Embryo Culture Research Articles

Assessing the clinical value of day 7 blastocysts: a predictive model for preimplantation genetic testing for aneuploidy (PGT-A) cycles.

To identify the benefit of extending embryo culture until day (D)7 based on patients and cycle characteristics. A retrospective cohort study was conducted including 25,120 blastocysts from 5278 PGT-A autologous cycles between 2017 and 2022. A theoretical cumulative live birth rate (CLBR) was calculated by binomial density function. An increase of ≥ 5% in theoretical CLBR was considered a tangible benefit when obtaining ≥ 1 euploid D7 blastocyst and ≤ 3 euploid blastocysts from D5/D6. A predictive model was built considering the number of embryos eligible for extended culture until D7, number of blastocysts already biopsied on D5/D6, and patient's age. Euploidy rates decreased for blastocysts biopsied on D5, D6, and D7 (55.6%, 39.7%, and 27.1%, P < 0.001, respectively). The probability of tangible benefit was increased with more embryos available for extended culture until D7, was decreased with higher D5/D6 blastocysts already biopsied and for older patients. The overall AUC of the final model in the validation sets was 0.75 (95% CI 0.72-0.78). Based on the predictive model, in poor cycles (< 1% tangible benefit), the benefit rate from extended culture was 0.3% and for moderate, good, and best cycles (1-10%, 10-20%, and ≥ 20% tangible benefit) were 4.4%, 14.0%, and 29.3%, respectively. An application of the predictive model is available online for external testing: https://artfertilityclinics.shinyapps.io/WET-D7/ . The predictive model provides a decision-making tool to objectively identify cycles that would benefit from extending embryo culture until D7.

Oviductal extracellular matrix hydrogels enhance in vitro culture of rabbit embryos and reduce deficiencies during assisted reproductive technologies.

In vitro embryo culture often falls short of mimicking the physiological dynamism occurring in the reproductive tract, prompting developmental plasticity in mammalian embryos with consequential genotypic and phenotypic deviations. Recent research highlights the potential of biological derivatives in in vitro culture to mitigate these effects, being the extracellular matrix (ECM) one of the most important components in retaining structural and biological signals derived from the native source tissue. Current bioengineering techniques could provide ECM-based biomaterials mimicking the native environment and offering optimal embryonic development. Rabbit oviducts (n = 24) were decellularized and solubilized to create tissue-specific ECM (OviECM) hydrogels. Following physicochemical characterization, these hydrogels were applied as coatings for the in vitro culture of two-cell embryos over 48h, along with embryos cultured under In vitro control conditions (n = 218/group), which were subsequently transferred to recipient females. A subset of embryos was recovered on day 6 for transcriptomic analysis (n = 75-80/group), while the remaining embryos were used to assess implantation and birth rates. Rabbit weights were monitored over 20 weeks post-delivery, with blood tests conducted at weeks 8 and 20. Bayesian inference methods were used for statistical analysis. Differences were considered relevant if P ≥ 0.8 (80%). No differences in embryo development and morphology were detected between the OviECM coating and In vitro control conditions. However, embryos cultured on these coatings exhibited upregulation of pathways involved in antigen presentation and immune system activation, as well as, increased cellular response to external stimulus and intracellular protein transport. The implantation and live birth rates were significantly higher in the coating group than in the In vitro control group (30.8% vs. 26.1% and 21.2% vs. 18.1%, respectively). During the first 20 weeks of life, the animals from the coating group showed higher weights than the In vitro control group P0 > 0.8. The animals of both experimental groups showed normal blood parameters. Implementation of OviECM coatings allows for improving in vitro conditions and decreases postnatal phenotypic deviations after assisted reproductive technology (ART). This study could initiate a new embryo culture techniques era to guarantee that ART is utilized in the most efficient and safest possible practice.

Novel application of metabolic imaging of early embryos using a light-sheet on-a-chip device: a proof-of-concept study.

Is it feasible to safely determine metabolic imaging signatures of nicotinamide adenine dinucleotide [NAD(P)H] associated auto-fluorescence in early embryos using a light-sheet on-a-chip approach? We developed an optofluidic device capable of obtaining high-resolution 3D images of the NAD(P)H autofluorescence of live mouse embryos using a light-sheet on-a-chip device as a proof-of-concept. Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. Indeed, in recent years, metabolic imaging has been investigated using highly advanced microscopy technologies such as fluorescence-lifetime imaging and hyperspectral microscopy. The potential safety of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67 h post metabolic imaging at the two-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system non-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). The microstructures were fabricated following a standard UV-photolithography process integrating light-sheet fluorescence microscopy into a microfluidic system, including on-chip micro-lenses to generate a light-sheet at the centre of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce two-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between the study groups. A convolutional neural network (ResNet 34) model using metabolic images was also trained. The optofluidic device was capable of obtaining high-resolution 3D images of live mouse embryos that can be linked to their metabolic activity. The system's design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet and fast imaging of the embryos (<2 s), while keeping a low dose of light exposure (16 J · cm-2 and 8 J · cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable of obtaining 30 times higher signal-noise-ratio images than images obtained with a confocal system (P < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (P > 0.05; Yate's chi-squared test). Additionally, embryos with inhibited metabolic activity showed a decreased blastocyst formation rate of 22.2% compared to controls, as well as a 47% reduction in metabolic activity measured by metabolic imaging (P < 0.0001; t-test). This indicates that the optofluidic device was capable of producing metabolic images of live embryos by measuring NAD(P)H autofluorescence, allowing a novel and affordable approach. The obtained metabolic images of two-cell embryos predicted blastocyst formation with an AUC of 0.974. N/A. The study was conducted using a mouse model focused on early embryo development assessing illumination at the two-cell stage. Further safety studies are required to assess the safety and use of 405 nm light at the blastocyst stage by investigating any potential negative impact on live birth rates, offspring health, aneuploidy rates, mutational load, changes in gene expression, and/or effects on epigenome stability in newborns. This light-sheet on-a-chip approach is novel and after rigorous safety studies and a roadmap for technology development, potential future applications could be developed for ART. The overall cost-efficient fabrication of the device will facilitate scalability and integration into future devices if full-safety application is demonstrated. This work was partially supported by an Ideas Grant (no 2004126) from the National Health and Medical Research Council (NHMRC), by the Education Program in Reproduction and Development (EPRD), Department Obstetrics and Gynaecology, Monash University, and by the Department of Mechanical and Aerospace Engineering, Faculty of Engineering, Monash University. The authors E.V-O, R.N., V.J.C., A.N., and F.H. have applied for a patent on the topic of this technology (PCT/AU2023/051132). The remaining authors have nothing to disclose.

Compatibility of dry incubator on in vitro production of bovine embryos

Embryo culture is crucial to achieve successful outcomes in in vitro production-embryo transfer for cattle. This study explored the innovative use of dry incubators for bovine embryo culture, building on their advantages in human medicine, such as reduced contamination risk, stable temperature control, and lower gas consumption. In this study, we examined changes in osmotic pressure, the in vitro developmental potential of IVP embryos including the cleavage rate, blastocyst development rate, blastocyst diameter, and blastocyst cell number, morphokinetics, and the transcriptional profile of the blastocysts between humidified and dry incubators. Our research demonstrates the feasibility of this approach, showing that although the osmotic pressure gradually increases over the culture period (on day 8: 271.7 vs. 299.0, respectively; P < 0.09), it did not negatively affect the blastocyst formation rate (62.4% vs. 69.8%) and the morphological quality of blastocysts (diameter: 237.4 vs. 242.8, total cell number: 189.2 vs. 242.8). Embryos cultured in dry incubators exhibited morphokinetics comparable to those cultured in conventional humidified incubators. Furthermore, RNA-seq revealed that while a few genes showed changes, the transcriptomic profiles of blastocysts cultured in dry incubators were largely similar to those of blastocysts cultured in humidified incubators. These findings highlight the considerable potential of dry incubators for the in vitro production of bovine embryos.

Curcumin enhances developmental competence and ameliorates heat stress in in vitro buffalo (Bubalus bubalis) embryos

Background and Aim: Buffalo is the principal dairy animal and plays a major role in the economic growth of the dairy industry, contributing nearly 50% of the country’s milk production. The Buffalo core body temperature is typically 38.5°C, but it can rise to 41.5°C in the summer, causing heat stress, which leads to the generation of reactive oxygen species or oxidative stress and affects the reproductive physiology of animals. Curcumin acts as an antioxidant, improves cellular development, and combats the effect of heat stress on in vitro-produced embryos. This study aimed to examine the impact of curcumin on developmental competence and the expression of important genes under normal and heat-stressed conditions during in vitro embryo production in buffalo. Materials and Methods: Group-1: All embryo production steps (i.e., in vitro maturation [IVM], in vitro fertilization [IVF], and in vitro culture [IVC]) were conducted at 38.5°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin. Group-2: All embryo production steps (i.e., IVM, IVF, and IVC) were carried out at 38.5°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin, but the early cleaved embryos were exposed to heat stress (39.5°C) for 2 h after 48 h of IVF and then cultured at 38.5°C for embryo production. Results: Blastocyst production was 16.63 ± 1.49%, 21.46 ± 0.67%, and 6.50 ± 1.17% at control, 5 μM and 10 μM of curcumin at 38.5°C, respectively, whereas at 39.5°C, it was 8.59 ± 1.20%, 15.21 ± 1.31%, and 3.03 ± 1.20% at control, 5 μM and 10 μM curcumin, respectively. The blastocyst rate was found to be significantly higher (p &lt; 0.05) at 5 μM curcumin compared with the control or 10 μM at 38.5°C and 39.5°C. The antioxidant, antiapoptotic, and pluripotency-related genes exhibited higher (p &lt; 0.05) expression in the presence of 5 μM curcumin compared to 10 μM or control at both temperatures. Conclusion: Curcumin supplementation in embryo culture media effectively enhances embryo production in vitro and mitigates the adverse effects of heat stress. Keywords: antioxidant, buffalo, curcumin, embryo, heat stress.

Effect of the addition of IGF-1 during in vitro culture on the embryonic development speed from different crossbreed bovine embryos.

Supplementation with insulin-like growth factor type 1 (IGF-1) during in vitro culture of bovine embryos has yielded mixed results, likely due to genetic variability among embryos. This work aimed to evaluate the effect of IGF-1 at two concentrations on the development speed embryos from primary F1 crossbreeds used in dual-purpose cattle farming in the Colombian low tropics. Specifically, we investigated the influence of IGF-1 and embryo breed on the blastocyst formation rate. Oocytes were sourced from non-pregnant cows: Bos taurus indicus (20 Brahman and 14 Gyr) and Bos taurus taurus (12 Holstein and 28 Romosinuano). Oocytes were fertilized with semen from specific bulls (Recoil for Holstein, Gabinete for Gyr, and UBER POI 1490 for Brahman). The resulting embryos from each crossbreed group were randomly distributed in three different cultured media with 50 ng/mL IGF-1, 100 ng/mL IGF-1, or no IGF-1 (control) for 7 days. Results showed that 50 ng/mL IGF-1 significantly increased embryo production by day 6 (25.9%±14.56%) compared to control (20.5%±11.84%) and 100 ng/mL IGF-1 (23.0%±9.54%) (p < 0.05). By day 7, both 50 ng/mL (42.6%±26.55%) and 100 ng/mL (49.7%±21.98%) IGF-1 groups exhibited significantly higher production rates compared to the control group (p < 0.001). The embryo breed also influenced development, with Gyr-Holstein (GxH) crossbreeds showing the highest production rates (p < 0.001). In conclusion, IGF-1 supplementation enhances in vitro embryo production, with the effect influenced by both breed and IGF-1 concentration. These findings suggest that breed-specific optimization of IGF-1 conditions is necessary to maximize embryonic development outcomes.

Ин витро култивиране на говежди ембриони

Embryo cultivation is the longest step during the in vitro embryo production process. In this study, the effect of adding 5% FCS or 3 mg/ml BSA to SOF and KSOM in vitro culture media was investigated. When adding FCS in SOF medium, a better effect on separation was observed (P &lt; 0.05), and the percentage of blastocysts was higher - 58.6%. When BSA was added to SOF medium, the percentage of blastocysts was 40.1% - significantly lower. When cultured in KSOM medium with the addition of FCS, the division rate was lower than when cultured in SOF+FCS and the proportion of blastocysts was also slightly lower. When BSA was added to the KSOM medium, the percentage of blastocysts was almost as in the SOF+BSA group. Аt the conclusion of the conducted experiment, the addition of FCS in KSOM and SOF media is beneficial for in vitro cultivation, resulting in a higher percentage of blastocysts.

Cryopreservation, cryoprotectants, and potential risk of epigenetic alteration.

The cryopreservation of gametes and embryos has increased notably over the past 20years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservationprocess is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming-crucial for the fate of the embryo-occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health.

Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

BackgroundIn vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%).MethodsBovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.ResultsUnder 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted.ConclusionOES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.

Non Invasive Preimplantation Testing for Aneuploidies in Assisted Reproduction: A SWOT Analysis.

The implementation of non-invasive PGT-A offers a new strategy to genetically assess the preimplantation embryo and to enhance IVF results. The extraction of DNA from the embryo culture medium has been sufficiently demonstrated, and the ability to obtain chromosomal information as a result is particularly interesting. As morphological criteria have proven to have a weak correlation with embryo ploidy status, this technique emerges as a promising alternative for embryo selection. It also appears reasonable that avoiding biopsy may enhance further embryo development. However, there are growing concerns regarding several aspects of this technique, such as the origin of this cell free DNA, the degree of representativeness of the whole embryo, the need for extended culture or the absence of standardized protocols. Despite the published data on good prognosis couples are promising, niPGT-A is yet to be considered a substitute for trophectoderm biopsy. The current SWOT analysis aims to summarize both resolved and unresolved issues, as well as limiting aspects of niPGT-A.

Bta-miR-665 improves bovine blastocyst development through its influence on microtubule dynamics and apoptosis.

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Comparison of Preimplantation Mouse Embryos with Different Genetic Backgrounds as Models for Evaluating Human Embryo Culture Media Composition.

Can the best culture medium be determined by mouse embryo testing? Zygotes/embryos of four different mouse strains: inbred Balb/c (n = 79) and C57Bl/6 (n = 95), F1 hybrid DBA/2*C57Bl/6 (n = 133), and outbred ICR (n = 69) were incubated in vitro in four culture media: 1-Step Culture Medium (VitaVitro), CSC medium (Irvine Scientific), Sage 1-Step medium (Origio), G-TL medium (Vitrolife). The embryos were cultured under standard conditions at atmospheric oxygen: 37℃; 6% CO2, 100% humidity. Standard MEA-test parameters (number of expanded blastocysts on E4.5), as well as additional characteristics (embryo development on E2.5-E4.5, number of hatching and hatched blastocysts on E4.5, embryo arrest and degeneration) were assessed for each of the strain-medium combinations. The results were compared across strains as well as across media. Embryo development depended on both the media and the mouse strain. Embryos from inbred and outbred mice were more sensitive to suboptimal culture conditions compared to the hybrid strain; this was reflected in reduced blastocyst and hatching rates, as well as an increased percentage of arrested and degraded embryos. The choice of optimal media depended strongly on the strain: CSC was better for Balb/c, Sage 1-step for C57Bl/6, while both were preferred for hybrids, and G-TL for outbreds. VitaVitro was quite good for hybrids and performed worse for other strains. Overall, the embryos of each strain behaved differently in different media, and it was not possible to make a real preference for any of them over human embryos based on the results of any single mouse strain. Only a pooled sample of different mouse strains can be used for comprehensive media MEA testing. MEA testing with any strain of mice is doomed to error due to their specific culture requirements. Hybrid mice are too reproductively efficient and cannot be used to distinguish media based on their subtle differences. Outbred and inbred mice are too sensitive and may be delayed or degraded in culture media that is well suited to the requirements of human embryos. Combined analysis of multiple mouse strains should be used to test media more fully and may serve as a model for the heterogeneity of human embryos in IVF clinics.

Current research status and clinical applications of noninvasive preimplantation genetic testing: A review.

Noninvasive preimplantation genetic testing (ni-PGT) is conducted by obtaining genetic information from embryos through the analysis of free DNA released by embryos in spent embryo culture medium or blastocoel fluid. Compared to conventional preimplantation genetic testing relying on trophectoderm biopsy, ni-PGT is characterized by its noninvasiveness. It has demonstrated early advancements in the detection of embryonic chromosomal aneuploidies and the diagnosis of monogenic diseases, showcasing considerable potential for clinical application. However, there are substantial controversies in the literature concerning the reliability of ni-PGT, the source of cell-free DNA, and maternal contamination. This paper elaborates on the principles, research advancements, effectiveness, and limitations of ni-PGT to provide a basis for clinical applications.

Real-time visualisation of developing chick embryos cultured in transparent plastic films from the blastoderm stage until hatching

Real-time visualisation of chick embryo development occurring inside an opaque eggshell is a major goal of developmental biology. This goal was partially achieved when 3-day-old embryos recovered from fertilised shelled eggs, preincubated in a conventional incubator, hatched after the egg contents had been transferred into shell-less embryo culture systems constructed of transparent plastic film placed in a plastic cup. However, the hatchable shell-less embryo culture systems described thus far cannot be used to visualise the dynamic morphological changes associated with the emergence of major organ systems during the first 3 days of embryo development, as the blastoderm area needs to be covered with an opaque membrane. Here, we present the development of a chick embryo culture system that enables omnidirectional real-time visualisation of the developing embryo, beginning from the newly laid blastoderm stage embryo until hatching.

Advanced maternal age affects their frozen-thawed embryo susceptibility to high oxygen environment

Preimplantation embryos can experience stress from laboratory interventions and a sub-optimal culture environment. Though research has demonstrated advanced maternal age impairs oocyte quality, the response of embryos derived from such oocytes to vitrification-thawing and culture in a high oxygen (O2) environment in the assisted reproductive technology laboratory is unknown. Therefore, in this study, embryos produced by in vitro fertilization (IVF) using oocytes from two- and eight-month-old Swiss albino mice were vitrified and thawed during their 6–8 cell stage. and cultured at low oxygen (5%) tension (LOT) and high oxygen (20%) tension (HOT). Embryo development, apoptosis, inner cell mass (ICM) outgrowth proliferation ability in vitro and pluripotency were assessed. Embryos from advanced maternal age cultured at HOT showed reduced fertilizing ability (p < 0.05), poor survival post-thawing (p < 0.05), and increased apoptosis (p < 0.01) in comparison to sibling embryos cultured at LOT. Importantly, the extended culture of vitrified-thawed embryos from advanced maternal age led to a significant (p < 0.001) reduction in complete ICM outgrowth formation at HOT in comparison to the LOT environment. The findings of this study suggest that HOT is detrimental to embryos from advanced maternal age, and importantly, vitrified-thawed embryos are more susceptible to stress, which could have negative implications, especially during the peri-implantation developmental period.

Lack of effect of cAMP modulation during pre-maturation in vitro on development to the blastocyst stage and expression of specific genes associated with lineage commitment

Pharmacological elevation of cAMP of cultured cumulus-oocyte complexes (COC) before or coincident with initiation of maturation has been reported to improve outcomes for various systems for in vitro production of embryos. Here it was hypothesized that artificial elevation of cAMP in the oocyte for a 2-h period of pre-maturation would improve developmental competence of matured oocytes and result in increased blastocyst yield and alter expression of genes important for embryonic differentiation. Treated COC were cultured for 2 h with dibutyryl cAMP (dbcAMP), a membrane-permeable form of cAMP, and 3-isobutyl-1-methylxanthine (IBMX), which inhibits phosphodiesterases that convert cAMP to ATP. Subsequently, oocytes were matured and fertilized and the resultant embryos cultured for 7.5 d. There was no effect of treatment on the percent of oocytes or cleaved embryos becoming blastocysts or on transcript abundance of EOMES, GATA4, NANOG, SOX2, or SOX17 in the blastocyst. Results do not support the use of pharmacological enhancers of cAMP concentrations in the oocyte for improving the blastocyst yield following in vitro oocyte maturation, fertilization, and embryo culture.

Quantification of elastic modulus variations during zebrafish embryo development using a 3D-printed microfluidic platform

Reliable indicators to assess embryo quality are critical for the in vitro fertilization. Increasing evidence suggests that elasticity is emerging as a potential marker to evaluate the early development of embryos. This paper introduces a 3D-printed microfluidic device to measure the elastic modulus of zebrafish embryos deformed in a circular constriction channel. Firstly, numerical simulation was performed to analyze the impact of inlet pressure, embryo size and constriction channel diameter on the maximum protrusion length of the embryo. Subsequently, the zebrafish embryos were deformed using the device to record the protrusion length which was automatically measured using U-net, before the power-law rheological model was employed to calculate the elastic modulus. Experiments showed the power-law exponent and embryo elasticity were stable as the inlet pressure was not less than 250 mbar. Embryo culturing after squeezes revealed that embryos could maintain normal development even after multiple squeezes at 150 mbar while higher pressure may be fatal. Afterward, the deformation of the yolk was found to increase elasticity by 60.4% compared to cases where only the chorion envelope was deformed. Finally, the elasticity variation of zebrafish embryos was measured for 17hours. It revealed that the elasticity initially increased from hour 1 to hour 7-10 and then returned to approximately the original value during culture from the cleavage to the segmentation stages. The system with the ability of precise and long-term assessment of embryo elasticity may find valuable application potentials in the mechanical evaluation and sorting of embryos.

Mitoquinone improves porcine embryo development through modulating oxidative stress and mitochondrial function

Oxidative stress caused by excess reactive oxygen species (ROS) is one of the main causes of low efficiency in in vitro production of embryos. These ROS can cause mitochondrial dysfunction and apoptosis, resulting in poor embryo development. Therefore, to prevent mitochondrial damage and apoptosis caused by ROS, we investigated the effects of mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, on the in vitro culture (IVC) of porcine embryos. Various concentrations of MitoQ (0, 0.01, 0.1, or 1 nM) were supplemented during the entire period of IVC. The results showed that supplementation with 0.1 nM MitoQ significantly increased the blastocyst formation rate, with a higher total cell number including trophectoderm cell number and higher transcript expression of lineage-specific transcription factors in blastocysts. In addition, the 0.1 nM MitoQ-treated group showed a significantly lower percentage and number of apoptotic cells in blastocysts with positively regulated transcript expression of apoptosis-related genes. Therefore, 0.1 nM MitoQ was suggested as optimal concentration for porcine IVC and used for further investigations. MitoQ treatment significantly reduced intracellular ROS levels and increased glutathione levels in Day 2 embryos, with upregulated the transcript expression of antioxidant enzymes-related genes. Furthermore, the MitoQ group exhibited a significantly higher mitochondrial quantity, mitochondrial membrane potential, and ATP content in Day 2 embryos, with increased transcript expression of mitochondrial biogenesis-related genes. Taken together, these findings reveal that MitoQ supplementation can enhance the developmental competence of porcine embryos by decreasing oxidative stress and improving mitochondrial function.

Evolution of Media Supporting the Development of Mammalian Preimplantation Embryos In Vitro.

Assisted reproductive technology has revolutionized our ability to genetically manipulate, maintain and rederive laboratory animals of biomedical importance; manipulate animal reproduction or genetics to boost production of farm animals; and improve human reproductive health. The media for in vitro manipulation and the culture of embryos play a critical role in the development of assisted reproductive technology. In this review, the evolution of culture media supporting embryo development in vitro from selected animal species, laboratory animals (mice and rats) and farm animals (pigs and cattle), will be discussed with a focus on the development of chemically defined media.