scholarly journals Does miRNA Expression in the Spent Media Change During Early Embryo Development?

2021 ◽  
Vol 8 ◽  
Author(s):  
Paul Del Rio ◽  
Pavneesh Madan
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Rna Extraction ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Total Rna ◽  
Implantation Period ◽  
Spent Media

Distinct miRNA populations have been detected in the spent media of in-vitro culture systems. However, profiling has been limited to media conditioned with blastocyst-stage embryos. Therefore, the aim of the study was to profile extracellular miRNAs throughout the pre-implantation period in bovine embryos. To achieve this, cumulus oocyte complexes were aspirated from ovaries, in-vitro matured, fertilized, and cultured under standard laboratory procedures to the 2-cell, 8-cell, or blastocyst stage of development. At each developmental stage, 25 μl of spent in-vitro culture media was collected, pooled to 300 μl, and processed for total RNA extraction. In-vitro culture media, which never came in contact with any embryos, were additionally processed for total RNA extraction to serve as a negative control. Following hybridization on a GeneChip miRNA 4.0 array, comparative analysis was conducted between spent media and control samples. In total, 111 miRNAs were detected in the spent media samples, with 56 miRNAs identified in blastocyst spent media, 14 miRNAs shared between 8-cell and blastocyst spent media, 7 miRNAs shared between all 3 conditions, and 6 miRNAs exclusive to 2-cell spent media. miRNA-mRNA target prediction analysis revealed that the majority of genes predicted to be regulated by the miRNAs identified in the study have roles in cellular process, metabolic process, and biological regulation. Overall, the study suggest that miRNAs can be detected in the spent media of in-vitro culture system throughout the pre-implantation period and the detected miRNAs may influence genes impacting early embryo development.

200 EFFECT OF EXOGENOUS PROGESTERONE DURING IN VITRO CULTURE ON EARLY EMBRYO DEVELOPMENT IN CATTLE

2008 ◽  
Vol 20 (1) ◽  
pp. 179
Author(s):  
M. Clemente ◽  
P. Lonergan ◽  
C. Borque ◽  
J. de La Fuente ◽  
D. Rizos
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Mineral Oil ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Significant Difference ◽  
The Media

Preimplatation embryos grown in vitro are sensitive to their environment, and the conditions of culture can affect developmental potential. Progesterone (P4) is the key hormone responsible for maintenance of pregnancy in mammals, and circulating levels in the early postconception period have been associated with pregnancy success. It is not clear whether P4 acts directly or indirectly on the embryo to alter gene expression and development. The aim of this study was to assess the effect of varying levels of exogenous P4 on the development of bovine zygotes to the blastocyst stage in vitro. A preliminary study was conducted to analyze the media used for culture (stock of P4, SOF, SOF + 1 × 10–7 M P4) on Days 1 (day of culture), 4, and 7 for P4 concentration in 25-μL droplets overlain with mineral oil or 500 μL in wells with or without mineral oil. P4 was measured using an ELISA kit, prepared for human serum or plasma (DE1561 Dimeditec Diagnostics GmbH, Kiel, Germany). Inter- and intra-assay coefficients of variation were 6.63 and 6.42%, respectively, and recovery was 95%. P4 concentration on Day 1 in all media was the expected (40 ng mL–1). However, on Days 4 and 7 in media under mineral oil, the level of P4 was nearly zero (0.1 to 1.6 ng mL–1) compared with the media without mineral oil, which remained unchanged (39 to 40 ng mL–1) through the 7 days of culture. Zygotes (n = 1467) were produced in 8 replicates by in vitro oocyte maturation and fertilization, and were cultured in groups of 40 to 50 in wells of 500 μL without mineral oil in (1) SOF supplemented with 5% fetal calf serum (control–), (2) SOF with ethanol (control+), (3) SOF with P4 0.1 × 10–7 M, (4) SOF with P4 1 × 10–7 M, and (5) SOF with P4 10 × 10–7 M at 39°C, 5% CO2 and 5% O2, with maximum humidity. No significant difference was found between groups in cleavage rate or blastocyst yield on Days 6, 7, and 8 (Table 1). These results indicate that the addition of P4 to the in vitro culture medium (SOF) did not enhance the development of bovine embryos to the blastocyst stage. However, further studies on the quality of these embryos in terms of gene expression are in preparation. Table 1. Effect of P4 on bovine in vitro early embryo development

86 EFFECT OF LACTATION ON EMBRYO DEVELOPMENT DURING THE POSTPARTUM PERIOD IN DAIRY COWS

2012 ◽  
Vol 24 (1) ◽  
pp. 155 ◽  
Author(s):  
V. Maillo ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Garrett ◽  
A. K. Kelly ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Embryo Development ◽  
Metabolic Profile ◽  
Reproductive Tract ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blood Samples ◽  
Early Embryo Development ◽  
Lactating Cows

The aim of this study was to examine the effect of lactation and associated metabolic profiles on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one age-matched primiparous Holstein cows were used. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. To characterise the metabolic profile of the cows, jugular blood samples were taken twice weekly starting 15 days before calving until Day 100 postpartum. At the same time, bodyweight (BW) and body condition score (BCS) were recorded. In Experiment 1, around Day 60 postpartum, the oestrous cycles of all cows were synchronized and sixty-five 2- to 4-cell in vitro-produced embryos were endoscopically transferred on Day 2 (Day 0 = oestrus) to the oviduct ipsilateral to the corpus luteum. On Day 7, the oviduct and uterus were flushed endoscopically and the number of embryos developing to the blastocyst stage was recorded. In Experiment 2, around Day 95 postpartum, cows were re-synchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterine horn ipsilateral to the corpus luteum. On Day 14, conceptuses were recovered by flushing the reproductive tract at slaughter and were measured. Jugular blood samples were taken daily from Day 0 to 7 (Exp. 1) or 14 (Exp. 2) to measure serum concentrations of progesterone. Data were analysed by ANOVA. Concentrations of NEFA and β-HB were higher (P ≤ 0.05) and glucose, insulin and IGF-1 were lower (P ≤ 0.05) in lactating compared with dry cows. BW and BCS were significantly higher in the non-lactating cows throughout the postpartum period. Recovery rates in both experiments were similar between groups (Exp. 1: 63.9 ± 7.2 vs 65.6 ± 8.6 and Exp 2: 33.3 ± 9.6 vs 39.8 ± 9.6 for dry and milking cows, respectively). In Exp. 1, of the structures recovered, significantly more developed to the blastocyst stage in the dry than in lactating cows (49.3 ± 3.8 vs 32.6.3 ± 4.4, respectively; P ≤ 0.05). Progesterone concentrations did not differ between groups. In Exp. 2, no differences were observed in terms of conceptus dimensions on Day 14 (n = 152). Progesterone concentrations were higher in lactating cows from Day 9 to 14 (P ≤ 0.05). In conclusion, this study provides evidence that at 60 days postpartum, the reproductive tract of lactating cows is compromised in its ability to support early embryo development compared with age-matched parous non-lactating cows; however, by 95 days postpartum there was no apparent difference in conceptus development, consistent with less metabolic stress as indicated by the metabolic profile. Funded by Science Foundation Ireland (SFI/07/SRC/B1156) and the Spanish Ministry of Science and Innovation (AGL2009-11810). VM was supported by an STSM award from the COST Action FAO7O2.

182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 205
Author(s):  
Y. Nagao ◽  
H. Yamamoto ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Laser Scanning ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mitochondrial Activity ◽  
Early Embryo Development

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

The Effect of CRH and Its Inhibitor, Antalarmin, on in Vitro Growth of Preantral Mouse Follicles, Early Embryo Development, and Steroidogenesis

Endocrinology ◽  
2013 ◽  
Vol 154 (1) ◽  
pp. 222-231 ◽  
Author(s):  
V. Dinopoulou ◽  
G. A. Partsinevelos ◽  
D. Mavrogianni ◽  
E. Anagnostou ◽  
P. Drakakis ◽  
...  
Keyword(s):  
Embryo Development ◽  
Early Embryo ◽  
In Vitro Growth ◽  
Early Embryo Development

P–181 Morphine regulates BMP4 growth factor and is involved in in-vitro early embryo development and PGCs formation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
I Muñoa ◽  
M Araolaza-Lasa ◽  
I Urizar-Arenaza ◽  
M Gianzo Citores ◽  
N Subiran Ciudad
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Environmental Factors ◽  
Embryo Development ◽  
Early Embryo ◽  
Epigenetic Changes ◽  
Morphine Treatment ◽  
Early Embryo Development

Abstract Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10–5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings: Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases Trial registration number Not applicable

P–187 Multinucleation and reverse cleavage, sings of early embryo self-repair mechanisms

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Munuer. Puigvert ◽  
V. Montalv Pallès ◽  
J Mass. Hernáez ◽  
A García-Faura ◽  
B Marquè. López-Teijón ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Live Birth Rate ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Control Group ◽  
Complete Fusion ◽  
Depth Analysis ◽  
Self Repair

Abstract Study question Have multinucleation and reverse cleavage any effect on embryo development and clinical outcomes on IVF treatments? Summary answer Embryos capable of repairing dysmorphisms and developing up to blastocyst stage keep intact their ability to become healthy babies. What is known already Time-lapse systems allow IVF laboratories to perform in-depth analysis of embryo development using the continuous monitoring tool. Some events that are impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered a negative factor when the embryo quality assessment is performed. However, it has been described the possibility that embryos have self-repair intrinsic methods. Study design, size, duration Retrospective study including data from 3,577 cycles with 21,274 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to day 5/6 between 2014 and 2019. Participants/materials, setting, methods Three embryo groups were considered: Control group, embryos without multinucleation or reverse cleavage (CG; n = 16,897); Multinucleation group, embryos with at least one blastomere multinucleated on D + 2/3 (MNC; n = 3,879) and Reverse Cleavage group, embryos undergoing complete fusion of two blastomeres on D + 2/3 (RC; n = 498). Single embryo transfer was performed on blastocyst stage. Clinical outcome rates were compared between groups and analyzed by Chi-square test. Main results and the role of chance As published by other groups, the 2.3% of our embryos showed at least one reverse cleavage event and we observed multinucleation in the 18.2% of the embryos. Blastocyst rate of dysmorphism groups was significantly lower (p &lt; 0.05) than Control group (MNC=20.0%; RC = 27.7%; CG = 58.0%). Once transferred, MNC and RC evolutive embryos showed significantly lower pregnancy (MNC=47.9%; RC = 46.8%; CG = 60.8%; p &lt; 0.05) and clinical pregnancy rates (MNC=39.4%; RC = 40.4% CG = 50.6%; p &lt; 0.05) than the Control group (p &lt; 0.05). However, during the post-implantational development the negative effect of dysmorphisms disappears, reaching values of live birth rate comparable to the Control group (MNC=28.3%; RC = 31.9% CG = 33.8%; p = 0.17). These results prove the importance of blastocyst culture and the inherent capability of the embryos to overcome some abnormal dynamics as multinucleation and reverse cleavage. Thus, these embryos showing the poor-prognosis events can be considered for transfer or vitrify. Limitations, reasons for caution There is a wide difference on sample size between groups despite the fact that the statistical analysis considers that into account. There are some ongoing pregnancies in all groups. Wider implications of the findings: When analyzing the development of embryos undergoing reverse cleavage and multinucleation, we hypothesize that these embryos could be showing a self-correction mechanism for some type of error detected. Embryos capable of repairing and developing up to blastocyst stage keep intact their ability to become healthy babies. Trial registration number Not applicable

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