphokinetic variables related to women and embryos were recorded and combined. Some embryos reached the expanded blastocyst stage on day 5 (BL Group, n = 210), some others did not (nBL Group, n = 365). RESULTS: The novel EmbryoMLSelection framework was developed following four-steps: Feature Selection, Rules Extraction, Rules Selection and Rules Evaluation. Six rules composed by a combination of 8 variables were finally selected, and provided a predictive power described by an AUC of 0.84 and an accuracy of 81%. CONCLUSIONS: We provided herein a new feature-signature able to identify with an high performance embryos with the best developmental competence to reach the expanded blastocyst stage on day 5. Clear and clinically relevant cut-offs were identified for each considered variable, providing an objective tool for early embryo developmental assessment.

Towards a more sustainable balance between optimal live birth rate and supernumerary embryos in ART treatments.

PURPOSE: To assess the relation between number of inseminated oocytes and cumulative live birth rate (CLBR) in order to provide guidance for limiting the number of surplus blastocysts. METHODS: The study was a retrospective, single-center cohort analysis of 1223 ART complete cycles. Cycles were stratified according to female age (≤ 34, 35-38, and 39-42 years) and number of inseminated oocytes (1-5, 6-10, and > 10). Inclusion criteria were indication for IVF/ICSI with own spermatozoa and blastocyst culture up to day 6 of all embryos. RESULTS: In patients younger than 35 years, insemination of more than ten oocytes produced an increase in overall blastocyst number, CLBR (40.3%, 54.3%, and 75.8%, respectively, for each oocyte group) and surplus embryo rate (12.9%, 27.8%, and 49.7% of cases for each group). Instead, in the middle age group, the use of more than ten oocytes was solely associated with an increase in the rate of surplus embryos (1.25%, 21.33%, and 28.68% of cases after stratification for oocyte number). In older patients, neither CLBR (9.1%, 23.9%, and 24.7%, respectively) nor rate of surplus embryos (2.0%, 7.1%, and 13.4% of cases for each group) were higher in cycles with more than ten inseminated oocytes. CONCLUSION: In women up to 38 years, sustainable CLBR are achieved while limiting the number of inseminated oocytes and the resulting blastocysts remaining unused. Based on this notion, novel treatment strategies could pursue high outcome rates, while alleviating the problems derived from surplus stored embryos.

The number of nuclei in compacted embryos, assessed by optical coherence microscopy, is a non-invasive and robust marker of mouse embryo quality.

Optical coherence microscopy (OCM) visualizes nuclei in live, unlabeled cells. As most cells are uninucleated, the number of nuclei in embryos may serve as a proxy of the cell number, providing important information on developmental status of the embryo. Importantly, no other non-invasive method currently allows for the cell number count in compacted embryos. We addressed the question of whether OCM, by providing the number of nuclei in compacted mouse embryos, may help evaluate embryo quality. We subjected compacted embryonic Day 3 (E3.0: 72 h after onset of insemination) mouse embryos to OCM scanning and correlated nuclei number and developmental potential. Implantation was assessed using an outgrowth assay (in vitro model meant to reflect embryonic ability to implant in vivo). Embryos with more cells at E3.0 (>18 cells) were more likely to reach the blastocyst stage by E4.0 and E5.0 (P ≪ 0.001) and initiate hatching by E5.0 (P < 0.05) than those with fewer cells (<12 cells). Moreover, the number of cells at E3.0 strongly correlated with the total number of cells in E4.0 and E5.0 embryos (ρ = 0.71, P ≪ 0.001 and ρ = 0.61, P ≪ 0.001, respectively), also when only E4.0 and E5.0 blastocysts were considered (ρ = 0.58, P ≪ 0.001 and ρ = 0.56, P ≪ 0.001, respectively). Additionally, we observed a strong correlation between the number of cells at E3.0 and the number of trophectoderm cells in E4.0 and E5.0 blastocysts (ρ = 0.59, P ≪ 0.001 and ρ = 0.57, P ≪ 0.001, respectively). Importantly, embryos that had more cells at E3.0 (>18 cells) were also more likely to implant in vitro than their counterparts with fewer cells (<12 cells; P ≪ 0.001). Finally, we tested the safety of OCM imaging, demonstrating that OCM scanning affected neither the amount of reactive oxygen species nor mitochondrial activity in the embryos. OCM also did not hinder their preimplantation development, ability to implant in vitro, or to develop to term after transfer to recipient females. Our data indicate that OCM imaging provides important information on embryo quality. As the method seems to be safe for embryos, it could be a valuable addition to the current repertoire of embryo evaluation methods. However, our study was conducted only on mouse embryos, so the proposed protocol would require optimization in order to be applied in other species.

A 3D "sandwich" co-culture system with vascular niche supports mouse embryo development from E3.5 to E7.5 in vitro.

BACKGROUND: Various methods for ex utero culture systems have been explored. However, limitations remain regarding the in vitro culture platforms used before implanting mouse embryos and the normal development of mouse blastocysts in vitro. Furthermore, vascular niche support during mouse embryo development from embryonic day (E) 3.5 to E7.5 is unknown in vitro. METHODS: This study established a three-dimensional (3D) "sandwich" vascular niche culture system with in vitro culture medium (IVCM) using human placenta perivascular stem cells (hPPSCs) and human umbilical vein endothelial cells (hUVECs) as supportive cells (which were seeded into the bottom layer of Matrigel) to test mouse embryos from E3.5 to E7.5 in vitro. The development rates and greatest diameters of mouse embryos from E3.5 to E7.5 were quantitatively determined using SPSS software statistics. Pluripotent markers and embryo transplantation were used to monitor mouse embryo quality and function in vivo. RESULTS: Embryos in the IVCM + Cells (hPPSCs + hUVECs) group showed higher development rates and greater diameters at each stage than those in the IVCM group. Embryos in the IVCM + Cells group cultured to E5.5 morphologically resembled natural egg cylinders and expressed specific embryonic cell markers, including Oct4 and Nanog. These features were similar to those of embryos developed in vivo. After transplantation, the embryos were re-implanted in the internal uterus and continued to develop to a particular stage. CONCLUSIONS: The 3D in vitro culture system enabled embryo development from E3.5 to E7.5, and the vascularization microenvironment constructed by Matrigel, hPPSCs, and hUVECs significantly promoted the development of implanted embryos. This system allowed us to further study the physical and molecular mechanisms of embryo implantation in vitro.

Evaluation of an automated dish preparation system for IVF and embryo culture using a mouse mode.

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures.

Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development†.

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer.

Culture conditions in the IVF laboratory: state of the ART and possible new directions.

In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

How and when to measure pH in IVF culture media: validation of a portable blood gas analyzer in two IVF culture dishes for time lapse and conventional incubators.

PURPOSE: Maintaining a stable pH at optimal level in human embryo culture media is crucial for embryo development but poses a challenge for all IVF laboratories. We validate analytically reliable conditions for pH measurement that are as close as possible to the embryo microenvironment during IVF. METHODS: This was a multicentric study. A Siemens EPOC portable blood gas analyzer was used. The analytical validation was carried out under the culture medium (Global Total HSA®) conditions of use (microdroplets, under oil overlay, in a IVF incubator with (EmbryoScope®) or without a time lapse system (K system G210+®) and using IVF dishes. The validation included repeatability ("within-run" precision), total precision (between-day precision), trueness based on inter-laboratory comparison, inaccuracy based on external quality assessment and comparison to the reference technique. We also assessed the pre-analytical medium incubation time required to obtain a target value. RESULTS: A measurement after an incubation period of 24 to 48 h is more representative of the pH to which the embryo will be exposed throughout the culture. The "within-run" and "between-day" precision show very low coefficients of variation (CV%): 0.17 to 0.22% and 0.13 to 0.34%, respectively, with IVF culture media. Trueness (% bias) range from - 0.07 to - 0.03%. We demonstrate good correlation between EPOC and reference pH electrode with an overestimation of 0.03 pH units of EPOC. CONCLUSION: Our method demonstrates good analytical performance for IVF laboratories wishing to implement a robust quality assurance system to monitor pH in embryo culture media. Compliance with stringent pre-analytical and analytical conditions is essential.

Zinc oxide nanoparticles functionalized with curcumin supplementation during in vitro embryo culture impaired in a concentration-dependent-manner blastocyst production in cattle.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 µM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 µM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 µM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

PURPOSE: To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. METHODS: We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. RESULT: After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. CONCLUSION: These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

Derivation of Bovine Primed Embryonic Stem Cells from Somatic Cell Nuclear Transfer Embryos.

Derivation of bovine embryonic stem cells from somatic cell nuclear transfer embryos enables the derivation of genetically matched pluripotent stem cell lines to valuable and well-characterized animals. In this chapter, we describe a step-by-step procedure for deriving bovine embryonic stem cells from whole blastocysts produced by somatic cell nuclear transfer. This simple method requires minimal manipulation of blastocyst-stage embryos, relies on commercially available reagents, supports trypsin passaging, and allows the generation of stable primed pluripotent stem cell lines in 3-4 weeks.

Randomized prospective study comparing conventional In Vitro Fertilization technique to Intravaginal Culture with the INVOCELL™ device for 3 and 5 days.

OBJECTIVE: The objective was to analyse and compare the formation and quality of the embryos developed using conventional in vitro fertilization (IVF) and IVC techniques with an INVOCell™ device. METHODS: Two groups were formed, with eight couples in each, one in culture for three days (D3) and another in culture for five days (D5), using intravaginal culture technique with an INVOcell device and a conventional in vitro fertilization technique. RESULTS: Embryo formation in Group D5 showed 46.7% (IVC) and 40% (IVF) of recovered blastocysts. In the group D5, the conventional IVF, better embryo development dynamics was observed, with 66% of expanded blastocysts, against 28% in the IVC. Group D3 showed 75% (IVC) and 53% (IVF) of embryo formation. Embryonic quality in Group D3 demonstrated that IVF embryos had a better synchrony in the number and quality of blastomeres. All embryos recovered in Group D3, in both techniques, did not show fragmentation. The pH of the medium contained in the INVOCell™ device in both Groups D5 and D3 showed no differences. The means were 7.26 and 7.25, respectively. The pH of the medium used in IVF was 7.29 in both groups. Microbiological analyzes of the culture media contained in the INVOCell™ devices used in Group D5 were negative. CONCLUSIONS: The results showed that the IVC technique, using the INVOCell™ device, provided a healthy and balanced environment for the development and obtaining of quality embryos with three and five days of culture.

Non-invasive evaluation of embryos using mid-infrared attenuated total reflection spectrometry of incubation medium: a preliminary study.

RESEARCH QUESTION: Can mid-infrared attenuated total reflection (MIR ATR) spectroscopy combined with machine learning methods be used as an additional tool to predict embryo quality and IVF treatment outcomes? DESIGN: Spent culture media was collected and analysed. MIR ATR absorbance spectra were measured using an ALPHA II spectrometer equipped with an attenuated total reflection (ATR) spectrometry accessory. Patient and treatment data and results were collected and analysed in combination with machine learning techniques to identify possible correlations. The main outcome measures were to define the characteristics of absorbance spectra of spent culture media and to distinguish the difference in absorbance between top- and low-quality embryos, day 3 and day 5 embryos and implanting embryos versus non-implanting embryos. RESULTS: Spent culture media of 227 embryos was collected and analysed. Absorbance peaks in the culture media were different between day 3 and day 5 embryos. Moreover, significant differences in P-values, spanning from 0.014 to 0.044 in absorbance peaks for day 3 embryos and 0.024 up to 0.04 for day 5 embryos, were seen between implanting and non-implanting embryos. Machine learning techniques offered a pregnancy prediction value of 84.6% for day 3 embryos. CONCLUSIONS: MIR ATR may offer an additional parameter for better selection of embryos based on the spectrometric absorbance and secretions of metabolites in the culture media.

Time-lapse monitoring technologies for the selection of bovine in vitro fertilized embryos with high implantation potential.

Over the years, the utilization of in vitro fertilization (IVF) in bovine embryo production has increased globally to accelerate the selection of cows with high genetic values. The selection of embryos with high implantation potential is a critical factor in establishing pregnancy. Time-lapse monitoring (TLM) has emerged as a new technique that allows frequent and non-invasive imaging of developing embryos. TLM is considered to have several advantages over the conventional morphological evaluation of embryos, which has been widely used in bovine embryo production. Establishing a novel embryo selection algorithm specifically for bovine IVF embryos is a critical challenge, but information on the association between morphokinetic data obtained using TLM and the implantation potential of embryos is still limited. This review outlines the potential application of TLM technology to improve the fertility of bovine IVF embryos, focusing on the results of human and bovine TLM studies that can be applied to select bovine embryos with high implantation potential. First, the progress of the TLM technology in bovine embryo production is summarized. The association between kinetic and morphological parameters and the developmental and implantation potential of human and bovine embryos is outlined. Finally, the benefits of evaluating blastocyst collapse and re-expansion as indicators of bovine embryo viability and the possible application of TLM to detect chromosomal abnormalities and determine embryo sex will be discussed.

The presence of partial compaction patterns is associated with lower rates of blastocyst formation, sub-optimal morphokinetic parameters and poorer morphologic grade.

BACKGROUND: Compaction is an important marker of embryonic genome activation and marks a critical step in the development to blastocyst. The objective of our study was to determine whether visualization of the embryonic compaction process through time-lapse imaging (TL) can assist in predicting the kinetics of embryo development as well as the likelihood for blastocyst formation, grade, or ploidy. METHODS: This study is a retrospective review of prospectively collected datafrom a single academic institution. Couples included were thosewho underwent preimplantation genetic testing for aneuploidy (PGT-A) following in vitro fertilization between Januaryand December 2020. Embryos were cultured in the Embrysocope. Embryo morphokinetic data was prospectively collected and analyzed.TL videos werelater reviewed in detail for compaction pattern. Embryo compaction patterns (CP) were categorized as follows: 1) full compaction (CP-F), 2) partial compaction with cell extrusion (P-ext), 3) partial compactionwith cell exclusion (P-exc) and 4) partial compactionwith both cell extrusion and exclusion (P-both). Assessment of embryo decompaction and re-compaction was evaluated. The association between CP, morphokinetic parameters,blastocyst formation, grade and ploidy were then analyzed. RESULTS: A total of 349 embryos were studied. Amongst embryos which progressed to morula (n = 281), the distribution of compaction patterns were: CP-F 45.6%, P-ext12.5%, P-exc29.5% and P-both 12.5%. Embryos exhibiting a CP-F were more likely to proceed to blastocyst compared with those that demonstrated partial compaction patterns (p = 0.006). When compared to CP-F, partial compaction patterns were significantly associated with poorer ICM and TE grades (P < 0.001). Of the 281 morula, 59.8% (n = 168) demonstrated at least one episode of decompaction and re-compaction. Of the 249 blastocysts formed, 200 were cryopreserved for future use after undergoing PGT-A evaluation. Of those, 42.5% were diagnosed as euploid, 39.0% as aneuploid, 9.0% as mosaic and 9.5% had no result. When compared to CP-F, partialCPs exhibited a significantly greater percentage of mosaic embryos (3.6% v. 15.6%, p = 0.032). Additionally, we found that a greater percentage of embryos demonstrating CP-F exhibited morphokinetics that fell into optimal ranges for embryo development when compared to those with partial compaction patterns. CONCLUSION: Time-lapse visualization of compaction patterns identified exclusions and/or extrusions as negative indicators of blastocyst formation and blastocyst grade. When compared to full compaction patterns, partial compaction patterns were associated with delayed embryonic development as well as lower rates of optimal kinetic development.

Combining Machine Learning with Metabolomic and Embryologic Data Improves Embryo Implantation Prediction.

This study investigated whether combining metabolomic and embryologic data with machine learning (ML) models improve the prediction of embryo implantation potential. In this prospective cohort study, infertile couples (n=56) undergoing day-5 single blastocyst transfer between February 2019 and August 2021 were included. After day-5 single blastocyst transfer, spent culture medium (SCM) was subjected to metabolite analysis using nuclear magnetic resonance (NMR) spectroscopy. Derived metabolite levels and embryologic parameters between successfully implanted and failed groups were incorporated into ML models to explore their predictive potential regarding embryo implantation. The SCM of blastocysts that resulted in successful embryo implantation had significantly lower pyruvate (p<0.05) and threonine (p<0.05) levels compared to medium control but not compared to SCM related to embryos that failed to implant. Notably, the prediction accuracy increased when classical ML algorithms were combined with metabolomic and embryologic data. Specifically, the custom artificial neural network (ANN) model with regularized parameters for metabolomic data provided 100% accuracy, indicating the efficiency in predicting implantation potential. Hence, combining ML models (specifically, custom ANN) with metabolomic and embryologic data improves the prediction of embryo implantation potential. The approach could potentially be used to derive clinical benefits for patients in real-time.

How to optimize culture media osmolality during Assisted Reproductive Technologies treatments.

OBJECTIVE: The objective of our study was to compare the osmolality in sequential and single step culture media, used for in vitro human embryo culture, covered with mineral oil and paraffin, in dry and humid incubators. METHODS: We performed a prospective observational study. A total of 120 Petri dishes, with 960 droplets of culture media, were evaluated. Each dish was prepared with 4 droplets of single step medium and sequential medium. Sixty dishes were covered with mineral oil and 60 with paraffin oil. Half were incubated in a dry incubator and half in a humid. Osmolality was measured on days 1, 3, 5, 7. ANOVA test was performed for statistical analysis. RESULTS: Osmolality results for single step and sequential medium, that were covered with both mineral and paraffin oil and placed in the dry incubator, significantly increased throughout the study time (D7>D5>D3). In the humid incubator, the results were similar for all periods. Osmolality was significantly lower in humid incubator, in all periods, when droplets were covered with both oils. When both culture media were placed in the humid incubator, no variation was detected, using both oils. However, when single step medium was placed in the dry incubator, covered with mineral oil, we observed a higher osmolality than the covered with paraffin oil. CONCLUSIONS: TWe can conclude that humid incubator is better for maintaining osmolality and paraffin oil protect single step media from evaporation in dry incubator.

Effects of fetal bovine serum on trophectoderm and primitive endoderm cell allocation of in vitro-produced bovine embryos.

Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.