Increased Blastocyst Formation Research Articles

Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

Impact of Group vs Individual Embryo Culture Strategies on Blastocyst and Clinical Outcomes.

Embryo culture is one of the most important steps in an assisted reproduction laboratory. Embryos can be cultured individually, one embryo per media drop, or in groups, culturing several embryos in the same media drop. Due to the controversy generated on this subject, we wondered which embryo culture method would have the best results in terms of quality and blastocyst formation rate. We designed a prospective randomized study comparing two different embryo culture strategies: group and individual embryo culture. The data were obtained from 830 embryos from 103 egg donation treatments. The zygotes were randomized into two groups: individual culture (group 1) or group culture (group 2). The embryos were cultured in 35-µl drops until day 5 when they were classified morphologically. We observed a significant increase in the blastocyst formation rate and in the usable embryo rate in individual culture on day 5 compared to group culture. However, good embryo quality (A/B blastocysts), implantation, and pregnancy rates were similar regardless of the type of embryo-culture. As a conclusion, individual culture may increase blastocyst formation rate and may benefit embryo quality on day 5. Our results support previous reports suggesting that individual culture could improve embryo development.

Interleukin-6 stimulates in vitro development of late-stage ovine embryos

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72–192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0–192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8–16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ± 1.52%) compared to the control (21.1 ± 1.11%), and 5 ng/ml (25.9 ± 2.98%) or 25 ng/ml (16.5 ± 0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ± 1.57%) and blastocyst formation (14.5 ± 1.28%) compared to the control group (65.8 ± 1.35% and 21.5 ± 0.97%, respectively). In 8–16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

C-X-C motif chemokine ligand 12 improves the developmental potential of bovine oocytes by activating SH2 domain-containing tyrosine phosphatase 2 during maturation†.

In vitro maturation of mammalian oocytes is an important means in assisted reproductive technology. Most bovine immature oocytes complete nuclear maturation, but less than half develop to the blastocyst stage after fertilization. Thus, inefficient in vitro production is mainly caused by a suboptimal in vitro culture process, in which oocyte quality appears to be the limiting factor. In our study, a potential maternal regulator, C-X-C motif chemokine ligand 12, was identified by analyzing transcriptome data. C-X-C motif chemokine ligand 12 supplementation promoted the developmental potential of oocytes by improving protein synthesis and reorganizing cortical granules and mitochondria during in vitro maturation, which eventually increased blastocyst formation efficiency and cell number after parthenogenesis, fertilization, and cloning. All these promoting effects by C-X-C motif chemokine ligand 12 were achieved by activating SH2 domain-containing tyrosine phosphatase 2, thereby promoting the mitogen-activated protein kinase signaling pathway. These findings provide an in vitro maturation system that closely resembles the maternal environment to provide high-quality oocytes for in vitro production.

O-234 Constant antioxidant supplementation increases blastocyst formation from oocyte donors

Abstract Study question Does constant supplementation of antioxidants to the culture media increase blastocyst formation from oocyte donors? Summary answer The constant supplementation of antioxidants to the culture media increases blastocyst formation from oocyte donors What is known already The use of a combination of various antioxidants in embryo culture media has recently been investigated to explore the potential benefit for embryo development and clinical outcomes. However, the concentration of antioxidants added to the culture has only been determined by its effect. In this study, we performed a repeated antioxidants supplementation to resemble a physiological oxidation reduction environment. Therefore, we investigated the effect of antioxidants added every 12 hours to the culture media on blastocyst formation and expansion in oocyte donors. Study design, size, duration This prospective study was conducted at CITMER, Mexico from April 2020 to November 2022.We included a total of 258 recipients from oocyte donors undergoing IVF/ICSI. Participants/materials, setting, methods A total of 2403 zygotes were divided into 4 groups and cultured in the following conditions until blastocyst stage: Group 1A: 563 zygotes 20% O2 with antioxidants every 12 hours, Group 1B: 1109 zygotes 20% O2 with antioxidants at the beginning, Group 2A: 339 zygotes 5% O2 with antioxidants every 12 hours, Group 2B: 392 zygotes 5% O2 with antioxidants at the beginning. Embryo development was assessed. Odds ratio and Fisher test were performed. p &amp;lt; 0.05=significant Main results and the role of chance For both oxygen tensions, we found a significant increase in the total blastocyst formation rate (day 5+day 6) when antioxidants were added repeatedly (1A: 54.7% vs 1B: 45.9%, p = 0.0007*; 2A: 58.7% vs 2A: 46.9%, p = 0.001*). In addition, the rate of expansion at days 5 + 6 was also significantly higher than in the groups where the antioxidants were added only at the beginning of the culture (1A: 35.9% vs 1B: 29.9%, p = 0.01*; 2A: 41.3% vs 2A: 31.6%, p = 0.006*). Limitations, reasons for caution Given this is a sibling zygotes study, patients are their own controls. Wider implications of the findings Constant supplementation of antioxidants to the culture media increases blastocyst formation from oocyte donors as well as the expansion rate, which may significantly improve clinical outcomes. Trial registration number 06162020-aox04

Developmental competence of IVF and SCNT goat embryos is improved by inhibition of canonical WNT signaling.

The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8-16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development.

The Effects of an In Vitro Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs

The aim of this study was to improve the production efficiency of Vietnamese native Ban pig embryos using somatic cell nuclear transfer (SCNT). Fibroblast cells from Ban pigs were injected into the enucleated cytoplasts of crossbred gilts, and the reconstructed embryos were subsequently cultured. In the first experiment, cytoplasts were isolated from oocytes matured in either a defined porcine oocyte medium (POM) or in TCM199 medium supplemented with porcine follicular fluid. Both media were supplemented with gonadotropic hormones, either for the first 22 h of in vitro maturation (IVM) or for the entire 44 h of IVM. In the second experiment, the reconstructed SCNT embryos were cultured with or without 50 μM chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.

Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

Maslinic Acid Supplementation during the In Vitro Culture Period Ameliorates Early Embryonic Development of Porcine Embryos by Regulating Oxidative Stress

As a pentacyclic triterpene, MA exhibits effective free radical scavenging capabilities. The purpose of this study was to explore the effects of MA on porcine early-stage embryonic development, oxidation resistance and mitochondrial function. Our results showed that 1 μM was the optimal concentration of MA, which resulted in dramatically increased blastocyst formation rates and improvement of blastocyst quality of in vitro-derived embryos from parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Further analysis indicated that MA supplementation not only significantly decreased the abundance of intracellular reactive oxygen species (ROS) and dramatically increased the abundance of intracellular reductive glutathione (GSH) in porcine early-stage embryos, but also clearly attenuated mitochondrial dysfunction and inhibited apoptosis. Moreover, Western blotting showed that MA supplementation upregulated OCT4 (p < 0.01), SOD1 (p < 0.0001) and CAT (p < 0.05) protein expression in porcine early-stage embryos. Collectively, our data reveal that MA supplementation exerts helpful effects on porcine early embryo development competence via regulation of oxidative stress (OS) and amelioration of mitochondrial function and that MA may be useful for increasing the in vitro production (IVP) efficiency of porcine early-stage embryos.

A combination containing natural extracts of clove, Sophora flower bud, and yam improves fertility in aged female mice via multiple mechanisms.

With society development, the age at which women choose to have children has been gradually delayed. To improve the reduced fertility in women at advanced maternal age, we developed a combination containing natural extracts from clove, Sophora flower bud and Chinese yam with a mass ratio 15:6:10 and named it as DACHAO. We then gavage DACHAO at a dose of 310 mg/kg BW to female mice at 10 month of age and investigated its effects on ovarian functions. Using MitoTracker probes, ROS, and JC-1 staining, we found that DACHAO treatment improved mitochondria functions in oocytes from aged mice. We also observed increased blastocyst formation when mature oocytes from control and DACHAO treated mice were for IVF and in vitro embryo culture. Cell counting and TUNEL assay further revealed increased cell numbers and decreased apoptosis in blastocysts of DACHAO group. After control or DACHAO treated mice being mated with fertile male mice, fertility test revealed a greater first litter size in the DACHAO group. Further studies demonstrated that DACHAO treatment could alleviate the retarded ovarian function in aged mice via changes in serum hormone levels, over-expression of antioxidant factors, under-expression of inflammation-related factors, and reduced apoptosis in the ovaries. Thus, the new combination DACHAO will be a good choice in clinic to improve ovarian functions for women at advanced maternal age.

Exogenous L-Glutathione Improves Vitrification Outcomes in Murine Preimplantation Embryos.

Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6-8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p &lt; 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p &lt; 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.

Oroxin A reduces oxidative stress, apoptosis, and autophagy and improves the developmental competence of porcine embryos in vitro.

Oroxin A (OA) is a flavonoid isolated from Oroxylum indicum (L.) Kurz that has various biological activities, including antioxidant activities. This study aimed to examine the viability of using OA in an in vitro culture (IVC) medium for its antioxidant effects and related molecular mechanisms on porcine blastocyst development. In this study, we investigated the effects of OA on early porcine embryo development via terminal deoxynucleotidyl transferase dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, quantitative reverse transcription PCR, and immunocytochemistry. Embryos cultured in the IVC medium supplemented with 2.5μM of OA had an increased blastocyst formation rate, total cell number, and proliferation capacity, along with a low apoptosis rate. OA supplementation decreased reactive oxygen species levels while increasing glutathione levels. OA-treated embryos exhibited an improved intracellular mitochondrial membrane potential and reduced autophagy. Moreover, levels of pluripotency- and antioxidant-related genes were upregulated, whereas those of apoptosis- and autophagy-related genes were downregulated by OA addition. In conclusion, OA improves preimplantation embryonic development by reducing oxidative stress and enhancing mitochondrial function.

O-063 Impact of group embryo culture vs individual embryo culture strategies on blastocyst rate and quality

Abstract Study question Does group embryo culture improve blastocyst formation compared to individual culture? Summary answer Individual culture provides an increased blastocyst formation rate. What is known already Embryo culture is one of the most important steps of an IVF treatment. Several studies pointed out that group embryo culture could help embryos with lower potential, through paracrine signaling, to achieve development and improve blastocyst formation rate. On the other hand, this issue continues to be controversial and other studies did not find differences in embryo development when using either method. Study design, size, duration A prospective randomized study was performed between January 2020 and December 2021. The data were obtained from 830 embryos that came from 103 egg donation treatments. We defined two groups: individual vs group culture. The number of embryos analyzed in the study was calculated to test a difference of 10% in the rate of blastocyst formation between the two groups. Participants/materials, setting, methods A total of 103 ICSI-egg donation cycles with normozoospermic males (fresh samples) were included. After fertilization assessment, zygotes were randomized into two groups; Group 1: individual culture (399 embryos), and Group 2: culture of 2-5 embryos per drop (431 embryos). The embryos were cultured in 35 µl drops of Global Total medium until day 5 when they were classified morphologically according to Gardner criteria (1998). The statistical analysis was performed using SPSS (version 20.0). Main results and the role of chance The rate of high-quality embryos on day 3 were similar in Group 1 compared to Group 2 (79.2% vs 81.4%, p = 0.432). However, we observed an increase in the blastocyst formation rate on day 5 (62.9% vs 56.1%, p = 0.048) and a trend for higher embryo quality (A/B blastocysts) (55.9% vs 52%, p = 0.266) in Group 1; compared to Group 2, respectively. Our data suggest that individual culture increases blastocyst formation rate and may benefit embryo quality on day 5. Our results challenge previous reports suggesting that culture in groups can improve embryo development. As hypothesis, we argue that some of the components that the embryos release to the culture environment during their division may affect the quality of the cohort potentially compromising further development up to the blastocyst stage. Limitations, reasons for caution The study is focused on laboratory outcomes until the blastocyst stage. Clinical outcomes were not analyzed. Wider implications of the findings Finding the most appropriate embryo culture conditions is essential to improve ART results. Time-lapse systems should be adapted to the use of both individual and group culture dishes. Larger randomized studies are needed in order to reach robust conclusions on the subject. Trial registration number NA

Supplementation with kaempferol relieves oxidative stress and enhances development of early bovine embryos in vitro.

Oxidative stress (OS) has been considered the principle cause of developmental failure of early embryos cultured in vitro; therefore, the addition of antioxidants is very important for improving in vitro culture (IVC) systems. Various antioxidants have been tested for IVC systems, but most have exhibited some side effects. Kaempferol (3,5,7-trihydroxy-2-[4-hydroxyphenyl]-4h-1-benzopyran-4-one, KAE) is a flavonoid with strong antioxidant activity and no obvious side effects. This study explored the effect of KAE on antioxidant capacity and developmental competence of bovine embryos after fertilization. KAE was added to bovine IVC medium and significantly reduced reactive oxygen species (ROS) in 2-, 4- and 8-cell stage embryos and increased blastocyst formation. In addition, the level of H3K9ac was increased, the apoptotic index was reduced and total cell numbers and trophectoderm cell numbers in day 7 blastocysts were increased significantly in KAE-treated embryos compared to control. Expression of the apoptotic gene, Bcl-2, was higher in blastocysts after KAE treatment, while expression of the endoplasmic reticulum (ER) stress genes, Bip and HDAC1, and the pro-apoptotic gene, Bax, were significantly lower in the KAE group. Thus, KAE significantly reduced ROS damage and improved development of IVC bovine embryos.

Effects of astaxanthin supplementation in fertilization medium and/or culture medium on the fertilization and development of mouse oocytes.

Objective We investigated the effect of supplementing fertilization medium and/or culture medium with astaxanthin (AST) on the two phases of in vitro fertilization: gamete fertilization and embryo development.Methods Mouse cumulus-oocyte complexes were divided into four groups with 5 µM AST added to the fertilization medium (group 3, n=300), culture medium (group 2, n=300), or both media (group 4, n=290). No AST was added to the control group (group 1, n=300).ResultsThe fertilization rate was significantly higher (p<0.001) in the groups using AST supplemented fertilization medium (group 3, 79.0%; group 4, 81.4%) than those without AST (group 1, 56.3%; group 2, 52.3%). The blastocyst rate calculated from the two-cell stage was significantly lower (p<0.001) in the groups using AST-supplemented embryo culture medium (group 2, 58.0%; group 4, 62.3%) than in those without AST (group 1, 82.8%; group 3, 79.8%). The blastocyst rate calculated from the number of inseminated oocytes was highest in group 3 (189/300, 63.0%) and lowest in group 2 (91/300, 30.3%) with statistical significance compared to other groups (p<0.001). There were significantly higher numbers of cells in the inner cell mass and trophectoderm, as well as significantly higher total blastocyst cell counts, in group 3 than in the control group.ConclusionAn increased blastocyst formation rate and high-quality blastocysts were found only in the fertilization medium that had been supplemented with AST. In contrast, AST supplementation of the embryo culture medium was found to impair embryo development.

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

The effects of embryo splitting on Cdx2, Sox2, Oct4, and Nanog gene expression in mouse blastocysts.

Embryo splitting is utilized in reproduction biotechnology. The blastomeres resulting from the splitting of an embryo in two-, four- or eight-cell stages can develop into separate embryos that are genetically similar to the other blastomeres. The present work studied the effects of splitting on embryo pluripotent gene expression (Cdx2, Sox2, Oct4, and Nanog) in mice. Two-cell embryos were isolated from stimulated mice. The embryos were grouped into "split" and "non-split" groups. The zona pellucida was removed from the split group and the blastomeres were distributed before being co-cultured with mouse embryo fibroblasts to the blastocyst stage. Normal (non-split) blastocysts were co-cultured in the same way. The 3.5-day-old blastomeres were collected as the control group. For molecular evaluation, real-time PCR was conducted to analyze changes in Cdx2, Sox2, Oct4, and Nanog gene expression. Moreover, the blastocyst formation rate, overall blastocyst rate, and the number of newborns were statistically analyzed. The findings showed that embryo splitting increased blastocyst formation, overall blastocysts, developmental potential embryos, and the number of infants. Furthermore, the split and non-split (control) groups showed equal expression of pluripotent genes (Cdx2, Sox2, Oct4, and Nanog) in the molecular analysis. It can be concluded that the growth and developmental potency of sister blastocysts derived from split two-cell stage mouse embryos are the same as those of normal blastocysts. So, there are no significant differences in gene expression between the split and non-split groups.

Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos.

The oocyte maturation process requires a high supply of energy, which generates reactive oxygen species (ROS), adversely affecting oocyte and embryo development. Balancing ROS by antioxidant supplementation is essential for maintaining oocyte maturation and embryonic quality in vitro. This study aimed to evaluate the impact of four antioxidants: β-mercaptoethanol (β-ME), chlorogenic acid (CGA), curcumin and sericin, when applied individually or in combinations, during oocyte maturation on development of porcine oocytes. Cumulus-oocyte complexes were collected, cultured in maturation medium supplemented with antioxidants for 44hr and subsequently subjected to in vitro fertilization (IVF) and culture for 7days. Combining all four (β-ME+CGA+curcumin+sericin) or three (β-ME+CGA+curcumin) antioxidants increased blastocyst formation rates. However, sericin supplementation alone, or in combination with β-ME or CGA, failed to improve blastocyst formation rates. The total cell numbers of blastocysts from the group supplemented with three antioxidants (β-ME+CGA+curcumin) were significantly higher than those from the other groups, except for the curcumin-supplement group. There were no differences in the maturation rates and proportions of oocytes with fragmented DNA between the antioxidant-supplemented and the non-supplemented control groups. In conclusion, supplementation with three antioxidants (β-ME+CGA+curcumin) during the maturation culture enhanced blastocyst formation and improved blastocyst quality.

Oviduct epithelial cells-derived extracellular vesicles improve preimplantation developmental competence of in vitro produced porcine parthenogenetic and cloned embryos.

Extracellular vesicles (EVs) carry bioactive cargoes involved in the early preimplantation development. This study investigated the effects of EVs obtained from an oviductal epithelial cell (OEC) conditioned medium on the developmental competence of in parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) porcine embryos. The OEC-EV-treated group showed significant increases in blastocyst formation and hatching rates compared to the control group (40.8% ± 2.2% and 20.1% ± 2.1% vs. 24.9% ± 2.0% and 5.3% ± 1.1%; p < 0.05), respectively. The 7 day OEC-EVs treatment group significantly increased blastocyst formation rate than the 3 day and 0 day-groups (45.0 ± 0.8 vs. 33.0 ± 0.7 and 26.7 ± 0.5; p < 0.05), respectively. SCNT revealed that the OEC-EV increased blastocyst formation rate compared to that of oviductal fluid EVs (OF-EVs) (35.4% ± 1.4% vs. 29.3% ± 1.3%; p < 0.05). Reactive oxygen species levels, apoptosis, and blastocyst lipid content were significantly decreased in the OEC-EVs group compared with the control group. OEC-EV group showed a significantly decreased BAX and increased BCL2, SOD1, POU5F1, SOX2, NANOG, GATA6, PNPLA2, LIPE, and MGLL gene expression than the control group (p < 0.05). In conclusion, OEC-EVs supplementation in embryo culture media improved the quality of porcine embryos, potentially helping porcine-cloned embryonic development possibly through transfer of messenger RNA and proteins to the early embryos.

Tricostatin A-treated round spermatid enhances preimplantation embryo developmental competency following round spermatid injection in mice.

It has been documented that the inefficacy of round spermatid injection (ROSI) might be caused by abnormal epigenetic modifications. Therefore, this study aimed to evaluate the effect of trichostatin A (TSA) as an epigenetic modifier of preimplantation embryo development in activated ROSI oocytes. Matured oocytes were collected from superovulated female mice. Testes were placed in human tubal fluid medium and masses were then cut into small pieces to disperse spermatogenic cells. Round spermatids were treated with TSA and subsequently injected into oocytes. The expression level of the development-related genes including Oct4, Sox2, Nanog, Dnmt and Hdac transcripts were evaluated using qRT-PCR. Immunohistochemistry was performed to confirm the presence of Oct-4 protein at the blastocyst stage. There was no statistically significant difference in fertilization rate following ROSI/+TSA compared with the non-treated ROSI and intracytoplasmic sperm injection (ICSI) groups. Importantly, TSA treatment increased blastocyst formation from 38% in non-treated ROSI to 68%. The relative expression level of developmentally related genes increased and Dnmt transcripts decreased in ROSI/+TSA-derived embryos, similar to the expression levels observed in the ICSI-derived embryos. In conclusion, our results indicate that spermatid treatment with TSA prior to ROSI would increase the success rate of development to the blastocyst stage and proportion of pluripotent cells.