In Vitro Fertilization Blastocysts Research Articles

Distinct properties of putative trophoblast stem cells established from somatic cell nuclear-transferred pig blastocysts

BackgroundGenetically modified pigs are considered ideal models for studying human diseases and potential sources for xenotransplantation research. However, the somatic cell nuclear transfer (SCNT) technique utilized to generate these cloned pig models has low efficiency, and fetal development is limited due to placental abnormalities.ResultsIn this study, we unprecedentedly established putative porcine trophoblast stem cells (TSCs) using SCNT and in vitro-fertilized (IVF) blastocysts through the activation of Wing-less/Integrated (Wnt) and epidermal growth factor (EGF) pathways, inhibition of transforming growth factor-β (TGFβ) and Rho-associated protein kinase (ROCK) pathways, and supplementation with ascorbic acid. We also compared the transcripts of putative TSCs originating from SCNT and IVF embryos and their differentiated lineages. A total of 19 porcine TSCs exhibiting typical characteristics were established from SCNT and IVF blastocysts (TSCsNT and TSCsIVF). Compared with the TSCsIVF, TSCsNT showed distinct expression patterns suggesting unique TSCsNT characteristics, including decreased mRNA expression of genes related to apposition, steroid hormone biosynthesis, angiopoiesis, and RNA stability.ConclusionThis study provides valuable information and a powerful model for studying the abnormal development and dysfunction of trophoblasts and placentas in cloned pigs.

Effects of insulin-like growth factor 1, leukemia inhibitory factor, and basic fibroblast growth factor on goat oocyte maturation and early embryonic development in vitro

In this study, we investigated the effects of insulin-like growth factor 1 (IGF-1), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) on the in vitro maturation of goat oocytes and early embryonic development of in vitro-fertilized (IVF) oocytes. We also aimed to prepare an efficient in vitro maturation culture system for goat oocytes as a technical basis for improving both the quality of goat oocytes and the efficiency of embryo production in vitro. The experimental results revealed that the addition of bFGF (40 ng/mL), LIF (20 ng/mL), and IGF-1 (20 ng/mL) to the maturation medium improved the maturation rate of goat oocytes. Compared with the control group, the in vitro maturation rate of oocytes (83.0% vs. 70.93%, P < 0.05) and the development rate of IVF blastocysts (45.88% vs. 35.81%, P < 0.05) in the bFGF, LIF, and IGF-1 (FLI) group were significantly increased. The total number of developing blastocysts (114.40 vs. 83.20, P < 0.05) and the ratio of inner cell mass cells (39.50 vs. 25.33, P < 0.05) were also significantly higher in the FLI group than in the control group. Evaluation of maternal gene expression revealed that FLI could significantly increase the expression levels of DNMT1, ZAR1, and NLRP5 and cumulus cell expansion genes PTX3, TNFAIP6, PTGS2, and HAS2 in mature oocytes (P < 0.01). We indirectly proved that bFGF, LIF, and IGF-1 support goat oocyte maturation and early embryonic development. In summary, this study established a reliable and efficient in vitro goat oocyte maturation culture system using bFGF, LIF, and IGF-1. The results of this study can serve as a technical basis for improving the quality of goat oocytes matured in vitro, potential of early embryonic development, IVF of goat embryos, and in vitro production of other livestock.

Image Processing Approach for Grading IVF Blastocyst: A State-of-the-Art Review and Future Perspective of Deep Learning-Based Models

The development of intelligence-based methods and application systems has expanded for the use of quality blastocyst selection in in vitro fertilization (IVF). Significant models on assisted reproductive technology (ART) have been discovered, including ones that process morphological image approaches and extract attributes of blastocyst quality. In this study, (1) the state-of-the-art in ART is established using an automated deep learning approach, applications for grading blastocysts in IVF, and related image processing techniques. (2) Thirty final publications in IVF and deep learning were found by an extensive literature search from databases using several relevant sets of keywords based on papers published in full-text English articles between 2012 and 2022. This scoping review sparks fresh thought in deep learning-based automated blastocyst grading. (3) This scoping review introduces a novel notion in the realm of automated blastocyst grading utilizing deep learning applications, showing that these automated methods can frequently match or even outperform skilled embryologists in particular deep learning tasks. This review adds to our understanding of the procedure for selecting embryos that are suitable for implantation and offers important data for the creation of an automated computer-based system for grading blastocysts that applies deep learning.

Non-invasive preimplantation genetic testing for conventional IVF blastocysts

BackgroundPrevious studies suggested that non-invasive preimplantation genetic testing (niPGT) for intracytoplasmic sperm injection (ICSI) blastocysts can be used to identify chromosomal ploidy and chromosomal abnormalities. Here, we report the feasibility and performance of niPGT for conventional in vitro fertilization (IVF) blastocysts.MethodsThis was a prospective observational study. In the preclinical stage, whole genome amplification and NGS were performed using the sperm spent culture medium (SCM). Then, trophectoderm (TE) biopsies and corresponding SCM derived from 27 conventional IVF monopronuclear embryos were collected. In the clinical stage, samples from 25 conventional IVF cycles and 37 ICSI cycles from April 2020–August 2021 were collected for performance evaluation.ResultsPreclinically, we confirmed failed sperm DNA amplification under the current amplification system. Subsequent niPGT from the 27 monopronuclear blastocysts showed 69.2% concordance with PGT results of corresponding TE biopsies. In the clinical stage, no paternal contamination was observed in any of the 161 SCM samples from conventional IVF. While maternal contamination was observed in 29.8% (48/161) SCM samples, only 2.5% (4/161) samples had a contamination ratio ≥ 50%. Compared with that of TE biopsy, the performances of NiPGT from 161 conventional IVF embryos and 122 ICSI embryos were not significantly different (P > 0.05), with ploidy concordance rates of 75% and 74.6% for IVF and ICSI methods, respectively. Finally, evaluation of the euploid probability of embryos with different types of niPGT results showed prediction probabilities of 82.8%, 77.8%, 62.5%, 50.0%, 40.9% and 18.4% for euploidy, sex-chromosome mosaics only, low-level mosaics, multiple abnormal chromosomes, high-level mosaics and aneuploidy, respectively.ConclusionsOur research results preliminarily confirm that the niPGT approach using SCM from conventional IVF has comparable performance with ICSI and might broadening the application scope of niPGT.

Methylation status of imprinted gene IGF2/ H19 DMR3 region in Goat (Capra hircus) blastocysts produced through parthenogenesis and in vitro fertilization

Parthenogenetic embryo production in livestock provides headway to understand the role of the maternal and paternal genome during early embryonic development and offers an ethically competent source of embryonic stem cells. IGF2 and H19 are crucial imprinted genes and play an essential role in mammalian embryonic and placental development. The current study evaluated the methylation status of the DMR3 region of IGF2/H19 genes in goat blastocysts produced through parthenogenesis and in vitro fertilization (IVF) using bisulfite sequencing. For this, immature oocytes of good quality were subjected to in vitro maturation and subsequently used for parthenogenetic and IVF embryo production. The blastocyst rate was significantly (p < 0.05) higher for parthenogenetic embryos (21.11 ± 2.51%) than for their IVF counterparts (8.23 ± 1.02%). Furthermore, we analyzed the methylation status of the DMR3 region of the IGF2/H19 genes in both types of blastocysts. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (p 0.05) in parthenogenetic blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively). We further analyzed the relative expression of IGF2/H19 in parthenogenetic and IVF blastocysts by qRT PCR. IGF2 (5.02-fold) gene was significantly upregulated, while H19 was significantly downregulated in parthenogenetic blastocyst compared to IVF blastocyst. The present study revealed a higher methylation status of the DMR3 region of IGF2/H19 in parthenogenetic blastocysts than the IVF blastocysts, which could upregulate IGF2 expression and the blastocyst rate.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 μg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 μg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

Bayesian Network Modeling of IVF Blastocyst Score Prediction

Embryo transfer may be performed at cleavage stage (on day 2-3) or at blastocyst stage (on day 5) in In-Vitro Fertilization (IVF) treatment. Elective single embryo transfer at blastocyst stage increases the pregnancy probability and reduces the number of multiple pregnancies. However, the extended culture of embryos in the laboratory may result in transfer cancelation if no high quality blastocyst develops by day 5. Predicting the blastocyst score of individual embryos may help physicians to decide whether or not to further culture the embryos in the laboratory. In this paper, we use Bayesian networks for predicting the blastocyst score by modeling the morphological evolution of IVF embryos. We propose a weighted nearest neighbor approach to adjust the frequency estimates in the conditional probability table. Experimental results show that the proposed method significantly increases the accuracy and reduces false positive rates in IVF data in comparison to the frequency estimate method. Our proposed model can also predict low quality blastocyst development with a 77.3% True Negative rate. Using this model can help preventing developmental failures of embryos during IVF treatment.

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.

Effects of laser zona thinning and artificial blastocoel collapse on the cryosurviving and hatching of buffalo (Bubalus bulalis) blastocysts of different ages

The objectives of this study were to investigate whether blastocoel collapse before vitrification induced by laser improves the cryo-survivability of buffalo in-vitro-fertilized (IVF) blastocysts and whether laser assisted hatching (LAH) promotes hatchability of fresh and frozen-thawed IVF blastocysts. The expanded blastocysts were harvested on Days 6–9 and randomly allocated into five groups as follows: (1) blastocysts were vitrified and thawed without any treatment; (2) blastocysts were vitrified after 15–20 μm zona pellucida (ZP) thinning opposite to the inner cell mass, and blastocoels were also blotted in order to outflow the blastocoelic fluid before vitrification; (3) ZP thinning was made immediately after thawing; (4) fresh blastocysts underwent LAH; and (5) as a control, fresh blastocysts without treatment. Results of the present study showed that the cryosurvival rates of vitrified Day 8 and Day 9 blastocysts in Group 2 were significantly (P < 0.01) higher in Group 2 than Group 1. The hatching rates of Day 8 and Day 9 blastocysts in Group 2 and Group 3 were also significantly (P < 0.01) higher compared with Group 1. Moreover, the hatching rate of Day 9 blastocysts in Group 4 was notably (P < 0.05) higher than Group 5. In conclusion, LAH promotes the hatching rates of Day 9 fresh and Days 8–9 vitrified blastocysts, and artificial blastocoel collapse before vitrification improves the cryosurvival rate of Days 8–9 IVF buffalo blastocysts.

Does in vitro fertilization affect the expression of miRNAs and their biogenesis pathway in preimplantation mouse embryos?

In vitro fertilization (IVF) is a well-accepted procedure which has been utilized for the treatment of infertile patients. As embryos at early stages of development are very vulnerable, the IVF conditions may influence genetic and epigenetic regulation of preimplantation mouse embryo. We assessed the effect of IVF on the expression of developmental and implantation related miRNAs (miR-21, miR-93, miR-24, and let-7a), their common presumptive target (Stat3), and miRNA biogenesis pathway genes (Drosha, Dgcr8, Exportin-5, Dicer, and Ago2). in vivo 8-cell and blastocysts were compared to IVF embryos. Expression levels of miRNAs, Stat3, and miRNA biogenesis pathway genes were evaluated by qRT-PCR in in vivo (n = 8) and IVF (n = 4) embryos. The expression levels of let-7a and Stat3 were significantly reduced in IVF blastocyst when compared with in vivo (p = .004 and p = .009, respectively). Nevertheless, the IVF procedure did not influence the expression levels of miRNA biogenesis pathway components in 8-cell and blastocyst embryos. Downregulation of let-7a and developmental related transcription factor, Stat3, in IVF mouse blastocysts may affect preimplantation development and implantation of embryos. Moreover, the genes of the miRNA biogenesis pathway were not changed in preimplantation mouse embryos through the IVF procedure.

Contrasting effects of heat shock during in vitro maturation on development of in vitro-fertilized and parthenogenetic bovine embryos.

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12hr of in vitro maturation (IVM), followed by 12hr at 38.5°C. Control group consisted of in vitro maturation for 24hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p<.01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48hpia. Heat shock had a negative impact (p<.01) on cleavage of IVF embryos, whereas no (p>.05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p<.05) the apoptotic index in Part blastocysts, but no effect (p>.05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p<.01) and up-regulated the expression of HSP70.1 (p<.01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p<.05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes.

RNA sequencing and transcriptome analysis of buffalo (Bubalus bubalis) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization.

Across farm animal species, the live birth rate obtained with somatic cell nuclear transfer (SCNT) embryos is only <2% compared with >40% obtained with in vitro fertilization (IVF) embryos, primarily due to incomplete nuclear reprogramming which results in aberrant embryonic gene expression. We used RNA sequencing to compare the global transcriptome profile of SCNT and IVF buffalo blastocysts. SCNT blastocysts expressed 17,061 transcripts, of which 941 were unique whereas, IVF blastocysts expressed 17,303 transcripts, of which 1,183 were unique. At ≥2-folds change (p < .05), 331 transcripts were differentially expressed in the two groups among which, 19 were unique, 188 were downregulated and 143 were upregulated in SCNT compared with IVF blastocysts. Many genes affecting pluripotency, trophectoderm development, developmental regulation, and epigenetic modifications were upregulated in SCNT compared with IVF blastocysts. Among the four functional categories analyzed, epigenetic regulators were the most affected. Most of the WNT signaling pathway genes were upregulated whereas, the inhibitors of this pathway, such as DKK1, were downregulated in SCNT blastocysts, suggesting that this pathway is overexpressed in SCNT embryos. Gene Ontology analysisrevealed that 25 biological processes, 20 molecular functions, and 24 cellular compartment categories were enriched in SCNT blastocysts. This data can help identify reprogramming errors for improving cloning efficiency.

Treatment of Buffalo (Bubalus bubalis) Somatic Cell Nuclear Transfer Embryos with MicroRNA-29b Mimic Improves Their Quality, Reduces DNA Methylation, and Changes Gene Expression Without Affecting Their Developmental Competence.

microRNA-29b (miR-29b) plays an important role in controlling DNA methylation in cells. We investigated its role during early embryonic development in buffalo embryos produced by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). miR-29b expression was highest at the 2-cell stage, decreased (p < 0.001) at the 4-cell stage, and remained low thereafter at the 8-cell, morula, and blastocyst stages, showing a similar pattern in cloned and IVF embryos. Treatment of reconstructed embryos with miR-29b mimic for 1 hour after 1 hour of electrofusion increased (p < 0.05) the total cell number and decreased (p < 0.05) the levels of apoptosis and DNA methylation compared with controls. It also increased (p < 0.05) the ratio of inner cell mass:trophectoderm cell numbers of blastocysts compared with controls to the levels observed in IVF blastocysts. However, the blastocyst rate was not affected by treatment with miR-29b mimic (29.0% ± 2.0% vs. 27.0% ± 2.0% for controls). The treatment decreased (p < 0.001) the expression of epigenetic-related genes, DNMT3A and DNMT3B, but not DNMT1, and increased (p < 0.05) that of pluripotency- (NANOG, OCT4, and SOX2) and development-related genes (FGF4 and GLUT1) in blastocysts compared with controls. Our results suggest that miR-29b mimic treatment of reconstructed embryos improves the quality, reduces the level of apoptosis and DNA methylation, and changes gene expression in SCNT blastocysts without affecting the blastocyst rate.

Selection of Reference miRNAs for Relative Quantification in Buffalo (Bubalus bubalis) Blastocysts Produced by Hand-Made Cloning and In Vitro Fertilization.

Very low birth rate and a high incidence of abnormalities in offspring born from cloned embryos, which have limited the application of cloning technology on a wide scale, are believed to be because of incomplete or aberrant nuclear reprogramming. MicroRNAs (miRNAs) are involved in regulating a wide range of biological processes including reprogramming and embryonic development. Selection of suitable reference miRNAs is critical for normalization of data for accurate relative quantification of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR), which is currently the most widely used technique for quantifying miRNAs. This study was aimed at identification of reference miRNAs suitable for normalization of qRT-PCR data from blastocyst-stage buffalo embryos produced by handmade cloning and in vitro fertilization (IVF). RNA isolated from cloned and IVF blastocysts was subjected to next-generation sequencing based on which, 12 highly and most consistently expressed miRNAs, which included miR-92a, miR-423, miR-151, Let-7a, miR-103a, miR-93, miR-16b, miR-25, miR-30e, miR-101, miR-127, and miR-197, were selected as candidates for identification of suitable reference miRNAs using three statistical algorithms namely geNorm, NormFinder, and BestKeeper. Based on consensus of the three algorithms, the combination of miRNAs found to be suitable as reference miRNAs were miR-127 and miR-103 for IVF blastocysts; miR-92a and miR-103 for cloned blastocysts, and miR-103, miR-423, and miR-93 across both IVF and cloned blastocysts. The data of this study can be very useful in miRNA expression analysis of blastocyst-stage cloned and IVF embryos.

GDF8 enhances SOX2 expression and blastocyst total cell number in porcine IVF embryo development

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.

Evaluation of porcine circovirus type 2 infection in in vitro embryo production using naturally infected oocytes

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation concentration of estradiol than that of the untreated medium (P < 0.05). Finally, the gene expression levels of P450scc and GDF9 were greater in ALC-treated oocytes and cumulus cells than those in untreated cells (P < 0.05). Therefore, in buffalo, our results suggest that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases the production of steroid hormones, leading to increased quality of matured oocytes and improved embryonic development in vitro.

Synergistic effect of cysteamine, leukemia inhibitory factor, and Y27632 on goat oocyte maturation and embryo development in vitro

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0–48.0%; P > 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P < 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P < 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P < 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P < 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P < 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.

Selenium supplementation during invitro maturation enhances meiosis and developmental capacity of yak oocytes.

The objectives of the present study were to investigate the effects of selenium (Se) supplementation during invitro maturation (IVM) on the developmental capacity of yak (Bos grunniens) oocytes. Nuclear maturation, DNA integrity, glutathione peroxidase (GSH-Px) activity, subsequent embryonic development, and gene expression after invitro fertilization (IVF) were evaluated. The Se concentrations in yak plasma and follicular fluid were 0.142 and 0.069μg/mL, respectively. The DNA damage in cumulus cells decreased significantly with 2 and 4μg/mL supplementation of sodium selenite to IVM medium (P<0.05). Total GSH-Px activity in oocytes increased in all Se supplementation groups, and the 2 and 4μg/mL groups were significantly higher than the control group (0μg/mL). However, the cleavage rate was not significantly different after Se supplementation (P>0.05). The IVF blastocyst formation rates of 0, 1 and 4μg/mL sodium selenite groups were 47.7%, 51.2% and 58.9%, respectively. The 2μg/mL sodium selenite group had the highest blastocyst formation rate (60.5%). Gene expression analysis revealed that the quantity of transcripts associated with selenoprotein and protein synthesis were high in the 2 and 4μg/mL groups. In conclusion, both GSH-Px activity of oocytes and DNA integrity of cumulus cells significantly increased with supplemental Se during oocyte IVM. Considering that embryonic development is responsive to Se supplementation, we inferred that appropriate Se concentrations during IVM were beneficial for yak oocyte maturation and subsequent development.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.