Culture Of Early Embryos Research Articles

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1-induced enhancement of mitochondrial activity and alleviation of proapoptotic events.

Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development-related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 μM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan-activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency-related factor levels were elevated (NANOG and octamer-binding transcription factor 4 [OCT4]), antioxidant-related genes were upregulated (nuclear factor-erythroid 2-related factor 2 [NRF2]), and apoptosis- (caspase 3 [CAS3]) and autophagy-related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

Probing morphological, genetic and metabolomic changes of in vitro embryo development in a microfluidic device.

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10-12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Recent advances in critical nodes of embryo engineering technology.

The normal development and maturation of oocytes and sperm, the formation of fertilized ova, the implantation of early embryos, and the growth and development of foetuses are the biological basis of mammalian reproduction. Therefore, research on oocytes has always occupied a very important position in the life sciences and reproductive medicine fields. Various embryo engineering technologies for oocytes, early embryo formation and subsequent developmental stages and different target sites, such as gene editing, intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis (PGD), and somatic cell nuclear transfer (SCNT) technologies, have all been established and widely used in industrialization. However, as research continues to deepen and target species become more advanced, embryo engineering technology has also been developing in a more complex and sophisticated direction. At the same time, the success rate also shows a declining trend, resulting in an extension of the research and development cycle and rising costs. By studying the existing embryo engineering technology process, we discovered three critical nodes that have the greatest impact on the development of oocytes and early embryos, namely, oocyte micromanipulation, oocyte electrical activation/reconstructed embryo electrofusion, and the in vitro culture of early embryos. This article mainly demonstrates the efforts made by researchers in the relevant technologies of these three critical nodes from an engineering perspective, analyses the shortcomings of the current technology, and proposes a plan and prospects for the development of embryo engineering technology in the future.

MTORC1/2 signaling is downregulated by amino acid-free culture of mouse preimplantation embryos and is only partially restored by amino acid readdition.

Development of the mammalian preimplantation embryo is influenced by autocrine/paracrine factors and the availability of nutrients. Deficiencies of these during in vitro culture reduce the success of assisted reproductive technologies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway integrates external and internal signals, including those by amino acids (AAs), to promote normal preimplantation development. For this reason, AAs are often included in embryo culture media. In this study, we examined how withdrawal and addition of AAs to culture media modulate mTORC1 pathway activity compared with its activity in mouse embryos developed in vivo. Phosphorylation of signaling components downstream of mTORC1, namely, p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6, and 4E binding protein 1 (4E-BP1), and that of protein kinase B (Akt), which lies upstream of mTORC1, changed significantly across stages of embryos developed in vivo. For freshly isolated blastocysts placed in vitro, the absence of AAs in the culture medium, even for a few hours, decreased mTORC1 signaling, which could only be partially restored by their addition. Long-term culture of early embryos to blastocysts in the absence of AAs decreased mTORC1 signaling to a greater extent and again this could only be partially restored by their inclusion. This failure to fully restore is probably due to decreased phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC2 signaling in culture, as indicated by decreased P-AktS473. mTORC2 lies upstream of mTORC1 and is insensitive to AAs, and its reduced activity probably results from loss of maternal/autocrine factors. These data highlight reduced mTORC1/2 signaling activity correlating with compromised development in vitro and show that the addition of AAs can only partially offset these effects.

Bioengineered microenvironment to culture early embryos.

The abnormalities of early post‐implantation embryos can lead to early pregnancy loss and many other syndromes. However, it is hard to study embryos after implantation due to the limited accessibility. The success of embryo culture in vitro can avoid the challenges of embryonic development in vivo and provide a powerful research platform for research in developmental biology. The biophysical and chemical cues of the microenvironments impart significant spatiotemporal effects on embryonic development. Here, we summarize the main strategies which enable researchers to grow embryos outside of the body while overcoming the implantation barrier, highlight the roles of engineered microenvironments in regulating early embryonic development, and finally discuss the future challenges and new insights of early embryo culture.

Development of microfluidic technology in reproductive researches

Reproduction is one of the most basic characteristics of organisms, and the guarantee of the continuation and evolution of a species. As the country with world's largest population, China has been gradually increasing its investment in research on the reproductive system in recent years, particularly in the field of basic research. The rapid development and wide application of the microfluidic technology since its birth are sufficient to explain its application prospect. Currently, infertility and birth defects are major problems in the field of reproduction. Micro-reproductive technologies, including microfluidic and organs-on-chips, are formed through the combination of a wide range of basic science and bioengineering technologies. In reproductive research, microfluidic technology display several advantages:flexible design of the microchannel shape and size to better simulate the physiological environment, the low consumption of microfluidic chip, and highly integrated microfluidic technology. Microfluidic technology has been applied to various processes including sperm vitality evaluation and screening, sperm chemotaxis, cumulus oophorus cell removal, zona pellucida removal, ootid localization and screening, fertilization, early embryo culture and reproductive organ simulation. This paper introduces the recent progress in reproductive research based on microfluidic technology and its application prospects.

The Domestic Dog Embryo: In Vitro Fertilization, Culture, and Transfer.

Advances in embryo technologies in the domestic dog have made significant strides in the past decade. This progress has been spurred by interests in taking advantage of the dog as a biomedical research model for human and companion animal medicine, developing assisted reproductive technologies to manage genetic diversity in endangered canids maintained ex situ, and improving breeding in rare or working breeds of dogs. Here, we focus on recent advancements and techniques for collection of in vivo-matured oocytes, in vitro fertilization (IVF), in vitro culture of early (≤8-cell) and advanced stage (≥16-cell) embryos, and embryo transfer.

Comparison between paraffin and mineral oil covering on early human embryo culture: a prospective randomized study

ABSTRACTThe oil overlay in microdrop culture systems prevents medium evaporation, helps to maintain appropriate pH and osmotic conditions and protects from microbial contamination. In the present study, we prospectively compared covering by Ovoil™, a paraffin oil, and LiteOil®, a mineral oil, on the in vitro development of human embryos and their suitability for transfer/freezing at day 3 and live birth rate. One hundred and one patients undergoing in vitro fertilization (IVF) treatment by intracytoplasmic sperm injection (ICSI) were enrolled in our study. After ICSI, 1237 oocytes were 1:1 randomly allocated into 2 groups according to the type of overlaying oil: Ovoil™ (616 oocytes) or LiteOil® (621 oocytes). Fertilization rate was assessed around 18 hours post-insemination (hpi) and embryos were checked for early cleavage at 25 hpi. Embryo morphology was recorded on days 2 and 3. A total of 437 (Ovoil™) and 438 day 3 embryos (LiteOil®) were analyzed. There were no differences between the two groups in terms of fertilization rate and occurrence of early cleavage. The proportion of top quality embryos (41.7% vs. 41.2%) and the final utilization rates (92.2% vs. 92.0%) were similar in Ovoil and LiteOil groups, respectively, at day 3. Live birth rate per transfer was essentially the same with Ovoil™ overlay (26.9%) when compared to LiteOil® (26.2%). Live birth rate in patients who simultaneously received embryos from both overlay types was 17.2%. Despite the different characteristics of these two oils regarding hydrocarbon saturation, packing and temperature storage, Ovoil™ and LiteOil® can be used in parallel in the same IVF protocol.Abbreviations: ART: assisted reproductive technologies; hpi: hours post-insemination; hSA: human serum albumin; HTF: human tubal fluid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; MII: metaphase II; MEA: mouse embryo assay; RT: room temperature.

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques.

Altered methylations of H19, Snrpn, Mest and Peg3 are reversible by developmental reprogramming in kidney tissue of ICSI-derived mice

Although the prevalence of Intracytoplasmic sperm injection (ICSI) has increased year by year, there remains concern about the safety of these procedures because of reports of the increased risk for imprinting disorders. Previous research has demonstrated that gonadotropin stimulation contributes to an increased incidence of epimutations in ICSI-derived mice. However, the epimutations in ICSI offspring after removing the effect of gonadotropin stimulation and the possibility that epimutations are reversible by developmental reprogramming has not been investigated. Our study is the first to investigate the effect of ICSI itself on methylation and exclude the effect of superovulation using the kidney tissues from the adult and old mice. We found reduced methylation and up-regulated expression of the imprinted genes, H19, Mest and Peg3, in adult ICSI mice, but the above alterations observed in adult mice were not detected in old ICSI mice. At the Snrpn DMR, methylation status was not altered in adult ICSI-derived mice, but hypermethylation and correlated down-regulated expression of Snrpn were observed in old mice. In conclusion, ICSI manipulation and early embryo culture resulted in alterations of methylation in differentially methylated region of H19, Mest, Peg3 and Snrpn, and the alterations were reprogrammed by developmental reprogramming.

Optimizing camel (Camelus dromedarius) oocytes in vitro maturation and early embryo culture after parthenogenetic activation

This study was undertaken to investigate on camel oocytes the effects of different in vitro maturation (IVM) time, supplementation of the IVM medium on oocyte maturation and the use of different in vitro culture media (KSOMaa, mSOF, TCM, and K-SICM) on embryo development to optimize in vitro embryo production procedures. Cumulus-oocyte complexes (COCs) were cultured for 28, 32, 36, 42, and 48h for IVM and were examined for cumulus expansion, meiotic maturation, and early embryo development after parthenogenetic chemical activation and IVC. The results showed that IVM of oocytes for 32 and 36h significantly increased embryonic development (P<0.05) compared with other treatments. Culturing the oocytes for longer durations (42 and 48h) permitted the entry and completion of meiosis II and significantly reduced embryonic developmental competence, as indicated by reduction in morula and blastocyst ratios (P <0.05). Moreover, supplementation of IVM medium with follicular fluid improved cumulus expansion when compared with fetal bovine serum supplementation. Culturing of parthenogenetic embryos in KSOMaa medium significantly improved morula and blastocyst development, compared with culturing in mSOF, TCM, and K-SICM media (P<0.05). We conclude that IVM of camel oocytes for a period of 32–36h would be sufficient to support early embryonic development after parthenogenetic activation and that KSOMaa is an efficient culture medium for in vitro camel embryo production.

M.C. Chang as a Mentor: A Memoir.

M.C. Chang as a Mentor: A Memoir.

Improvements to In Vitro Culture Media for Use in Bovine IVF

In assisted reproduction of cattle the design and preparation of in vitro culture media has been instrumental in supporting the development of bovine oocytes and embryos. In vitro production (IVP) involves three main sequential steps: oocyte aspiration and in vitro maturation (IVM); in vitro fertilization (IVF); and in vitro culture (IVC) of early pre-implantation embryos. Research on IVC media has aimed to emulate the crucial elements that are present in the oviduct and uterus of female cattle by supplementing with protein and growth factors. As a result, IVP has proven a reliable methodology with an acceptable, if far from remarkable, blastocyst transfer rate of typically 40%. Moreover, the addition to media of defined extracts has gradually replaced that of fetal calf serum, leading to improved embryo viability and cryotolerance. Nevertheless, major advances in the composition of novel media that may facilitate a further increase of blastocyst rate have yet to be realized. Currently, there is considerable variation in the constituents of IVC media used in bovine assisted reproduction. Hence, significant innovations are required in order to not only achieve a greater success rate of IVP and to enhance the cryotolerance of bovine IVF-derived embryos but also to provide a defined IVC medium that may be recognized as a point of reference across the cattle breeding industry. This review examines the progress made to date in IVP to enhance embryo quality and points to future areas for investigation of a ‘gold standard’ IVC medium. This is a fully defined medium that supports consistently the attainment of a bovine IVP blastocyst rate similar to that achieved using a conventional medium supplemented with serum.

Influence of cysteamine supplementation during in vitro culture of early stage caprine embryos on blastocyst production

The objective of this study was to evaluate the effect of cysteamine supplementation on embryo development in mCR2 media. The COCs (1251) were matured in TCM–199 medium containing FSH (5µg/ml), LH (10µg/ml), follicular fluid (10%), FBS (10%) with 3 mg/ml BSA for 27h at 38.5°C and 5% CO2 in an incubator. Matured oocytes were co-cultured with 1×106 spermatozoa/ml collected from a Sirohi buck in fertTALP (10% FBS+ 4mg/ml BSA and 10 µg/ml heparin) for 18 h in incubation. After 18 h of sperm-oocytes, co-incubation of oocytes with sperms were washed in embryo development medium to remove sperm cells adhered to zonapellucida. Presumptive zygotes (1,171) were selected and randomly divided into 2 groups. Group 1, the presumptive zygotes (610) were cultured in mCR2aa as a control for 12 days. Group 2, the presumptive zygotes (561) were culture in mCR2aa medium supplemented with 100 µM cysteamine for 10 days. The percentage of cleavage, morula, and blastocyst production in groups 1 and 2 were 36.39% and 31.71% (cleavage); 21.62% and 30.89% (morula); 4.95% and 8.98% (blastocyst) respectively. In conclusion, results indicated that the addition of cysteamine to the IVC medium stimulates caprine embryo development and blastocyst production.

Advances in Conifer Somatic Embryogenesis Since Year 2000.

This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

Imprinting methylation and assisted reproductive technology

Assisted reproductive technology (ART) is an important treatment for infertile people of reproductive age and is also known as fertility treatment. The processes of ART involves the isolation, handling and culture of early embryos, which may result in alterations in genomic methylation at specific loci and influence the proper establishment and maintenance of genomic imprints. Recent studies have identified an increased incidence of imprinting disorders via ART. In this article, we reviewed that the ART may be prone to induction of imprinting methylation errors during embryonic development. Further studies are necessary to elucidate the safety of ART in this field.

The effect of macromolecule and growth factor combinations on in vitrodevelopment of bovine embryos

This study was conducted to determine the effects of different macromolecule sources added to synthetic oviduct fluid (SOF) culture medium supplemented with growth factors on the development of bovine embryos and blastocyst morphology. Zygotes were distributed into 5 treatment groups. Cleavage, morula, and blastocyst rates were evaluated under a stereomicroscope. Trophectoderm (TE) and inner cell mass (ICM) cells were determined by differential staining method. It was found that bovine serum albumin (BSA), either alone or in combination with growth factors, as compared to the control or polyvinyl-alcohol (PVA) resulted in higher embryo yield and faster development during early bovine embryo culture. The quality of bovine embryos, based on the number of blastocyst cells and the ratio of ICM to total blastomeres, was affected by the sources of macromolecules and their combinations with growth factors. Growth factors supplemented to SOFaa media with BSA and PVA significantly increased the number of ICM cells and the ratio of ICM cells to total number of cells. In conclusion, replacing BSA with PVA depressed the blastocyst rate and cell numbers, and the number of blastomeres and ICM and TE cell numbers were affected by both the type of macromolecule and the growth factor supplements.