Blastocyst Activation Research Articles

Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development.

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.

DNA Methylation and Histone Modification Are the Possible Regulators of Preimplantation Blastocyst Activation in Mice.

Under ovarian hormone control, dormant blastocysts obtain implantation capacity (known as blastocyst activation) through their global gene expression. After the activated blastocysts communicate with the receptive uterus, the implantation-competent blastocysts start the implantation. Although dormant and activated blastocysts have different gene expression levels, the regulatory mechanisms underlying these transcriptions remain unclear. Hence, this study aimed to analyze epigenetic marks in dormant and activated blastocysts. In mice, blastocyst dormancy is artificially induced by daily progesterone injection without estrogen supplementation after peri-implantation ovariectomy; when estrogen is administered concomitantly, blastocyst activation and implantation occur. These phenomena demonstrate a mouse model of delayed implantation. We collected dormant and activated blastocysts from a delayed implantation mouse model. RNA-seq, methylated DNA immunoprecipitation (MeDIP)-seq, and chromatin immunoprecipitation (ChIP)-seq for H3K4 me3 and H3K27 me3 were performed using dormant and activated blastocysts. Cell cycle-related transcripts were affected during blastocyst activation. DNA methylations were accumulated in downregulated genes in the activated blastocysts. Histone H3 trimethylations were globally altered between the dormant and activated blastocysts. Dormant and activated blastocysts have unique methylation patterns on DNA and histone H3, with high correlation to gene expression. DNA methylation and histone modification can regulate preimplantation blastocyst activation.

Astaxanthin Supplementation Improves the Subsequent Developmental Competence of Vitrified Porcine Zygotes.

Cryopreservation of embryos has been confirmed to cause oxidative stress as a factor responsible for impaired developmental competence. Currently, astaxanthin (Ax) raises considerable interest as a strong exogenous antioxidant and for its potential in reproductive biology. The present study aimed to investigate the beneficial effects of Ax supplementation during in vitro culture of vitrified porcine zygotes and the possible underlying mechanisms. First, the parthenogenetic zygotes were submitted to vitrification and then cultured in the medium added with various concentrations of Ax (0, 0.5, 1.5, and 2.5 μM). Supplementation of 1.5 μM Ax achieved the highest blastocyst yield and was considered as the optimal concentration. This concentration also improved the blastocyst formation rate of vitrified cloned zygotes. Moreover, the vitrified parthenogenetic zygotes cultured with Ax exhibited significantly increased mRNA expression of CDX2, SOD2, and GPX4 in their blastocysts. We further analyzed oxidative stress, mitochondrial and lysosomal function in the 4-cell embryos and blastocysts derived from parthenogenetic zygotes. For the 4-cell embryos, vitrification disturbed the levels of reactive oxygen species (ROS) and glutathione (GSH), and the activities of mitochondria, lysosome and cathepsin B, and Ax supplementation could fully or partially rescue these values. The blastocysts obtained from vitrified zygotes showed significantly reduced ATP content and elevated cathepsin B activity, which also was recovered by Ax supplementation. There were no significant differences in other parameters mentioned above for the resultant blastocysts. Furthermore, the addition of Ax significantly enhanced mitochondrial activity and reduced lysosomal activity in resultant blastocysts. In conclusion, these findings revealed that Ax supplementation during the culture period improved subsequent embryonic development and quality of porcine zygotes after vitrification and might be used to ameliorate the recovery culture condition for vitrified embryos.

Fibrin-mediated growth restriction of early-stage human trophoblasts is switched to growth promotion through fibrinolysis.

Does fibrin promote trophoblast growth in human and mouse blastocysts during early embryo implantation? Mouse blastocysts were unaffected by fibrin; however, human blastocysts were significantly suppressed by fibrin in trophoblast growth and then switched to growth promotion through increased fibrinolysis with urokinase-type plasminogen activator (uPA) activity. Fibrin(ogen) plays an important role in various physiological processes and is also critical for maintaining feto-maternal attachment during pregnancy. The addition of fibrin to embryo transfer media has been used to increase implantation rates in human ART; however, its mechanism of action' in vitro has not yet been characterized. Vitrified mouse and human blastocysts were warmed and individually cultured in vitro for up to 120 and 168 h, respectively, on a fibrin substrate. Blastocysts were cultured at 37°C in 6% CO2, 5% O2 and 89% N2. Blastocyst development and related fibrinolytic factors were analyzed. ICR strain mouse embryos were purchased from a commercial supplier. Human blastocysts were donated with informed consent from two fertility centers. Mouse and human blastocysts cultured on fibrin-coated plates were compared to those on non-coated and collagen-coated plates in vitro. Trophoblast growth and fibrin degradation were assessed based on the cell area and fibrin-free area, respectively. Fibrinolytic factors were detected in supernatants using plasminogen-casein zymography. The fibrinolytic activity of blastocysts was investigated using a selective uPA inhibitor, exogenous uPA, plasminogen activator inhibitor-1 (PAI-1) inhibitor and fibrin degradation products (FDPs). Fibrinolysis-related mRNA expression level was detected using quantitative real-time PCR. Fibrin did not affect the developmental speed or morphology of mouse blastocysts, and a large fibrin-degrading region was observed in the attachment stage. In contrast, fibrin significantly suppressed the outgrowth of trophoblasts in human blastocysts, and trophoblasts grew after the appearance of small fibrin-degrading regions. uPA was identified as a fibrinolytic factor in the conditioned medium, and uPA activity was significantly weaker in human blastocysts than in mouse blastocysts. The inhibition of uPA significantly reduced the outgrowth of trophoblasts in mouse and human blastocysts. Human blastocysts expressed PLAU (uPA), PLAUR (uPA receptor), SERPINE1 (PAI-1) and SERPINB2 (PAI-2), whereas mouse blastocysts were limited to Plau, Plaur and Serpine1. In a subsequent experiment on human blastocysts, the addition of exogenous uPA and the PAI-1 inhibitor promoted trophoblast growth in the presence of fibrin, as did the addition of FDPs. This model excludes maternal factors and may not be fully reproduced in vivo. Donated human embryos are surplus embryos that may inherently exhibit reduced embryonic development. In addition, donated ART-derived embryos may exhibit weak uPA activity, because women with sufficient uPA-active embryos may not originally require ART. The present study used orthodox culture methods, and results may change with the application of recently developed protocols for culture blastocysts beyond the implantation stage. The present results suggest that the distinct features of trophoblast outgrowth in human blastocysts observed in the presence of fibrin are regulated by a phenotypic conversion induced by contact with fibrin and FDPs. Mouse embryos did not exhibit the human phenomenon, indicating that the present results may be limited to humans. The present study was supported by the Department of Obstetrics and Gynecology at the Hamamatsu University School of Medicine and Kishokai Medical Corporation. None of the authors have any conflicts of interest to declare. N/A.

Maturation of bovine oocytes under low culture temperature decreased glutathione peroxidase activity of both oocytes and blastocysts.

It is known that the basic variable in the cellular environment is temperature and low temperature decreases cellular metabolism rate. Also, low cellular metabolic activity reduces oxidative stress, resulting in low ROS production. The aim of this study was therefore to investigate the effect of 36.5°C (low) and 38.5°C (conventional) incubation temperatures during IVM on glutathione peroxidase activity of oocytes and blastocysts following fertilization. Bovine oocytes were matured in medium-199 for 22 hours at either 36.5°C or 38.5°C and they were subjected to in vitro fertilization (IVF). Putative zygotes were then transferred randomly into SOFaa embryo culture media with or without antioxidant (a mixture of GSH and SOD) until development to the blastocyst stage. Glutathione peroxidase enzyme (GSH-Px) activity was lower (p⟨0.05) in oocytes matured at low temperature than those of conventional temperature. Similarly, GSH-Px activity was lower (p⟨0.05) in blastocysts, which were obtained from oocytes matured at low temperature and cultured in antioxidants-supplemented embryo media. The GSH-Px activity of blastocysts, obtained from oocytes matured in low temperature, cultured in antioxidants-free embryo media was similar to blastocysts obtained from oocytes matured in conventional temperature, cultured in antioxidants-supplemented embryo media. The results of the present study show that decreasing the in vitro maturation temperature decreases antioxidant enzyme activity in both oocyte and blastocyst. Additionally, maturation of bovine oocytes at 36.5°C incubation temperature may provide an optimal thermal condition for the enzymatic antioxidant system of both oocytes and blastocyst.

Metabolic activity of human blastocysts correlates with their morphokinetics, morphological grade, KIDScore and artificial intelligence ranking.

Is there a relationship between blastocyst metabolism and biomarkers of embryo viability? Blastocysts with higher developmental potential and a higher probability of resulting in a viable pregnancy consume higher levels of glucose and exhibit distinct amino acid profiles. Morphological and morphokinetic analyses utilized in embryo selection provide insight into developmental potential, but alone are unable to provide a direct measure of embryo physiology and inherent health. Glucose uptake is a physiological biomarker of viability and amino acid utilization is different between embryos of varying qualities. Two hundred and nine human preimplantation embryos from 50 patients were cultured in a time-lapse incubator system in both freeze all and fresh transfer cycles. A retrospective analysis of morphokinetics, morphology (Gardner grade), KIDScore, artificial intelligence grade (EmbryoScore), glucose and amino acid metabolism, and clinical pregnancies was conducted. ICSI was conducted in all patients, who were aged ≤37 years and previously had no more than two IVF cycles. Embryos were individually cultured in a time-lapse incubator system, and those reaching the blastocyst stage had their morphokinetics annotated and were each assigned a Gardner grade, KIDScore and EmbryoScore. Glucose and amino acid metabolism were measured. Clinical pregnancies were confirmed by the presence of a fetal heartbeat at 6 weeks of gestation. Glucose consumption was at least 40% higher in blastocysts deemed of high developmental potential using either the Gardner grade (P < 0.01, n = 209), KIDScore (P < 0.05, n = 207) or EmbryoScore (P < 0.05, n = 184), compared to less viable blastocysts and in blastocysts that resulted in a clinical pregnancy compared to those that failed to implant (P < 0.05, n = 37). Additionally, duration of cavitation was inversely related to glucose consumption (P < 0.05, n = 200). Total amino acid consumption was significantly higher in blastocysts with an EmbryoScore higher than the cohort median score (P < 0.01, n = 185). Furthermore, the production of amino acids was significantly lower in blastocysts with a high Gardner grade (P < 0.05, n = 209), KIDScore (P < 0.05, n = 207) and EmbryoScore (P < 0.01, n = 184). Samples were collected from patients who had ICSI treatment and from only one clinic. These results confirm that metabolites, such as glucose and amino acids, are valid biomarkers of embryo viability and could therefore be used in conjunction with other systems to aid in the selection of a healthy embryo. Work was supported by Virtus Health. D.K.G is contracted with Virtus Health. The other authors have no conflict of interest to declare. N/A.

Transcriptome analysis of blastocysts acquiring implantation competency in mice

Blastocyst activation, referring to the blastocyst acquiring the implantation competency, is the determining factor for implantation into the receptive uterus. It involves the process of embryonic cell differentiation to contact and initiate dialog with the uterine cells. Before the application of -omics approaches to this biological event, only the cellular morphological changes and a small number of molecules were known to regulate this process. This review aims to discuss the recent exploration for global molecular changes during the blastocyst activation in mouse model, based on a transcriptional evaluation strategy.

A role for Msx genes in mammalian embryonic diapause.

Mammalian embryonic diapause is a reproductive phenomenon defined by the reversible arrest in blastocyst development and metabolic activity within the uterus which synchronously becomes quiescent to implantation. This natural strategy, evident in over 130 species across eight orders, can temporally uncouple conception from delivery until conditions are favorable for the survival of the mother and newborn. While the maternal endocrine milieu has been shown to be important for this process, the local molecular mechanisms by which the uterus and embryo achieve quiescence, maintain blastocyst survival and then resumes blastocyst activation with subsequent implantation in response to endocrine cues remains unclear. Here we review the first evidence that the proximal molecular control of embryonic diapause is conserved in three unrelated mammalian species which employ different endocrine programs to initiate diapause. In particular, uterine expression of muscle segment homeobox (Msx) genes Msx1 or Msx2 persists during diapause, followed by downregulation with blastocyst reactivation and implantation. Mice (Mus musculus) with conditional inactivation of Msx1 and Msx2 in the uterus fail to achieve diapause and reactivation. Remarkably, the mink (Neovison vison) and tammar wallaby (Macropus eugenii) share this pattern of MSX1 or MSX2 expression as in mice during delay - it persists during diapause and is rapidly downregulated upon implantation. Therefore, these findings were the first to provide evidence that there are common conserved molecular regulators in the uterus of unrelated mammals during embryonic diapause.

86 Effect of invitro culture conditions on mitochondria functions in mouse embryos

Mitochondria provide the energy for oocyte maturation, fertilisation, and embryo formation via oxidative phosphorylation. Consequently, any adverse influence on mitochondrial function may negatively affect the development of pre-implantation embryos especially because there is no mitochondrial DNA (mtDNA) replication until post-implantation. Studies in the field of mitochondrial dynamics have identified an intriguing link between energy demand/supply balance and mitochondrial architecture, which may suggest that inappropriate culture conditions may inhibit mitochondrial functions, which may negatively affect embryo development. We wanted to check whether invitro culture (IVC) conditions of mouse embryos affect mitochondrial functionality. The IVC as well as naturally matted (NM) mouse embryos at the 2-cell and blastocyst stage were subjected to mitochondrial analysis (distribution, organisation, and mitochondrial membrane potential), and expression of mRNA and proteins involved in regulation of mitochondria functions, as well as number of mtDNA copies, were evaluated. Significance level was set at 0.05. We observed that the mitochondria in 2-cell IVC embryos were less numerous and localised mainly in the pericortical region of the cytoplasm, whereas mitochondria in NM embryos were numerous and homogeneously distributed in both blastomeres. Drastic differences were observed in blastocysts. Mitochondria in the IVC group were fragmented, rounded, and aggregated mainly in the perinuclear region of the cells, whereas mitochondria of NM blastocysts were numerous and created an elongated mitochondrial network along the cells. Time-lapse analysis showed reduced mitochondrial and mitochondrial membrane activity in IVC blastocysts. Moreover, our results indicate the IVC group had reduced mRNA expression of mitofusin 1, mitofusin 2, and optic atrophy 1 responsible for mitochondrial fusion. Additionally, mtDNA copy number for IVC blastocysts (398 887.45±30 608.65) was significantly lower than that of NM blastocysts (593 367.12±66 540.32; P&amp;lt;0.02). Furthermore, no significant differences were found in mtDNA copy number of IVC 2-cell embryos when compared with NM embryos. The results obtained clearly showed that IVC conditions affect proper mitochondrial functionality and hence embryo development.

Minimally invasive uterine aspiration 24 hours ahead of embryo transfer characterizes the compromised RIF uterine microenvironment and is predictive of reproductive outcome

Repeat implantation failure (RIF) is particularly challenging to treat in ART, resulting in limited success even when adequate preparation of the endometrium is established and a transfer is performed with a high grade euploid blastocyst. The objective of this study was to utilize a multidisciplinary approach to decipher the complexity of RIF through investigations of the maternal molecular components ahead of an embryo transfer. Research study. Patients were recruited with IRB consent 24 hours prior to a programmed frozen embryo transfer (FET) with a euploid blastocyst. Uterine secretions were collected by gentle aspiration (∼2-5ul) under ultrasound guidance and grouped according to reproductive outcomes: Failed euploid FET (RIF patients, ≥3 prior IVF failures) and Positive live birth FET (maternally age-matched patients; mean 36.6 ±3.8 years). Total and small RNA (n = 22) was isolated for sequencing on the NovaSEQ 6000 (Illumina). Reads were aligned to hg38 using GSNAP and analyzed with edgeR (FDR cutoff of 5%; P<0.01). Metabolite analysis (n = 20) was performed by UHPLS-MS (Thermo) using MassMatrix and Maven (Princeton Univ). Proteomic analysis (n = 6) involved FASP digestion and LC-MS/MS, with protein identifications generated by Mascot (v 2.6) and Scaffold (v 4.8.9) (α of 0.05; fold change >1.5 or <0.5). A unique uterine microenvironment was observed for RIF patients and negative implantation outcomes 24 hours prior to an embryo transfer (P<0.05). An interplay of several biological processes were evident in RIF failed aspirates with a focused interest on 13 significantly reduced transcripts, 7 significantly increased maternal miRNAs, 12 significantly decreased amino acids and 16 proteins of significantly altered abundance (P<0.05). Specific examples included decreased expression of PLA2G4D (P<0.0001) which regulates the eicosanoid pathway, thereby impacting downstream synthesis of prostaglandins like PGE2. Decreased expression of TET1 (P<0.0001), an epigenetic regulator required for DNA methylation. Increased expression of miR-17, a known negative regulator of VEGFA, required for successful implantation (P<0.01). Decreased quantities of arginine, essential for blastocyst activation and trophectoderm motility (P<0.05). Lastly, an increased abundance of SERPING1, a protein associated with inflammation, which regulates complement activation (P<0.05). Analysis of uterine secretions 24 hours prior to FET, allowed for an in-depth molecular characterization of the compromised RIF uterine microenvironment and was predictive of reproductive outcome. The negative influence on key miRNAs and gene transcription levels, in addition to altered amino acid and protein concentrations, were all identified as critical contributors to poor RIF outcomes. Ongoing investigations into the relationships of these molecular networks will lead to the possibility of more effective clinical interventions for this difficult patient population.

Blastocyst activation engenders transcriptome reprogram affecting X-chromosome reactivation and inflammatory trigger of implantation

Implantation of the blastocyst into the uterus is the gateway for further embryonic development in mammals. Programming of blastocyst to an implantation-competent state known as blastocyst activation is the determining factor for implantation into the receptive uterus. However, it remains largely unclear how the blastocyst is globally programmed for implantation. Employing a delayed implantation mouse model, we show here that the blastocyst undergoes extensive programming essential for implantation. By analyzing the transcriptional profile of blastocysts with different implantation competency, we reveal the dynamic change in the biosynthesis, metabolism, and proliferation during blastocyst reactivation from diapause. We also demonstrate that reactivation of the X chromosome, one of the most important events during periimplantation of female embryonic development, is not completed even in blastocysts under conditions of dormancy, despite long term suspension in the uterus. Moreover, the mural trophectoderm (TE), but not the polar TE, differentiates to be more invasive through the weakened cell-cell tight junctions and extracellular matrices (ECMs). By analyzing the differentially expressed profile of secretory proteins, we further demonstrate that the blastocyst functions as a proinflammatory body to secrete proinflammatory signals, such as TNFα and S100A9, thereby triggering embryo-uterine attachment reaction during implantation. Collectively, our data systematically and comprehensively disclose the programming of blastocyst reactivation from diapause for implantation and uncover previously undefined roles of blastocyst during implantation.

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P&amp;lt;0.05. We have shown that CDS FBS significantly improved (P&amp;lt;0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P&amp;lt;0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P&amp;lt;0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Ice nucleating agents allow embryo freezing without manual seeding

Embryo slow freezing protocols include a nucleation induction step called manual seeding. This step is time consuming, manipulator dependent and hard to standardize. It requires access to samples, which is not always possible within the configuration of systems, such as differential scanning calorimeters or cryomicroscopes. Ice nucleation can be induced by other methods, e.g., by the use of ice nucleating agents. Snomax is a commercial preparation of inactivated proteins extracted from Pseudomonas syringae. The aim of our study was to investigate if Snomax can be an alternative to manual seeding in the slow freezing of mouse embryos. The influence of Snomax on the pH and osmolality of the freezing medium was evaluated. In vitro development (blastocyst formation and hatching rates) of fresh embryos exposed to Snomax and embryo cryopreserved with and without Snomax was assessed. The mitochondrial activity of frozen-thawed blastocysts was assessed by JC-1 fluorescent staining. Snomax didn't alter the physicochemical properties of the freezing medium, and did not affect embryo development of fresh embryos. After cryopreservation, the substitution of manual seeding by the ice nucleating agent (INA) Snomax did not affect embryo development or embryo mitochondrial activity. In conclusion, Snomax seems to be an effective ice nucleating agent for the slow freezing of mouse embryos. Snomax can also be a valuable alternative to manual seeding in research protocols in which manual seeding cannot be performed (i.e., differential scanning calorimetry and cryomicroscopy).

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births.

SUMMARYMost in vitro culture conditions are less‐than‐optimal for embryo development. Here, we used a transcriptional‐profiling database to identify culture‐induced differences in gene expression in porcine blastocysts compared to in vivo‐produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria‐related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro‐produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246–258, 2016. © 2016 The Authors.

MicroRNA and Embryo Implantation.

In mammals, implantation involves interactions between an activated blastocyst and a receptive endometrium. There are controversies on the role of microRNAs in preimplantation embryo development. The actions of endometrial microRNAs on implantation are beginning to be understood. Review of literature on microRNAs in preimplantation embryos and endometrium. Emerging evidence suggests a role of microRNAs in blastocyst activation and implantation. Differential expression of microRNAs is found between receptive and non-receptive endometria. Members of the let-7, miR-200, miR-30 families, and the miR-17-92 clusters are more commonly found to be associated with endometrial receptivity. Experimental studies show that the targets of the differentially expressed microRNAs affect endometrial receptivity, decidualization, and embryo implantation. Free and exosome/microvesicle containing microRNAs have been detected in human and ovine uterine luminal fluid (ULF). They may serve as mediators of embryo-endometrium dialog. Some observations suggest that the microRNAs in ULF may be used as biomarkers in infertility treatment. MicroRNAs in endometrium and blastocysts are involved in the implantation process.

Update of Wnt signaling in implantation and decidualization

Embryonic development into an implantation-competent blastocyst, synchronized uterine transformation into a receptive stage, and an intimate cross-talk between the activated blastocyst and the receptive uterus are essential for successful implantation, and therefore for subsequent pregnancy outcome. Evidence accumulating during recent years has underlined the importance of the Wnt signaling pathway in mammalian implantation and decidualization. Herein, this review focuses on the current state of knowledge regarding Wnt signaling in multiple implantation and decidualization events: pre-implantation embryo development, blastocyst activation for implantation, uterine development, and decidualization.

In vitro Development of Chimeric Bovine Embryos Created by Combining a Single Blastomere of SCNT Embryos with an IVF Embryo

In this study, we produced chimeric embryos from a single blastomere of somatic cell nuclear transfer (SCNT) bovine embryo and in-vitro fertilized (IVF) bovine embryo, and examined the rate of blastocyst development and the contribution of the SCNT blastomere to the blastocyst in order to improve cloning efficiency. We produced SCNT embryos from bovine fibroblasts carrying a luciferase gene under the control of the β-actin promoter. At 96 hours post fusion, chimeric embryos were produced by transferring a single blastomere of a 16-cell SCNT embryo to the perivitelline space of an IVF embryo. At 4 days after production of the chimeric embryos, half of the embryos reached the blastocyst stage, which is the same as that of IVF embryos. Furthermore, luciferase activity in blastocysts from the chimeric embryos was detected in both the inner cell mass and

UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst.

The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development.