Effects of stearic acid on the embryo cryopreservation in mouse.

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 µg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 µg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 µg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

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.

Meta-analysis to determine efficacy of colony-stimulating factor 2 for improving pregnancy success after embryo transfer in cattle.

Results have been inconsistent as to whether addition of colony stimulating factor 2 (CSF2) to culture medium improves embryo competence for establishment of pregnancy in cattle and humans. The purpose of the current study was to use all available experiments in cattle concerning effects of CSF2 on pregnancy success after transfer into recipient cattle. The approach was to perform a meta-analysis of all published data sets as well as data from an unpublished experiment described for the first time here. Meta-analysis failed to support the hypothesis that addition of CSF2 to embryo culture medium improves competence of bovine blastocysts to increase pregnancy or calving rates after transfer into recipient females. Thus, its general use as a culture medium additive to increase pregnancy success after embryo transfer is not recommended.

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.

NRF2 attenuation aggravates detrimental consequences of metabolic stress on cultured porcine parthenote embryos.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

The unbearable ignorance of the composition of IVF culture media.

Culture media play an essential role in the success of IVF. Their composition has undergone major modifications over the 45 years since the birth of Louise Brown. Most IVF programmes now rely on commercially produced media, which they buy in small vials, guaranteed to be sterile and non-embryotoxic. Unfortunately, information about the components of the culture media and their concentrations is no longer available. Arguing that culture media recipes are proprietary, relevant commercial interests have stopped labelling their products with this vital information. Given the critical role that is played by culture media in the success of IVF, as well as the subsequent health of the children who are born after IVF, this information should not remain a 'company secret'. Clinicians and scientists working in IVF must insist that the labelling of culture media includes all of the constituents and their concentrations. Only in this way can we monitor the influence of culture media on IVF outcomes, innovate and continue to advance the field of IVF.

Developmental Toxicity Using the Rat Whole Embryo Culture.

In vivo and in vitro experiments have been used to investigate the effect of drugs, chemical agents, growth factors, vitamins, etc., on embryonic development. Alternative tests have been developed to determine whether drugs or other compounds have toxic or teratogenic effects on a particular organ or tissue. The rat whole embryo culture method is useful for studying the mechanism of normal embryo development. The explanted rat conceptuses grow in the culture environment at the same rate as in utero development. Furthermore, the considerable advantage of explanting rat embryos is that it allows direct observation of embryogenesis. The rat whole embryo culture is run by explanting rat embryos on gestation day 9.5 for rats. The conceptuses are then cultured on a rotating platform in a mixture of culture medium with appropriate gassing for 48 hours. The maternal serum is used as the culture medium, and chemicals or other agents to be evaluated separately are added to this medium. At the end of the culture period, growth and development of the embryos are evaluated morphologically.

Long-term culture induces Bax-dependent apoptosis in rat preimplantation embryos.

Although rat preimplantation embryos are necessary for producing genetically modified rats, their in vitro culture remains a challenge. Rat zygotes can develop from the one-cell stage to the blastocyst stage in vitro; however, long-term culture reduces their developmental competence via an unknown mechanism. In this study, we examined how in vitro conditions affect rat preimplantation embryos, which may explain this reduced competence. Comprehensive gene expression analysis showed that genes related to apoptosis and energy metabolism were differentially expressed in rat embryos cultured long-term in vitro compared with those developed in vivo. Furthermore, we found that the expression of Bak1 and Bax, which are responsible for mitochondrial outer membrane permeabilization, were more upregulated in embryos cultured in vitro than those developed in vivo. Similarly, apoptosis-dependent DNA fragmentation was also exacerbated in in vitro culture conditions. Finally, gene disruption using CRISPR/Cas9 showed that Bax, but not Bak1, was responsible for these effects. These findings suggest that long-term in vitro culture induces Bax-dependent apoptosis through the mitochondrial pathway and may provide clues to improve the long-term culture of rat preimplantation embryos for genetic engineering research.

Does endometriosis inflict harm on embryos? A systematic review of embryo morphokinetics analysed by time lapse monitoring in women with endometriosis.

Endometriosis has been shown to be associated with unfavorable development and maturation of oocytes, as well as aberrancies in embryonal development, including arrest after fertilization, following in vitro fertilization (IVF). Time-lapse monitoring (TLM) enables continuous and non-invasive monitoring of embryo morphokinetics during the IVF process and might be useful in the assessment of embryos from women with endometriosis. In this review, five eligible studies were evaluated to determine if embryo morphokinetics assessed under TLM differ in patients with endometriosis and subsequently predict blastocyst quality, implantation and success of pregnancy. The studies showed overall inferior morphokinetic parameters of embryos from endometriosis patients when compared to controls, independent of the severity of endometriosis. Embryos with optimal early morphokinetic parameters (t2, s2, t5, tSB, tEB) and late developmental events (compaction, morulation, and blastulation) had better implantation rates than those who had suboptimal ranges. However, due to few studies available with mostly retrospective data, the validity of these findings and their generalizability for clinical practice needs to be further assessed. Prospective studies with larger sample sizes are needed to determine whether using TLM for embryo selection in endometriosis improves pregnancy and live birth outcomes.

Considerations for future modification of The Association for the Study of Reproductive Biology embryo grading system incorporating time-lapse observations.

The Association for the Study of Reproductive Biology (ASEBIR) Interest Group in Embryology (in Spanish 'Grupo de Interés de Embriología') reviewed key morphokinetic parameters to assess the contribution of time-lapse technology (TLT) to the ASEBIR grading system. Embryo grading based on morphological characteristics is the most widely used method in human assisted reproduction laboratories. The introduction and implementation of TLT has provided a large amount of information that can be used as a complementary tool for morphological embryo evaluation and selection. As part of IVF treatments, embryologists grade embryos to decide which embryos to transfer or freeze. At the present, the embryo grading system developed by ASEBIR does not consider dynamic events observed through TLT. Laboratories that are using TLT consider those parameters as complementary data for embryo selection. The aim of this review was to evaluate review time-specific morphological changes during embryo development that are not included in the ASEBIR scoring system, and to consider them as candidates to add to the scoring system.

Mammalian embryo culture media: now and into the future.

For over 70years, since the culture of the first mammalian embryo in vitro , scientists have undertaken studies to devise and optimise media to support the manipulation and culture of gametes and embryos. This area of research became especially active in the late 1970s onwards following the successful birth of the first human in vitro fertilised embryo. This review summarises some of the key advances in mammalian embryo culture media over time based on a greater understanding of the biochemical milieu of the reproductive tract. It highlights how learnings from studies in mice and agricultural species have informed human culture media compositions, in particular the inclusion of albumin, growth factors, cytokines, and antioxidants into contemporary culture media formulations, and how these advances may then in turn help to inform and guide development of in vitro culture systems used in other arenas, in particular agriculture. Additionally, it will highlight how the introduction of new technologies, such as timelapse, can influence current trends in media composition and usage that may see a return to a single step medium.

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.

Effect of food supplementation on in vitro embryo production and growth performance in prepubertal Nelore heifers.

In vitro embryos production from prepubertal heifers can help contribute to breeding programs; however, strategies are necessary to increase their embryo production. The aim of this study was to investigate the effects of two nutritional plans on oocyte recovery, embryo production and growth performance of prepubertal Nelore heifers. Thirty-four Nelore heifers with age of 6.5 months were divided into two feeding treatments (NP1 and NP2). The NP1 diets served as the control and NP2 diets were formulated to contain an average of 1.22-fold more energy than NP1. After 3 months of supplementation, the animals underwent follicular aspiration (ovum pick-up, OPU) every 21 d for 3 months and embryos were produced in vitro. Wither height, chest depth, body weight and subcutaneous fat of animals were measured. The number of retrieved and viable oocytes per OPU were 1.49-fold and 1.42-fold greater in NP2 heifers (p = 0.018 and p = 0.049, respectively) than those in NP1 heifers. Heifers administered NP2 produced 29.7% blastocysts, a percentage higher than NP1 animals that produced 24.40% embryos (p < 0.05). Consequently, females in the NP2 treatment showed improved body development. These results indicate a positive effect of a higher energy diet on assisted reproduction and body development in prepubertal heifers.

Time-lapse observation of mouse preimplantation embryos using a simple closed glass capillary method.

Time-lapse observation is a popular method for analyzing mammalian preimplantation embryos, but it often requires expensive equipment and skilled techniques. We previously developed a simply and costly embryo-culture system in a sealed tube that does not require a CO2 incubator. In the present study, we developed a new time-lapse observation system using our previous culture method and a glass capillary. Zygotes were placed in a glass capillary and sunk in oil for observation under a stereomicroscope. Warming the capillary using a thermoplate enabled most of the zygotes to develop into blastocysts and produce healthy offspring. This time-lapse observation system captured images every 30 min for up to 5 days, which confirmed that the developmental speed and quality of the embryos were not affected, even with fluorescence. Overall, this new system is a simple time-lapse observation method for preimplantation embryos that does not require dedicated machines and advanced techniques.

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.

Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 µM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 µM niacin had an increased cleavage rate (p < .05). In addition, 300 µM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 µM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 µM increased glutathione levels in day two embryos. On day seven, 300 µM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 µM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

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.