Journal articles on the topic 'Pre-implantation embryo'
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Niakan, K. K., J. Han, R. A. Pedersen, C. Simon, and R. A. R. Pera. "Human pre-implantation embryo development." Development 139, no. 5 (February 7, 2012): 829–41. http://dx.doi.org/10.1242/dev.060426.
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Flores, Diana, Manoj Madhavan, Savannah Wright, and Ripla Arora. "Mechanical and signaling mechanisms that guide pre-implantation embryo movement." Development 147, no. 24 (November 6, 2020): dev193490. http://dx.doi.org/10.1242/dev.193490.
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ABSTRACTHow a mammalian embryo determines and arrives at its attachment site has been studied for decades, but our understanding of this process is far from complete. Using confocal imaging and image analysis, we evaluate embryo location along the longitudinal oviductal-cervical axis of murine uteri. Our analysis reveals three distinct pre-implantation phases: embryo entry, unidirectional movement of embryo clusters and bidirectional scattering and spacing of embryos. We show that unidirectional clustered movement is facilitated by a mechanical stimulus of the embryo and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, depends on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. Finally, we demonstrate that uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site necessary for the success of a pregnancy.
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Morris, D. G., P. Humpherson, H. J. Leese, and J. M. Sreenan. "Protein and energy metabolism in the pre-implantation cattle embryo." BSAP Occasional Publication 26, no. 2 (September 2001): 443–46. http://dx.doi.org/10.1017/s0263967x0003408x.
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AbstractThere is no information on the metabolism of the cattle embryo during the period from day 8 to 16 a period of greatest embryonic loss. In this study the rate of protein synthesis and phosphorylation was measured in 13 to 15 day old cattle embryos. The rate of glucose utilisation and amino acid uptake/efflux by day 14 to 16 embryos was also measured. Protein synthesis and phosphorylation activity when expressed per unit of protein decreased with increasing embryo size and age. Similarly the rate of glucose utilisation was greatest for the earlier day 14 embryos. Embryos differed in their requirement for different amino acids. The pattern of uptake/efflux was similar to that of the earlier day 7 embryo. This study suggests that the metabolic rate of cattle embryos expressed per unit of protein content tends to decrease with increasing age and size from the initial burst of activity at day 13 around the time that expansion of the embryo begins.
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Rajhans, R., G. S. Kumar, and G. T. Sharma. "292 EXPRESSION PROFILES OF STRESS AND METABOLIC MARKER GENES DURING IN VITRO PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS." Reproduction, Fertility and Development 18, no. 2 (2006): 253. http://dx.doi.org/10.1071/rdv18n2ab292.
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An increased understanding of the pre-implantation embryo developmental stage, with respect to physiological interaction of embryo with its micromilieu both in vivo and in vitro, is imperative to comprehend the events of pre-implantation development.The objective of the present study was to examine the temporal expression of heat shock protein 70 (Hsp-70) and glucose transporter 1 (Glut1) genes in pre-implantation-stage buffalo embryos produced under the standard in vitro production (IVP) system. Embryos were produced from slaughterhouse ovaries employing standard in vitro embryo production protocol, and presumptive zygotes produced following IVM/IVF were cultured in vitro in mSOF under mineral oil; FCS (10%) was added at 48 h post-insemination (hpi).The time series of development at stages being zygote (18-20 hpi), 2-1 cell (48 hpi), 8-16 cell (94-96 hpi), morula (120-144 hpi), and blastocyst (168-192 hpi), pre-implantation embryos conforming to the above developmental pattern were considered as 'fast-cleaving embryos', and all the embryos that did not conform to the above developmental timing were regarded as 'slow-cleaving embryos'. Pools of immature oocytes (IM, 120), Matured oocytes (MO, 120), 8-16 cell stages (8-16, 70), morula (M, 28), and blastocyst (B, 9) were collected and prepared for total RNA isolation and RT-PCR for the specific transcripts, with �-actin as loading control. The total RNA content ranged from 2.5 to 5.0 ng per oocyte/embryo. Presence of Hsp 70 and Glut1 gene transcripts was assessed in different stages of buffalo pre-implantation embryos using primers designed from bovine Hsp 70 and Glut1 by using the OLIGO program. RT was standardized using the embryo equivalent of 1-10 oocytes/embryo as the template as described by Arcellana-Panlilio and Schultz 1993 (Methods Enzymol. 225, 303-328) with PCR conditions being 59�C and 62�C for 45 s with 39 cycles for Glut1 and Hsp 70 gene transcripts, respectively. Amplicons were subjected to restriction digestion and sequencing (Acc. No. AJ812563, AJ812564). The expression of Hsp 70 throughout pre-implantation development in the fast-cleaving embryos indicated their maternal and zygotic origin, but transcripts of the Hsp 70 gene, represented by a single 488-bp amplicon, were not detected in slow-cleaving embryos, suggesting altered zygotic expression. Glut1 expression was prominent from the 8-16 cell stage, indicating a metabolic shift from pyruvate to glucose after the pre-compaction stage. For slow-cleaving embryos, transcripts of the Glut1 gene, represented by a single 327-bp amplicon, were absent during morula- and blastocyst-stage embryos, indicating the poor developmental competence of these embryos, which morphologically appeared normal. These transcription patterns reflect the embryonic response to the in vitro culture conditions and also correlate with the embryo quality and the speed of development of the pre-implantation buffalo (Bubalus bubalis) embryos.
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O'Neill, C. "013.The regulation of survival of the pre-implantation embryo." Reproduction, Fertility and Development 16, no. 9 (2004): 13. http://dx.doi.org/10.1071/srb04abs013.
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There are many aspects of the regulation of the growth and survival of the pre-implantation embryo that remain enigmatic. The increasing production of such embryos by assisted reproductive technologies (ART) in human medicine, animal production and conservation biology has highlighted the relatively poor viability of such embryos. Many embryos fail to survive past the normal time of implantation. Population biology theory predicts that any circumstance that results in high death rates within a population creates a potential for genetic selection. This occurs if the surviving individuals have a genetic make up that preferentially favours survival. Since ART clearly favours the survival of some embryos over others, it is a high priority to develop a sound understanding of those factors that normally govern embryo survival and how they may be affected by ART. It raises the question, do embryos that survive ART have a genetic make up that favours their survival compared to the proportion of the population that does not survive? It is now demonstrated that autocrine and paracrine factors are essential for embryo survival and that these act via the 1-o-phosphatidylinositol-3-kinase (PI3K) survival signalling pathway (1). PI3K activates many downstream pro-survival and anti-apoptotic mediators. ART changes the expression of some of these mediators. Pharmacological and genetic moderation of their expression can influence embryo survival, highlighting potential targets for genetic selection through ART. Studies in appropriate models will allow rational approaches to safety assessment of ART and spawn new strategies for media and procedural design. (1) Lu, D. P., Chandrakanthan, V., Cahana, A., Ishii, S., O'Neill, C. (2004) J. Cell Sci. 117, 1567–1576.
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Matsumoto, K., A. Uenoyama, T. Matsuoka, K. Saeki, Y. Hosoi, and A. Iritani. "228 EXPRESSION OF zag1 IN MOUSE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 17, no. 2 (2005): 264. http://dx.doi.org/10.1071/rdv17n2ab228.
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Embryonic gene activation (EGA) first occurs during the second half of the mouse 1-cell embryo (Latham KE 1999 Int. Rev. Cytol. 193, 71–124). Moreover, precise regulation of EGA is considered to be essential for normal embryo development. To understand the molecular basis for the regulation of EGA, we have focused on the identification and functional characterization of genes activated at the late 1-cell stage of the mouse embryo. Recently, we have identified and isolated a novel gene, termed zag1 (zygotic activating gene 1), transcribed specifically at the EGA, using a fluoro-differential display method with oocytes and embryos at 15 h post-insemination. Messenger RNA of zag1 expressed at lower level in the oocyte than that in the embryo at 15 h post-imsemination. In this study, we investigated the potential function of zag1 by analysis of mRNA expression and protein distribution in mouse tissues and pre-implantation embryos. Nucleotide sequence analysis of zag1 cDNA revealed that the open reading frame of 1726 bps encodes a protein of 575 amino acids with a predicted molecular mass of 66 kDa. The deduced amino acid sequence indicated that zag1 protein might be a soluble protein with a bipartite nuclear targeting sequence, a NACHT NTP domain, and an APT/GTP binding site motif as a predicted functional domain. Two μg of Poly(A)+ RNA from various tissues of adult mice were subjected to Northern blot analysis using the mouse zag1 cDNA probe. We detected this gene abundantly expressed in mouse testis and ovary by approximately 2- to 3-fold compared with one in other mouse tissues (heart, liver, kidney, lung, brain, skeletal muscle, and spleen). zag1 transcript and protein, as assessed by RT-PCR and immunoblotting, respectively, were slightly present in ovulated oocytes, gradually decreased in the early 1-cell embryos, but re-expressed in the late 1-cell and early 2-cell stage embryos which coincided with the mouse EGA. Subsequent to microinjection of an expression vector encoding zag1-enhanced green fluorescent protein (EGFP), fused protein into male pronucleus of 1-cell embryos was detected in the nuclei of 2-cell embryos. These findings suggest that zag1 may be functionally associated with early embryonic development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
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Li, Geng, Karam Khateeb, Erin Schaeffer, Bao Zhang, and Hasan Khatib. "Genes of the transforming growth factor-beta signalling pathway are associated with pre-implantation embryonic development in cattle." Journal of Dairy Research 79, no. 3 (June 12, 2012): 310–17. http://dx.doi.org/10.1017/s0022029912000210.
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One of the main factors affecting cattle fertility is pre-implantation development of the bovine embryo, which is a complex process regulated by various signal-transduction pathways. The transforming growth factor-β (TGF-β) signalling system, which is responsible for many biological processes including cell proliferation, differentiation and apoptosis, also is involved in embryo development. We hypothesized that altered expression of TGF-β genes in pre-implantation bovine embryos is associated with morphological abnormalities of these embryos. To test this hypothesis, we produced embryos in vitro and classified them at the blastocyst stage as either normally developed blastocysts or degenerates (growth-arrested embryos). The expression patterns of 25 genes from the TGF-β pathway were assessed using quantitative real time PCR. Ten genes showed differential expression between the two embryo groups, four genes displayed similar expressional profiles, and 11 genes had no detectable expression. An altered expression profile was statistically significant for 10 of the 14 expressed genes, and all were up-regulated in degenerate embryos vs. blastocysts. Furthermore, genomic association analysis of the cows from which embryos were produced revealed a significant association of ID3 and BMP4 polymorphisms—two of the most significant differentially expressed genes—with fertilization rate and blastocyst rate, respectively. Taken together, we conclude that TGF-β pathway genes, especially BMP4 and ID3 play a vital function in the regulation of pre-implantation embryo development at both embryo and maternal levels. Hence, these genes may be suitable as genetic markers for embryo development and fertility in cattle.
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Maganha, Juliana, Evelise de Souza Rocha, Marcos Antônio Fernandes Brandão, Vera Maria Peters, and Martha de Oliveira Guerra. "Embryo development alteration in rats treated with lapachol." Brazilian Archives of Biology and Technology 49, no. 6 (November 2006): 927–34. http://dx.doi.org/10.1590/s1516-89132006000700010.
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Lapachol, a naphthoquinone extracted from plants of the genus Tabebuia (family Bignoneaceae), showed multiple therapeutic activities. Pregnant Wistar rats were treated with Lapachol from the 1st to the 4th (pre-implantation period) and from 5th to 7th (implantation period) post insemination day (PID). Mothers were sacrificed on the 5th or on the15th PID. Number of corpora lutea, preimplantation embryo, blastocysts, live and dead fetuses and resorptions were counted. There were no signs of maternal toxicity. The number and the morphology of embryos, during oviduct development (pre-implantation period), did not seem to be affected by this drug, but during the implantation period, lapachol was toxic causing the death of embryos and intrauterine growth retardation.
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Fu, Bo, Hong Ma, and Di Liu. "Endogenous Retroviruses Function as Gene Expression Regulatory Elements During Mammalian Pre-implantation Embryo Development." International Journal of Molecular Sciences 20, no. 3 (February 12, 2019): 790. http://dx.doi.org/10.3390/ijms20030790.
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Pre-implantation embryo development encompasses several key developmental events, especially the activation of zygotic genome activation (ZGA)-related genes. Endogenous retroviruses (ERVs), which are regarded as “deleterious genomic parasites”, were previously considered to be “junk DNA”. However, it is now known that ERVs, with limited conservatism across species, mediate conserved developmental processes (e.g., ZGA). Transcriptional activation of ERVs occurs during the transition from maternal control to zygotic genome control, signifying ZGA. ERVs are versatile participants in rewiring gene expression networks during epigenetic reprogramming. Particularly, a subtle balance exists between ERV activation and ERV repression in host–virus interplay, which leads to stage-specific ERV expression during pre-implantation embryo development. A large portion of somatic cell nuclear transfer (SCNT) embryos display developmental arrest and ZGA failure during pre-implantation embryo development. Furthermore, because of the close relationship between ERV activation and ZGA, exploring the regulatory mechanism underlying ERV activation may also shed more light on the enigma of SCNT embryo development in model animals.
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Lutz, J., S. Johnson, K. Duprey, P. Taylor, H. Vivanco, M. Ponce-Salazar, M. Miguel, and C. Youngs. "7 Pregnancy from a vitrified-warmed alpaca pre-implantation embryo." Reproduction, Fertility and Development 32, no. 2 (2020): 128. http://dx.doi.org/10.1071/rdv32n2ab7.
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The alpaca (Vicugna pacos) is a ruminant livestock species in the South American camelid family. There are more than 9 million South American camelids globally that make important contributions to the livelihoods of rural farmers through conversion of low quality roughages to high quality food and fibre. Reproductive biotechnologies for alpacas are not well developed compared with those for other ruminant livestock species. In particular, embryo cryopreservation technologies are lacking. The objective of this study was to evaluate under field conditions a vitrification protocol originally developed for old world camels that we adapted for use in alpacas. Potential donors were evaluated for follicular development using a 7.5-MHz ultrasound probe. Hembras (sexually mature female alpacas) with ovarian follicles 7-10mm in diameter were behaviour tested to determine sexual receptivity, and receptive females were naturally mated to a proven herd sire. At the time of breeding, non-superovulated donors (n=4) received 30μg gonadorelin. Embryos were nonsurgically collected 7 days after breeding and handled at 20°C. Diameter of harvested embryos (n=4 quality grade 1 hatched expanded blastocysts) was measured using an eyepiece reticle. All recovered embryos were placed individually into 0.5-mL drops of vitrification solution (VS1: 1.4M glycerol) for 5min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6M ethylene glycol) for 5min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6M ethylene glycol) for 20s, and 0.05-mL drops of VS3 for 20s while loading into open-pulled straws (OPS). Each OPS was plunged directly into liquid nitrogen for storage for 29 days. At warming, each OPS was submerged into a 1-mL drop of warming solution 1 (WS1: 0.5M galactose) for 1min followed by 1min in WS2 (0.25 M galactose) for 5min before being incubated at 37°C in 5% CO2 in humidified air for 21h in 1mL of Syngro holding medium supplemented with 10% (vol/vol) alpaca serum. Embryos that grew during culture (n=2) were transferred individually into synchronous recipients, and embryos that did not appear to grow (n=2) were transferred together as a pair. Prior to embryo transfer, potential recipients were evaluated ultrasonographically as described previously. Hembras with ovarian follicles 7-10mm in diameter were behaviour tested, and sexually receptive females received 30μg gonadorelin 6 days before embryo transfer. Final selection of recipients (n=3) was based on presence of a corpus luteum and nonreceptive behaviour to a herd sire 24h before transfer. Pregnancy was detected ultrasonographically, and fetal heartbeat was detected 29 days post-transfer in one of the three recipients. Ultrasound at 177 days post-transfer revealed that the pregnancy, generated from a 400μm×375μm vitrified-warmed embryo that had grown in culture, was still ongoing. If this pregnancy results in the birth of a live cria (newborn alpaca), it would represent-to the best of our knowledge-the world's first cria born from a cryopreserved alpaca pre-implantation embryo. It would also demonstrate the potential utility of this protocol under field conditions.
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Savostina, G. V., S. G. Perminova, A. V. Timofeeva, and M. A. Veyukova. "Modern Methods for Assessment of the Implantation Potential of Embryos in Assisted Reproductive Programs." Doctor.Ru 20, no. 8 (2021): 12–18. http://dx.doi.org/10.31550/1727-2378-2021-20-8-12-18.
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Objective of the Review: To analyse the modern methods for assessment of the implantation potential of embryos in assisted reproductive programs. Key Points. We present the study results for selection of a most optimal embryo for transfer, using visual assessment of embryo quality, preimplantation genetic aneuploidy testing, analysis of metabolomic, proteomic, transcriptomic profiles of culture media and embryo blastocele. We have paid special attention to assessment of small non-coding RNA (sncRNA) in embryo culture medium. Conclusion. Due to the high sensitivity, objectivity and biomarker resistance to degradation, the most promising non-invasive method to assess the implantation potential of an embryo is analysis of the sncRNA profile in embryo culture media. Keywords: aneuploidy, pre-implantation genetic testing, small non-coding RNAs, proteomic analysis, metabolomic analysis.
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Hobbs, JG, and PL Kaye. "Glycine uptake in pre-implantation mouse embryos: kinetics and the effects of external." Reproduction, Fertility and Development 2, no. 6 (1990): 651. http://dx.doi.org/10.1071/rd9900651.
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The kinetic parameters (with standard errors) describing glycine uptake by mouse 2-cell embryos and blastocysts were determined by non-linear regression. Uptake at both stages was best described by a combination of a non-saturable component and a single saturable uptake system. During development, the rate constants for both components increased, as would be expected from the known increases in surface area, from 9.4 +/- 3.7 pL per 10 min per embryo and 128 +/- 12 fmol per 10 min per embryo in 2-cell embryos to 38.9 +/- 2.1 pL per 10 min per embryo and 258 +/- 15 fmol per 10 min per embryo in blastocysts. In contrast to earlier reports, there was no change in Km, which was 88 +/- 13 microM in 2-cell embryos and 115 +/- 10 microM in blastocysts. Reducing the external [Na+] from 230 mM increased Km for both stages. This effect on Km appeared to be related to [Na+]-2 or [Na+]-3. Vmax was increased in embryos of both stages by increasing [Na+] from 60 to 100 mM. However, whilst further increases to 400 mM were without major effect on uptake by 2-cell embryos, they inhibited uptake by blastocysts. This may result from osmotic effects on trophectodermal transport in the blastocysts. These results suggest that during development to blastocysts, the gly-system that operates in 2-cell embryos may be modified to a less restricted system with a similar Km and a complex dependence on [Na+].
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Li, Shuai, and Wipawee Winuthayanon. "Oviduct: roles in fertilization and early embryo development." Journal of Endocrinology 232, no. 1 (January 2017): R1—R26. http://dx.doi.org/10.1530/joe-16-0302.
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Animal oviducts and human Fallopian tubes are a part of the female reproductive tract that hosts fertilization and pre-implantation development of the embryo. With an increasing understanding of roles of the oviduct at the cellular and molecular levels, current research signifies the importance of the oviduct on naturally conceived fertilization and pre-implantation embryo development. This review highlights the physiological conditions within the oviduct during fertilization, environmental regulation, oviductal fluid composition and its role in protecting embryos and supplying nutrients. Finally, the review compares different aspects of naturally occurring fertilization and assisted reproductive technology (ART)-achieved fertilization and embryo development, giving insight into potential areas for improvement in this technology.
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Duan, Kui, Chen-Yang Si, Shu-Mei Zhao, Zong-Yong Ai, Bao-Hua Niu, Yu Yin, Li-Feng Xiang, Hao Ding, and Yun Zheng. "The Long Terminal Repeats of ERV6 Are Activated in Pre-Implantation Embryos of Cynomolgus Monkey." Cells 10, no. 10 (October 9, 2021): 2710. http://dx.doi.org/10.3390/cells10102710.
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Precise gene regulation is critical during embryo development. Long terminal repeat elements (LTRs) of endogenous retroviruses (ERVs) are dynamically expressed in blastocysts of mammalian embryos. However, the expression pattern of LTRs in monkey blastocyst is still unknown. By single-cell RNA-sequencing (seq) data of cynomolgus monkeys, we found that LTRs of several ERV families, including MacERV6, MacERV3, MacERV2, MacERVK1, and MacERVK2, were highly expressed in pre-implantation embryo cells including epiblast (EPI), trophectoderm (TrB), and primitive endoderm (PrE), but were depleted in post-implantation. We knocked down MacERV6-LTR1a in cynomolgus monkeys with a short hairpin RNA (shRNA) strategy to examine the potential function of MacERV6-LTR1a in the early development of monkey embryos. The silence of MacERV6-LTR1a mainly postpones the differentiation of TrB, EPI, and PrE cells in embryos at day 7 compared to control. Moreover, we confirmed MacERV6-LTR1a could recruit Estrogen Related Receptor Beta (ESRRB), which plays an important role in the maintenance of self-renewal and pluripotency of embryonic and trophoblast stem cells through different signaling pathways including FGF and Wnt signaling pathways. In summary, these results suggest that MacERV6-LTR1a is involved in gene regulation of the pre-implantation embryo of the cynomolgus monkeys.
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Shi, Yu, Mingcheng Cai, Kun Du, Xue Bai, Lipeng Tang, Xianbo Jia, Shiyi Chen, Jie Wang, and Songjia Lai. "Dynamics of Known Long Non-Coding RNAs during the Maternal-to-Zygotic Transition in Rabbit." Animals 11, no. 12 (December 19, 2021): 3592. http://dx.doi.org/10.3390/ani11123592.
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The control of pre-implantation development in mammals undergoes a maternal-to-zygotic transition (MZT) after fertilization. The transition involves maternal clearance and zygotic genome activation remodeling the terminal differentiated gamete to confer totipotency. In the study, we first determined the profile of long non-coding RNAs (lncRNAs) of mature rabbit oocyte, 2-cell, 4-cell, 8-cell, and morula embryos using RNA-seq. A total of 2673 known rabbit lncRNAs were identified. The lncRNAs exhibited dynamic expression patterns during pre-implantation development. Moreover, 107 differentially expressed lncRNAs (DE lncRNAs) were detected between mature oocyte and 2-cell embryo, while 419 DE lncRNAs were detected between 8-cell embryo and morula, consistent with the occurrence of minor and major zygotic genome activation (ZGA) wave of rabbit pre-implanted embryo. This study then predicted the potential target genes of DE lncRNAs based on the trans-regulation mechanism of lncRNAs. The GO and KEGG analyses showed that lncRNAs with stage-specific expression patterns promoted embryo cleavage and synchronic development by regulating gene transcription and translation, intracellular metabolism and organelle organization, and intercellular signaling transduction. The correlation analysis between mRNAs and lncRNAs identified that lncRNAs ENSOCUG00000034943 and ENSOCUG00000036338 may play a vital role in the late-period pre-implantation development by regulating ILF2 gene. This study also found that the sequential degradation of maternal lncRNAs occurred through maternal and zygotic pathways. Furthermore, the function analysis of the late-degraded lncRNAs suggested that these lncRNAs may play a role in the mRNA degradation in embryos via mRNA surveillance pathway. Therefore, this work provides a global view of known lncRNAs in rabbit pre-implantation development and highlights the role of lncRNAs in embryogenesis regulation.
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Pock, Tim, Katharina Schulte, Stefan Schlatt, Michele Boiani, and Verena Nordhoff. "GM-CSF perturbs cell identity in mouse pre-implantation embryos." PLOS ONE 17, no. 2 (February 10, 2022): e0263793. http://dx.doi.org/10.1371/journal.pone.0263793.
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Growth factors became attractive candidates for medium supplementation to further improve the quality of embryo culture and to mimic in vivo nutrition. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine influencing the maternal-fetal interface and supporting placental development in mouse and human. It is expressed in epithelial cells of the endometrium under the regulation of estrogens. The factor is already in clinical use and a large clinical trial showed that, if supplemented to an embryo culture medium, it leads to increased survival of embryos, especially in women with previous miscarriages. Animal and cell culture studies on isolated trophectoderm cells support an effect mainly on cellular expansion. Aim of this study was to investigate, if the supplementation of GM-CSF either in a human ART medium or in a mouse optimized medium, leads to a change in cell number and cell lineages in the early pre-implantation mouse embryo. Our data shows that mouse GM-CSF increased total cell numbers with increasing concentrations. This increase of cell number has not been found in embryos cultured in ART media with or without human GM-CSF (hGM-CSF) or in a mouse medium supplemented with different concentrations of hGM-CSF. The changes were caused by a marked difference in TE and primitive endoderm cell numbers but not due to a change in epiblast cell numbers. Additionally, results show an ectopic expression of NANOG among trophectoderm cells in both, human ART media (with and without GM-CSF) and at increasing concentrations in the mouse and the human GM-CSF supplemented media. In conclusion, we could show that GM-CSF has an effect on cell identity in mice, which might probably also occur in the human. Therefore, we would like to rare awareness that the use of supplements without proper research could bare risks for the embryo itself and probably also in the post-implantation phase.
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Sutton-McDowall, M., D. Feil, R. Robker, J. Thompson, and K. Dunning. "92 UTILISATION OF ENDOGENOUS FATTY ACID STORES FOR ENERGY PRODUCTION IN BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 158. http://dx.doi.org/10.1071/rdv24n1ab92.
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Current embryo culture media are based on the carbohydrate metabolism of embryos. However, little is known about the metabolism of endogenous lipids. This is surprising given the high intracellular lipid densities of embryos of some species and the potential for ATP production via β-oxidation. L-carnitine is a β-oxidation co-factor that is absent in most culture media. The aim of this study is to investigate the influence of carnitine supplementation ± carbohydrates on bovine embryo development. Abattoir-derived cattle cumulus–oocyte complexes were cultured and fertilised (Sutton-McDowall et al. 2006 Biol. Reprod. 74, 881–888). Post-fertilisation (24 h), presumptive zygotes were transferred into an amino acid-free cleavage media ± carbohydrates (glucose, lactate and pyruvate) ±5 mM carnitine and cultured for 4 days. The absence of carbohydrates during culture resulted in embryos arresting at the 2- and 4-cell stages. Remarkably, +carnitine significantly increased development to the morula stage compared with +carbohydrates alone (20.4 ± 3% vs 4.7 ± 2.5% morula development; P < 0.001). The combination of carbohydrates and carnitine supplementation further improved embryo development, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +carnitine group compared with the +carbohydrates group (+carbohydrates = 3.1 ± 1.9 vs +carbohydrates +carnitine = 43.8 ± 9.1% morula development; P < 0.05). The beneficial effects of carnitine supplementation on embryo development were reversed when embryos were cultured in presence of etomoxir, a non-reversible inhibitor of the rate-limiting enzyme of β-oxidation (development to 8-cell stage; +carnitine = 33.9 ± 8% vs +carnitine +etomoxir = 19.2 ± 4.9%; P < 0.05). Intracellular lipid content of embryos +carnitine was determined by culturing presumptive zygotes in media -carbohydrates ± carnitine for 24 h. Lipid content of embryos was determined by measuring BODIPY 493/503 dye fluorescence. Carnitine supplementation reduced fluorescence intensity 1.8-fold (P < 0.001). Adenosine triphosphate and ATP:ADP levels were measured in embryos after 24 h of culture ± carbohydrates ± carnitine. While there was a trend for +carnitine to increase ATP levels (P = 0.09), ADP levels were higher and ATP:ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared with embryos cultured in –carnitine. This indicates +carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. We have shown carnitine supplementation supports pre-compaction embryo development and there is an additive effect of +carnitine +carbohydrate on early embryo development. This is most likely through increased β-oxidation levels within embryos. Current disparities between in vivo and in vitro embryo production, in particular increased lipid content (Romek et al. 2010 Theriogenology 74, 265–276) and decreased developmental potential of in vitro-produced embryos, may be an artefact resulting from limited lipid oxidation in vitro.
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Fontana, Vanina, Virginia Choren, Liliana Vauthay, Juan Carlos Calvo, Lucrecia Calvo, and Monica Cameo. "Exogenous interferon-γ alters murine inner cell mass and trophoblast development. Effect on the expression of ErbB1, ErbB4 and heparan sulfate proteoglycan (perlecan)." Reproduction 128, no. 6 (December 2004): 717–25. http://dx.doi.org/10.1530/rep.1.00335.
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Implantation is a crucial event in human pregnancy. The participation of cytokines in the implantation process has been widely documented, although the role of many of these molecules is still a matter of controversy. In a previous report from our laboratory, we demonstrated that addition of interferon-γ to the culture medium produces deleterious effects on mouse embryo development. In this study we investigated the effect of this cytokine on outgrowing embryo morphology and on the expression of epidermal growth factor receptors (ErbBs) and heparan sulfate proteoglycan (perlecan) in mouse embryos culturedin vitro. Morphological assessment of inner cell mass and trophoblast development was carried onin-situfixed and stained outgrowths. Localization of ErbB1, ErbB4 and perlecan on pre- and peri-implantation embryos was investigated by immunocytochemistry. Addition of interferon-γ produced a deleterious effect on both inner cell mass and trophoblast morphology. Immunostaining demonstrated that ErbB1, ErbB4 and perlecan are present on pre-implantation embryos and blasto-cysts; interferon-γ altered the expression of ErbB4 and Perlecan at the blastocyst stage. We propose that the effects produced by this cytokine could be related to the altered acquisition of adhesion competence and low implantation rates observed in certain reproductive immunological disorders.
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Glinkina, Zh I., A. F. Sayfitdinova, O. A. Pavlova, O. A. Leontyeva, A. N. Panina, N. K. Bichevaya, and I. V. Boroznyak. "Analysis of Concordance of Pre-implantation Aneuploidy Genetic Testing Results Obtained Using Next Generation Sequencing on Illumina Platform in Cells of Various Parts of Trophoblast." Doctor.Ru 21, no. 5 (2022): 18–24. http://dx.doi.org/10.31550/1727-2378-2022-21-5-18-24.
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Study Objective: To study trophoblast (TB) cells taken from various sections of the embryo using Next Generation Sequencing (NGS) on Illumina platform, and to compare data in order to identify the degree of discordance between various samples from one embryo. Study Design: Comparative study. Materials and Methods. For the study, we used human embryos at early development stages, which originated from artificial insemination of germ cells taken from healthy donors with normal karyotype within the scope of the in vitro fertilisation program. We selected 14 human embryos originating from insemination of oocytes of 10 donors aged 20 to 32 years old with sperm taken from 9 donors from the semen bank of the International Centre for Reproductive Medicine. Two embryos underwent degradation during defrosting. For 12 embryos, we performed a repeated TB cells biopsy from two independent sections: one biopsy from TB adjacent to inner cell mass (ICM) cells and the other TB biopsy from blastocyte pole opposite to the embryoblast. Study Results. A comparison of molecular karyotype of TB cells taken from various sections of blastocyte in 12 embryos, 36 samples (3 sample for each embryo), demonstrated partial discordance only in one observation. In initial study, molecular karyotype of an embryo showed trisomy 16 syndrome: Seq(16)x3,(XY)x1. In the follow-up study, we found an additional deletion in the form of mosaicism in chromosome 7 section adjacent to ICM of the embryo: Seq(16)x3,(7q21.3 -> 7q36.3)x[0.5]). All other results demonstrated complete concordance irrespective of a TB section in question or a laboratory where sequencing was performed. Conclusion. It may be concluded that pre-implantation aneuploidy genetic testing of 5-day-old embryos using Next Generation Sequencing on Illumina platform allows obtaining reliable information on chromosomal abnormalities and can be successfully used to identify aneuploidy in pre-implantation embryos. Keywords: pre-implantation aneuploidy genetic testing, Next Generation Sequencing, in vitro fertilisation, aneuploidy, embryo mosaicism, chromosomal pathology.
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Nguyễn Minh, Tâm, Bắc Nguyễn Duy, Tùng Nguyễn Thanh, and Trường Đặng Tiến. "Kết quả áp dụng quy trình xét nghiệm di truyền trước làm tổ bệnh Hemophilia A." VietNam Military Medical Unisversity 47, no. 5-2022 (June 2022): 42–49. http://dx.doi.org/10.56535/jmpm.v2022050804.
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Objectives: To evaluate the results of applying the pre-implantation genetic testing in families carrying the Hemophilia A gene. Subjects and methods: Using linkage genetic analysis technique for pre-implantation genetic testing of 16 families with Hemophilia A gene who were performed embryogenation and embryo biopsy at the Andrology and Fertility Hospital of Hanoi, from September 2018 to December 2021. Results: 117 blastocysts of 16 couples classified according to morphology included 85 good embryos (72.65%), 30 fair embryos (25.64%), 2 poor embryos (1.71%). Among 115 embryos of a good and fair type which were performed a biopsy and genetic diagnosis, there were 70 normal embryos (60.87%), 26 carried embryos (22.61%), and 19 disease affected embryos (16.52%). Conclusion: Hemophilia A gene carrier status was successfully diagnosed in 115 embryos of 16 couples participating in the study. * Keywords: Pre-implantation genetic testing; Hemophilia A.
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Sharma, Parvati. "Interaction of Pre-implantation Embryo with Endometrial Cells." Acta Scientific Agriculture 3, no. 7 (June 14, 2019): 132–33. http://dx.doi.org/10.31080/asag.2019.03.0530.
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Liu, Weimin, and William S. B. Yeung. "LET-7 REGULATES PRE-IMPLANTATION MOUSE EMBRYO DEVELOPMENT." Fertility and Sterility 116, no. 3 (September 2021): e279. http://dx.doi.org/10.1016/j.fertnstert.2021.07.748.
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Dietrich, J. E., and T. Hiiragi. "Stochastic patterning in the mouse pre-implantation embryo." Development 134, no. 23 (October 24, 2007): 4219–31. http://dx.doi.org/10.1242/dev.003798.
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Schulz, Laura Clamon, and R. Michael Roberts. "Leptin Localization in the Pre-Implantation Mouse Embryo." Biology of Reproduction 81, Suppl_1 (July 1, 2009): 242. http://dx.doi.org/10.1093/biolreprod/81.s1.242.
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Kradolfer, D., J. Knubben, V. Flöter, J. Bick, S. Bauersachs, and S. E. Ulbrich. "139 SEX-SPECIFIC GENE EXPRESSION IN PORCINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 28, no. 2 (2016): 199. http://dx.doi.org/10.1071/rdv28n2ab139.
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X-Chromosome inactivation in female mammals starts during early blastocyst stage with expression of the X-inactive specific transcript (XIST), which coats and silences the inactive X chromosome. However, this compensation is not complete in blastocysts, as a large number of X-linked transcripts are more highly expressed in female embryos than in males. Furthermore, the process of X chromosome inactivation is altered in IVF and cloned porcine embryos, possibly explaining problems of embryo survival with these techniques. The aim of this study was to gain more insights into the transcriptional dynamics of the porcine pre-implantation embryo, with a particular focus on sex-specific differences. RNA sequencing (RNA-Seq) was performed for individual blastocysts at 8, 10, and 12 days after ovulation, and the temporal development of sex-specific transcripts was analysed. German Landrace sows were cycle synchronized and inseminated with sperm of the same Pietrain boar. On Days 8, 10, and 12 post-insemination, sows were slaughtered and embryos were removed from the uterus using 10 mL of PBS (pH 7.4) per horn. Single embryos were shock frozen in liquid nitrogen and stored at –80°C until the extraction of RNA and DNA (AllPrep DNA/RNA Micro Kit, Qiagen, Valencia, CA, USA). Using the isolated DNA, the sex of the embryos was determined and 5 female and male embryos, respectively, were analysed per stage. Illumina TruSeq Stranded mRNA libraries (Illumina Inc., San Diego, CA, USA) were sequenced on a HiSEqn 2500 (Illumina Inc.), and 15 to 25 million 100-bp single-end reads were generated per sample. Reads were filtered and processed using Trimmomatic and mapped to the porcine genome assembly Sscrofa10.2 with TopHat2. Mapped reads were counted by the use of QuasR qCount based on the current National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) GFF3 annotation file. Statistical analysis of count data was performed with the BioConductor R (https://www.bioconductor.org/) package DESEqn 2. At all 3 stages, we found 7 Y-linked transcripts that were highly expressed in male embryos (EIF2S3, EIF1AY, LOC100624590, LOC100625207, LOC100624329, LOC102162178, LOC100624937). On the other hand, 47 X-linked transcripts showed increased expression in female blastocysts, most of them at all 3 time points. However, a small number of genes (DDX3X, LAMP2, and RPS6KA3) were more highly expressed in females at Days 8 and 10 but more highly expressed in males at Day 12. Three X-linked genes (OFD1, KAL1, and LOC100525092) were more highly expressed in male embryos, although only at a low fold change of 1.2 to 1.4. Furthermore, expression of 8 transcripts located on autosomes was higher in females. In conclusion, our study expands the current knowledge of sex-specific gene expression in 8- to 12-day-old porcine blastocysts, a critical time period during pre-implantation embryo development.
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Tong, Terry YY, Victor HH Goh, Chee Fei Tain, Helen Mok, and Shirley Tsang. "Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos." Canadian Journal of Physiology and Pharmacology 78, no. 6 (June 1, 2000): 453–56. http://dx.doi.org/10.1139/y00-008.
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Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ng·mL-1 oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ng·mL-1 oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.Key words: culture, embryonic development, oestradiol, rat.
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Al Naib, A., M. Wallace, L. Brennan, T. Fair, and P. Lonergan. "143 METABOLOMIC ANALYSIS OF BOVINE PRE-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 22, no. 1 (2010): 230. http://dx.doi.org/10.1071/rdv22n1ab143.
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Metabolomics is the study of small molecules or metabolites present in biological samples. The metabolism of the bovine embryo is marked by a transition from the oocyte and early stage embryo, which are entirely dependent on TCA cycle activity for the generation of ATP, toward a significantly greater input of glycolysis during morula compaction and blastocyst formation. The aim of this study was to describe the changes in metabolic profiles of bovine embryos across pre-implantation development. Five pools of 100 embryos at the 2- to 4-cell, 8-cell, 16-cell, morula, and blastocyst stages (i.e. 500 embryos in total per developmental stage) were produced by IVM, IVF, and IVC. Each pool was snap frozen and stored at -80°C until analysis. Extraction of metabolites was performed using 6% perchloric acid. 1H spectra were acquired on a 600-MHz Varian NMR spectrometer operating at 25°C (Varian Inc., Palo Alto, CA, USA). All spectra were processed, baseline corrected, and integrated into bins of 0.02 ppm width. The water region was excluded and data were normalized to the sum of the spectral integral. Data were analyzed using multivariate statistics. Principal component analysis (PCA), an unsupervised pattern recognition technique, was performed initially to assess variation and expose any trends or outlying data. Partial least squares-discriminant analysis (PLS-DA) was subsequently performed to define the maximum separation between the different developmental stages. Data were visualized by constructing principal component scores and loadings plots, where each point on the score plot represented an individual sample and each point on the loadings plot represented a single 1H NMR spectral region. The quality of all models was judged by the goodness-of-fit parameter (R2) and the predictive ability parameter (Q2). PCA analysis of all the data showed distinct separation of the 2- to 4-cell, and 8-cell (i.e. pre-embryonic genome activation, EGA) from 16-cell, morula and blastocyst (i.e. post-EGA) extracts. Pair-wise comparisons between successive developmental stages were performed. PCA analysis showed separation of 2- to 4-cell and 8-cell extracts. A PLS-DA was built with an R2 of 0.97 and a Q2 of 0.55. The main discriminating metabolites were acetate, acetoacetate, and an unidentified peak at 1.25 ppm. Similarly, PCA analysis showed separation of 8-cell and 16-cell embryo extracts. A PLS-DA model was built with an R2 of 0.99 and a Q2 of 0.92. The discrimination was due to higher levels of acetate and an unidentified peak in 8-cell extracts and the appearance of a new peak in 16-cell extracts, tentatively assigned to oxalacetate. PCA analysis showed no separation of the 16-cell, morula, and blastocyst extracts. In conclusion, 1H NMR spectroscopy allows the simultaneous measurement of small-molecular-weight molecules in complex biological samples. Given that the metabolome is downstream of gene function it may represent a superior measure of cellular activities compared to transcriptomic and proteomic approaches. Supported by Science Foundation Ireland (07/SRC/B1156).
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Jones, D., M. Paczkowski, and T. Kuehl. "104 EMBRYO MANIPULATION TECHNIQUES ALTER PRE-IMPLANTATION DEVELOPMENT AND GENE EXPRESSION IN MOUSE EMBRYOS." Reproduction, Fertility and Development 28, no. 2 (2016): 182. http://dx.doi.org/10.1071/rdv28n2ab104.
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Micromanipulation techniques are commonly employed with human embryos; however, the long-term effects on growth and development of embryos are unknown. Subjective techniques, such as embryo grading, may not be sensitive enough to show differences in developmental potential and more objective assessments are needed. The objective of this experiment was to evaluate whether manipulation techniques and cryopreservation alter morphologic and gene expression endpoints of embryos. Two-cell mouse embryos were cultured to blastocyst stage and divided into 6 treatment groups: nonmanipulated control (UNMAN; n = 252), laser-assisted hatching (LAH; n = 124), LAH with slow cooling cryopreservation (LAHcryo; n = 78), simulated biopsy (BIOP; n = 90) performed by laser disruption of 1/8 of the embryo’s cellular volume, BIOP with slow cooling cryopreservation (BIOPcryo; n = 91), and BIOP with vitrification (BIOPvit; n = 66). After manipulation or thaw, embryos were cultured 28 h to blastocyst stage. Fully hatched and hatching blastocysts were collected for cell counts and quantitative PCR to assay absolute transcript abundance for Plac8, Glut1, Oct4, and Cox2, 18s rRNA, and Ppia using standard curves generated from serial dilutions of digested plasmids. Data were analysed using ANOVA with Duncan’s post hoc test and significance was defined as P < 0.05. Manipulation groups differed in developmental stage at 28 h postmanipulation (P = 0.03), with a larger percentage of embryos completely hatched in LAH (46%) and BIOP (45%) groups compared to UNMAN (11%). Cryopreservation reduced percentages of completely hatched embryos (LAHcryo = 26%; BIOPcryo = 31%; BIOPvit = 19%). The number of cells per embryo also varied (P < 0.001); UNMAN and LAH groups had similar numbers of blastomeres (UNMAN = 71; LAH = 74), whereas BIOP groups had reduced cell counts (BIOP = 53; BIOPcryo = 56; BIOPvit = 48). Patterns of gene expression varied between groups; the BIOP group had more cDNA per cell (UNMAN = 125 ng cell–1; BIOP = 202 ng cell–1; P = 0.004), whereas BIOPcryo group did not show this effect (BIOPcryo = 111 ng cell–1). Transcript abundance of Cox2, Oct4, and Glut1 per embryo was significantly decreased (P < 0.05) in biopsied embryos that were cryopreserved, either by slow-cooling or vitrification (Cox2: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268; Oct4: UNMAN = 266, BIOP = 181, BIOPcryo = 95, BIOPvit = 97; Glut1: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268). The data suggests that micromanipulation alters developmental timelines and gene expression of embryos. While manipulations differ in degree and nature of effect, our investigation implies all manipulations have some effect on the transcriptome of the developing embryo. Differences in cDNA quantity in the BIOP group may indicate a stress response or recovery attempt that becomes blunted in biopsied embryos which are subsequently cryopreserved. Thus, our study suggests that cryopreservation of biopsied embryos may come at a price and offers scientific support for only judiciously selected and medically indicated embryo biopsies.
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Bazzano, María Victoria, Gisela Belén Sarrible, Martín Berón de Astrada, and Evelin Elia. "Obesity modifies the implantation window and disrupts intrauterine embryo positioning in rats." Reproduction 162, no. 1 (July 1, 2021): 61–72. http://dx.doi.org/10.1530/rep-21-0015.
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Obesity is a chronic disease that impairs female reproduction. When gestation is achieved, maternal obesity can cause offspring’s health complications. We intended to evaluate the effects of maternal pre-conceptional obesity on uterine contractile activity, embryo implantation and offspring development. Using cafeteria diet-induced obesity as an animal model, we found that maternal obesity delays embryo transport from the oviduct to the uterus and alters the intrauterine embryo positioning. Adrenergic receptor (AR) signaling is involved in embryo positioning, so all AR isoforms were screened in the pre-implantation uteri. We found that the β2AR is the dominant isoform in the rat uteri and that obesity causes its upregulation. Although β2AR activation is known to induce uterine relaxation, higher spontaneous contractile activity was detected in obese dams. Uteri from obese dams showed a higher sensitivity to salbutamol (a selective agonist of β2AR) than controls, consistent with the higher β2AR levels detected in those animals. Despite this, in obese dams, some embryos were still in the oviduct at the predicted time of initial embryo attachment, embryo implantation is successfully carried out since the total number of fetuses on gd 18.5 were similar between control and obese dams. These findings show that obesity is modifying the implantation window. Moreover, we found that maternal obesity resulted in macrosomia in the offspring, which is an important predictor of fetal programming of postnatal health. Hence, our results show that maternal obesity prior to pregnancy not only disturbs the implantation process, but also affects offspring development.
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Govindasamy, Niraimathi, Binyamin Duethorn, Hatice O. Oezgueldez, Yung S. Kim, and Ivan Bedzhov. "Test-tube embryos - mouse and human development in vitro to blastocyst stage and beyond." International Journal of Developmental Biology 63, no. 3-4-5 (2019): 203–15. http://dx.doi.org/10.1387/ijdb.180379ib.
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Mammalian embryogenesis is intrauterine and depends on support from the maternal environment. Therefore, in order to directly study and manipulate early mouse and human embryos, fine-tuned culture conditions have to be provided to maintain embryo growth in vitro. Over time, the establishment and implementation of embryo culture methods have come a long way, initially enabling the development of few pre-implantation stages, expanding later to support in vitro embryogenesis from fertilization until blastocyst and even ex utero development beyond the implantation stages. Designing culture conditions that enable near physiological development of early embryos without maternal input, especially during the peri- and post-implantation stages, requires overcoming numerous experimental challenges, and it is still far from optimal. Nevertheless, embryo culture methods are an essential cornerstone of both assisted reproductive technologies and basic research, and these methods provide a platform to understand life’s greatest miracle – the development of a new organism.
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Travnickova, Ivona, Pavlina Hulinska, Zbysek Sladek, Mariusz T. Skowronski, and Marie Machatkova. "Changes of the zona pellucida patterns during oocyte maturation, fertilization and embryo development in mammals: mini-review." Medical Journal of Cell Biology 10, no. 1 (March 1, 2022): 23–28. http://dx.doi.org/10.2478/acb-2022-0004.
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Abstract The mammalian zona pellucida (ZP) is an extracellular matrix that surrounds immature and mature oocytes and early embryos until the stage of a blastocyst and its implantation. This mini-review summarizes basic information on the ZP and its morphologic and functional changes during in vitro oocyte maturation and fertilization and in vivo pre-implantation embryo development.
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Teson, J., K. Lee, L. Spate, and R. S. Prather. "142 DYNAMICS OF Tet FAMILY DURING PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 183. http://dx.doi.org/10.1071/rdv24n1ab142.
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One of the key regulators of gene expression in mammals is DNA methylation. The Tet family (Tet1–3) is suggested to be involved in regulating the level of methylation by hydroxylating a methyl group from 5-methylcytosine to form 5-hydroxymethylcystosine. This hydroxylation alters the 3-dimensional structure of the DNA and results in altered gene expression. Previous studies conducted in the mouse have shown that Tet1 is important for inner cell mass specification by regulating the apparent level of methylation on a specific promoter region in blastocysts and Tet3 is related to the apparent paternal DNA demethylation after fertilization by hydroxylating the paternal genome. The objective of this study was to investigate the expression profile of the Tet family in porcine oocytes and pre-implantation-stage embryos derived from IVF and somatic cell nuclear transfer (SCNT). The RNA was isolated from donor cells, germinal vesicle (GV), MII and 2-cell and blastocyst stage embryos (20 oocytes or embryos per group). Levels of mRNA for each Tet gene were measured by quantitative real-time RT-PCR. The levels of each mRNA transcript were compared to YWHAG, a housekeeping gene that shows a constant level of expression throughout pre-implantation embryo development and normalized to the GV stage. The analysis was repeated with 3 biological replications and 2 experimental replications. Differences in gene expression were compared by ANOVA and P < 0.05 was considered significant. No difference was found in the levels of the Tet family members between GV and MII stage oocytes. Compared with GV stage oocytes, up-regulation of Tet3 at the 2-cell stage was detected in both IVF and SCNT embryos, 4.7 and 6.2 fold, respectively. A dramatic increase in Tet1 was also observed at the blastocyst stage in IVF and SCNT embryos when compared with the GV stage, 65.7 and 79.7 fold increases, respectively. Interestingly, the level of Tet3 was down-regulated in blastocyst embryos at a 25 or more fold decrease compared with GV. The level of Tet2 remained constant throughout embryo development. Embryos (2-cell and blastocyst) compared from IVF and SCNT showed no difference in Tet expression levels. Donor cells had significantly lower levels of Tet2 and Tet3 when compared with GV. Our results indicate that the Tet family shows a dynamic expression profile during porcine pre-implantation embryo development. High expression of Tet3 in 2-cell stage embryos suggests its importance during the post-activation demethylation process. The increase of Tet1 transcript in blastocysts suggests that Tet1 is involved in regulating the type of methylation at the blastocyst stage. These results are consistent with results from previous mouse studies. There was no misregulated expression of the Tet family in SCNT embryos compared with IVF embryos, thus indicating successful reprogramming of the Tet family after SCNT. Lower levels of Tet2 and Tet3 would indicate that Tet1 is important for maintaining type of methylation in donor cells. This is the first report on the profile of the Tet family during porcine pre-implantation embryo development and further studies are needed to clarify their role during this stage.
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Sullivan-Pyke, Chantae, Sneha Mani, Eric A. Rhon-Calderon, Teri Ord, Christos Coutifaris, Marisa S. Bartolomei, and Monica Mainigi. "Timing of exposure to gonadotropins has differential effects on the conceptus: evidence from a mouse model†." Biology of Reproduction 103, no. 4 (June 25, 2020): 854–65. http://dx.doi.org/10.1093/biolre/ioaa109.
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Abstract Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications.
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AGRESTA, Franca, Claire GARRETT, Sharyn STOCK-MYER, David GARDNER, and Kate STERN. "Is the Ploidy Status of the Embryo Impacted by Gonadotrophin Dose?" Fertility & Reproduction 04, no. 03n04 (September 2022): 172. http://dx.doi.org/10.1142/s2661318222740838.
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Background: There has been much debate and concern about whether the dose of exogenous gonadotrophins used for controlled ovarian stimulation (COS) adversely affects the selection of leading follicles and potentially impacts the quality of the eggs retrieved and the embryos created. The ploidy status of the embryo, as assessed by pre-implantation genetic testing (PGT), is used as a measure of embryo quality.
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Isom, S. C., J. R. Stevens, R. Li, L. D. Spate, W. G. Spollen, and R. S. Prather. "143 TRANSCRIPTIONAL PROFILING BY HIGH-THROUGHPUT SEQUENCING OF PORCINE PRE- AND PERI-IMPLANTATION EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 184. http://dx.doi.org/10.1071/rdv24n1ab143.
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Significant embryo mortality occurs at or around the time of implantation or attachment in virtually all mammalian species studied to date, even in naturally conceived embryos. Embryos resulting from assisted reproductive technologies (ART) are even more susceptible to peri-implantation failure. Herein we describe our effort to characterise the transcriptomes of embryonic disc (ED) and trophoblast (TE) cells from porcine embryos derived from AI, IVF, parthenogenetic oocyte activation (PA) and somatic cell nuclear transfer (NT) on Days 10, 12 and 14 of gestation. The IVF, PA and somatic cell NT embryos were generated using in vitro–matured oocytes, cultured overnight in vitro and then transferred at the 1- to 2-cell stage into appropriately synchronized recipient gilts. On the appropriate collection day, embryos were flushed from the uterus and ED was separated from TE by mechanical dissection. Double-stranded cDNA from the collected samples was sequenced using the GAII platform from Illumina (San Diego, CA, USA). The resulting sequencing reads were aligned to a custom swine transcriptome database (see Isom et al. 2010). A generalized linear model was fit for each of 41 693 genomic regions, for ED and TE samples separately, accounting for embryo type, gestation day and their interaction and using total lane read count as a normalizing offset. Genes with significant embryo type differences (controlling the false discovery rate at 0.10) were subsequently tested for differences between IVF and each of AI, PA and NT. Those genes with significant post hoc differences (either up- or down-regulated compared with IVF) were characterised in terms of gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using a gene set enrichment test. Bone morphogenetic protein signalling was down-regulated (KEGG; P = 0.0099; adjusted to control for FDR at 0.05) in the ED of IVF embryos when compared with AI embryos. In TE cells from IVF embryos, ubiquitin-mediated proteolysis and ErbB signalling (adj P = 0.031 for both pathways) were aberrantly regulated when compared with AI embryos. Of particular interest is the observation that expression of genes involved in chromatin modification (GO:BiologicalProcess; q-value = 0.00005) and epigenetic regulation of transcription (q = 0.00007) was very significantly disrupted in inner cell mass cells from NT embryos compared with IVF embryos. Surprisingly, no such disruption of the epigenetic machinery was observed in the TE cells from NT embryos. In summary, we have used high-throughput sequencing technologies to compare gene expression profiles of various ART embryo types during peri-implantation development. We expect that these data will provide important insight into the root causes of (and possible opportunities for mitigation of) suboptimal development of embryos derived from ART. Funding was received from NIH R01 RR013438 and Food for the 21st Century (RSP) and the Utah Agricultural Experiment Station (UTA00151 and UTA00560 for S. C. Isom and J. R. Stevens, respectively).
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Parashchuk, V. Y., A. S. Lutsky, and N. G. Gryshchenko. "The effectiveness of different protocols of preparation of the endometrium when transferring vitrified/warmed embryos." HEALTH OF WOMAN, no. 2(118) (March 29, 2017): 30–32. http://dx.doi.org/10.15574/hw.2017.118.30.
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Currently, there is a tendency toward increasing usage of criocycles in programs of in vitro fertilization. This approach allows to achieve pregnancy as well as prevent the development of ovarian hyperstimulation syndrome and utilize the pre-implantation genetic diagnostics. The objective: to improve the efficiency of in vitro fertilization treatment cycles with transferring of cryopreserved/warmed embryos into the uterus. Patients and methods. The study of 824 treatment cycles (in natural menstrual cycle, in cycle with hormone replacement therapy, after egg donation, in natural cycle without embryo transfer in «fresh» cycle, in cycle with hormone replacement therapy without embryo transfer in «fresh» cycle). Vitrification and cryopreservation method. Results. The analysis of the study results has shown that the implantation rate of frozen embryos in the natural cycle is higher than in the cycle with hormone replacement therapy. The implantation rate of frozen embryos after the use of agonist gonadotropin-releasing hormone as a trigger of final maturation of the agonist is higher than that of human chorionic gonadotropin, which allows us to consider vitrification as an effective tool that provides great flexibility of conducting controlled ovarian stimulation cycles and prevention of ovarian hyperstimulation syndrome. Conclusion. The effectiveness of frozen embryo transfer protocols gives us grounds to assert that the optimum cycle for the transfer is a cycle without stimulation. Key words: in vitro fertilization, the endometrium, frozen embryo implantation.
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Yang, Luhan, Claudia Baumann, Rabindranath De La Fuente, and Maria M. Viveiros. "Bisphenol Exposure Disrupts Cytoskeletal Organization and Development of Pre-Implantation Embryos." Cells 11, no. 20 (October 14, 2022): 3233. http://dx.doi.org/10.3390/cells11203233.
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The endocrine disrupting activity of bisphenol compounds is well documented, but less is known regarding their impact on cell division and early embryo formation. Here, we tested the effects of acute in vitro exposure to bisphenol A (BPA) and its common substitute, bisphenol F (BPF), during critical stages of mouse pre-implantation embryo development, including the first mitotic division, cell polarization, as well as morula and blastocyst formation. Timing of initial cleavage was determined by live-cell imaging, while subsequent divisions, cytoskeletal organization and lineage marker labeling were assessed by high-resolution fluorescence microscopy. Our analysis reveals that brief culture with BPA or BPF impeded cell division and disrupted embryo development at all stages tested. Surprisingly, BPF was more detrimental to the early embryo than BPA. Notably, poor embryo development was associated with cytoskeletal disruptions of the actomyosin network, apical domain formation during cell polarization, actin ring zippering for embryo sealing and altered cell lineage marker profiles. These results underscore that bisphenols can disrupt cytoskeletal integrity and remodeling that is vital for early embryo development and raise concerns regarding the use of BPF as a ‘safe’ BPA substitute.
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Filby, A., M. Mitchell, A. Georgiou, and M. Lane. "128. A ROLE FOR SIRTUIN 3 IN THE DEVELOPING MAMMALIAN EMBRYO." Reproduction, Fertility and Development 21, no. 9 (2009): 47. http://dx.doi.org/10.1071/srb09abs128.
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Pre-implantation embryo development relies critically on the balance between cytoplasmic and mitochondrial metabolism for the generation of metabolic intermediates such as NAD+. SIRT3 is a mitochondrial sirtuin with NAD+-dependant deacetylase activity that, targets glutamate dehydrogenase (GDH). In this study we characterised SIRT3 mRNA, protein and activity through pre-implantation development and determined whether modulation of SIRT3 activity influenced GDH activity. Embryos (zygotes, 2-cell, 8-cell and blastocyst stages) were recovered from female CBA/C57Bl6 mice following ovarian stimulation and mating with CBA/C57Bl6 males. Expression of SIRT3 mRNA was measured using real-time RTPCR, protein localisation examined using immunohistochemistry and SIRT3 activity measured using a Fluor-de-Lys SIRT3 fluorescentassay. Functional GDH activity was assessed in 2-cell embryos indirectly by measuring glutamine oxidation, following culture from zygote to 2-cell in the presence of nicotinamide, (a sirtuin inhibitor), G1.2 media, or simpleG1 media, compared to in vivo controls. SIRT3 mRNA was detected at all stages of development, with significantly greater levels expressed in the blastocyst. SIRT3 protein was localised predominantly around the nucleus of zygote and 2-cell embryos, and was mainly cytoplasmic in 8-cell embryos and blastocysts. SIRT3 activity remained constant throughout pre-implantation development, and tended to increase at the blastocyst stage. Glutamine oxidation was reduced for embryos cultured in G1.2 media relative to in vivo controls (0.14 pmol/e/hr vs 0.21pmol/e/hr), and this was further reduced by the addition of nicotinamide (0.07pmol/e/hr). Embryo culture in perturbing simpleG1 increased glutamine metabolism (0.33pmol/e/hr). In conclusion, SIRT3 mRNA, protein and activity was detected throughout pre-implantation development. Modulation of sirtuins by nicotinamide decreased glutamine metabolism, likely as a result of decreased deacetylation, thus decreased activity of GDH. SIRT3 can translocate to the mitochondria during cellular stress, thus the increased glutamine metabolism in simpleG1 conditions may be caused by translocation of SIRT3 to mitochondria, potentially increasing GDH deacetylation and enzymatic activity.
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Wu, Xiaoli, Sumit Sandhu, Nehal Patel, Barbara Triggs-Raine, and Hao Ding. "EMG1 is essential for mouse pre-implantation embryo development." BMC Developmental Biology 10, no. 1 (2010): 99. http://dx.doi.org/10.1186/1471-213x-10-99.
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Hupalowska, Anna, Agnieszka Jedrusik, Meng Zhu, Mark T. Bedford, David M. Glover, and Magdalena Zernicka-Goetz. "CARM1 and Paraspeckles Regulate Pre-implantation Mouse Embryo Development." Cell 175, no. 7 (December 2018): 1902–16. http://dx.doi.org/10.1016/j.cell.2018.11.027.
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Chin, P. Y., J. G. Thompson, and S. A. Robertson. "Programming embryo development with a pre-implantation inflammatory insult." Journal of Reproductive Immunology 86, no. 1 (August 2010): 39. http://dx.doi.org/10.1016/j.jri.2010.06.075.
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Cram, D., and H. Leese. "Session 35: The Health of the Pre-Implantation Embryo." Human Reproduction 25, Supplement 1 (June 1, 2010): i52—i53. http://dx.doi.org/10.1093/humrep/de.25.s1.35.
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Alyafee, Yusra, Qamre Alam, Abeer Al Tuwaijri, Muhammad Umair, Shahad Haddad, Meshael Alharbi, Hayat Alrabiah, Maha Al-Ghuraibi, Sahar Al-Showaier, and Majid Alfadhel. "Next-Generation Sequencing-Based Pre-Implantation Genetic Testing for Aneuploidy (PGT-A): First Report from Saudi Arabia." Genes 12, no. 4 (March 24, 2021): 461. http://dx.doi.org/10.3390/genes12040461.
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Recently, high-throughput next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies techniques came into use. This technique is essential for successful embryo transfer and accomplishing pregnancy, thus reducing the time and cost of additional cycles. In this study, we describe our first experience in introducing an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) service using next-generation sequencing in King Abdulaziz Medical City located in Riyadh, Saudi Arabia. Our main goal was to report the successful implementation of this new technology in clinical practice and highlight the factors that may affect the results. In total, 200 blastomere biopsies were obtained from 36 in vitro fertilization (IVF) cycles from Saudi couples suffering from prolonged infertility or recurrent embryo transfer failure. NGS-based PGT-A was performed in all embryos. The results were analyzed in five age groups, showing that aneuploidy rates increased with maternal age. Moreover, the results also showed that complex abnormal embryos with (2–5) aneuploidy are the most common type of embryos. Additionally, our data showed that chromosome 16-related abnormality was the most frequent abnormality detected among all reported abnormalities. In conclusion, our study suggests that NGS-based PGT-A is an applicable and reliable technique for routine-based embryo screening, especially for couples suffering from recurrent miscarriages or multiple embryo transfer failures.
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Ortega, N., V. Ahola, A. Plaza-Reyes, J. Schell, P. Kumar, A. Jouneau, V. Duranthon, and F. Lanner. "56 Mammalian pre-implantation embryos at the single cell level: The bovine as a model for early human embryonic development." Reproduction, Fertility and Development 32, no. 2 (2020): 153. http://dx.doi.org/10.1071/rdv32n2ab56.
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Single-cell transcriptomics and gene editing on human pre-implantation embryos have proved that mechanisms previously identified and well characterised in mouse pre-implantation development may not hold true in the human embryo. However, ethical and legal concerns limit the availability of surplus human embryos for research, resulting in the need for novel animal models. Bovine embryos share morphological and temporal resemblance with human early development; still, key molecular and cellular developmental mechanisms need to be further explored. In the present study, we performed a comparative single-cell RNA sequencing analysis across multiple pre-implantational developmental stages of invitro-derived bovine, human (Petropoulos et al. 2016 Cell 165, 1012-1026; https://doi.org/10.1016/j.cell.2016.03.023), and mouse embryos (Cheng et al. 2019 Cell Rep. 26, 2593-2607, https://doi.org/10.1016/j.celrep.2019.02.031; Posfai et al. 2017 eLife 6, e22906, https://doi.org/10.7554/eLife.22906; Chen et al. 2016 Genome Res. 26, 1342-1354). In total 497 blastomeres corresponding to embryonic days (E) E4-E8 were prepared using Smart sEqn 2, Illumina HiSEqn 2500 sequencing with 50-bp single-end reads. Embryonic stage was defined by cell numbers and morphology as multicellular stage (Mu, ~E4), morulae (M; E5), early blastocysts (EB, ~6), mid-expanded blastocysts, (MB, ~E7), and late expanded/hatched blastocysts (LB, ~E8). We found top differentially expressed genes elucidating how lineage specification and pluripotency is controlled in the early bovine embryo. Our transcriptomic data showed that the first lineage segregation in the pre-implantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and trophectoderm (TE) profiles. Different from the mouse, where the first lineage segregation of TE-ICM is suggested to occur at the morula stage due to CDX2-OCT4 antagonism, we found that the expression of OCT4 protein cattle expanded blastocysts is not restricted to the ICM, similar to what has been reported to occur in the human embryo. Interestingly, we also found that the second lineage segregation, which segregates the epiblast from the primitive endoderm within the ICM, starts in bovine at E8 expanded hatched late blastocysts, suggesting a potential difference with humans, where TE-ICM and epiblast-primitive endoderm has previously been reported to occur almost concurrently. We are now complementing our findings with genome editing in bovine embryos by generating knockout embryos of different target genes part of an evolutionarily conserved signaling pathway to study their effects in the early pre-implantation embryo. We aim to elucidate how early lineage specification and pluripotency establishment occurs while offering theoretical support for efficient derivation and culture of bovine embryonic stem cells.
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Choi, Y. H., D. L. Hartman, and K. Hinrichs. "134 VIABILITY OF EQUINE BLASTOCYSTS SUBJECTED TO BIOPSY FOR PRE-IMPLANTATION GENETIC DIAGNOSIS." Reproduction, Fertility and Development 21, no. 1 (2009): 166. http://dx.doi.org/10.1071/rdv21n1ab134.
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Pre-implantation genetic diagnosis of embryos is commonly performed in humans but has not been developed in the horse. To our knowledge, only one previous report of transfer of equine embryos after biopsy is available (Huhtinen et al. 1997 Theriogenology 48, 361); in that study, three pregnancies were achieved after transfer of 14 embryos. Failure of pregnancy after biopsy of equine embryos has been attributed to difficulty in penetrating the equine embryonic capsule, and possible effects of capsule damage on subsequent embryo survival. Recent developments in intracytoplasmic sperm injection (ICSI) and embryo culture make it possible to produce equine blastocysts in vitro. In vitro-produced equine embryos lack a capsule, but form one when transferred to the uterus. This study was designed to examine the viability of equine blastocysts subjected to biopsy. In Study 1, in vitro- and in vivo-produced embryos were vitrified after biopsy, as would be needed while awaiting results of genetic testing. In vitro-produced blastocysts (n = 5) were obtained from slaughterhouse-derived oocytes that were matured, fertilized by ICSI, and cultured 7 to 8 days. In vivo-recovered blastocysts (n = 9) were obtained by uterine flush of inseminated mares 6.5 days after ovulation. The biopsy procedure was conducted using a Piezo drill in modified PBS without calcium and magnesium. Two to three biopsy manipulations were conducted on each embryo to ensure that an adequate number of cells was obtained. After biopsy, embryos were vitrified as described by Eldridge-Panuska et al. (2005 Theriogenology 63, 1308). Four vitrified in vivo-produced blastocysts were warmed and cultured for 2 days. Of these, two appeared viable, showing a 50- to 100-μm increase in diameter, and two did not grow. The remaining 5 in vivo- and 5 in vitro-produced vitrified blastocysts were shipped to a transfer facility, warmed and transferred transcervically to recipient mares. None of these embryos established pregnancy. In Study 2, three Day-6 in vivo-recovered blastocysts were biopsied then shipped by air (6 h transport time) for transfer to recipient mares. All three embryos established pregnancies and showed a normal heartbeat at 25 days. These results indicate that cells may be obtained from in vitro and in vivo-produced equine blastocysts by biopsy with the Piezo drill. In vivo-produced blastocysts transferred directly after biopsy were viable (3/3). While some blastocysts vitrified after biopsy showed growth in vitro, vitrified embryos did not produce pregnancies after transfer. To our knowledge, this is the first report of normal pregnancy rate after transfer of biopsied equine embryos. Further studies are needed to improve survival of embryos vitrified after biopsy. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.
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Nishii, Kiyomasa, Yasushi Kobayashi, and Yosaburo Shibata. "Absence of connexin43 and connexin45 does not disturb pre- and peri-implantation development." Zygote 24, no. 3 (July 21, 2015): 457–64. http://dx.doi.org/10.1017/s0967199415000386.
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SummaryGap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43−/−Cx45−/− embryos by Cx43+/−Cx45+/− intercrossing, because mice deficient in Cx43 (Cx43−/−) exhibit perinatal lethality and those deficient in Cx45 (Cx45−/−) exhibit early embryonic lethality. Wild-type, Cx43−/−, Cx45−/−, and Cx43−/−Cx45−/− blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43−/−Cx45−/− embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45−/− mutation proved lethal. The Cx43−/−Cx45−/− embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.
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Lavranos, TC, and RF Seamark. "Addition of steroids to embryo-uterine monolayer co-culture enhances embryo survival and implantation in vitro." Reproduction, Fertility and Development 1, no. 1 (1989): 41. http://dx.doi.org/10.1071/rd9890041.
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Co-culture with a mixed cell monolayer established from trypsinized uterine tissue increased the numbers of embryos developing in a minimum essential medium beyond the hatching blastocyst stage in the 5-day period of culture to 56.0% (343/613) compared with 30.2% (93/308) for embryos cultured in medium alone. The inclusion of progesterone (3.2 x 10(-6), 3.2 x 10(-5), or 3.2 x 10(-4) M) or oestradiol (3.7 x 10(-5) M) to the co-cultures increased the mean percentage of embryos developing to the advanced stages to 72.7-78.4%. The addition of progesterone (3.2 x 10(-6) M) together with oestradiol (3.7 x 10(-5) M) resulted in additional improvement to a mean of 86.5% (787/910, P less than 0.001) but the combination was without significant effect on embryos cultured in media alone. Blastocyst viability was not impaired by co-culture as assessed in embryo survival following surgical transfer to pseudopregnant recipients. This study confirms the feasibility of establishing co-culture systems to facilitate investigation of pre-implantation events in vitro and highlights a role of steroid hormones in enhancing the capacity of uterine cells to support pre-implantation-stage embryos.
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Bauersachs, S., V. Zakhartchenko, S. E. Ulbrich, F. Sinowatz, H. D. Reichenbach, H. Blum, and E. Wolf. "102 EVIDENCE FOR PRE-IMPLANTATION ORIGIN OF PLACENTAL FAILURE IN BOVINE CLONE PREGNANCIES." Reproduction, Fertility and Development 21, no. 1 (2009): 151. http://dx.doi.org/10.1071/rdv21n1ab102.
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Placental abnormalities account for a high proportion of pregnancy loss after transfer of cloned (SCNT) bovine embryos. The high rate of pregnancy failure has been linked to the finding of abnormal placental formation and function. Because bovine SCNT embryos were shown to exhibit altered trophoblast differentiation (Arnold DR et al. 2006 Reproduction 132, 279–290), we hypothesized that placental abnormalities in bovine clone pregnancies may originate from disturbed embryo-maternal communication in the pre-implantation period. To test this hypothesis, we evaluated the response of the endometrium to SCNT v. IVF embryos. The SCNT embryos were produced from fibroblast cultures derived from 4 different fetuses to exclude specific effects of a particular donor cell culture. After SCNT or IVF, embryos were cultured under identical conditions. Two SCNT or IVF blastocysts (grade 1) were transferred per recipient heifer (Day 8 of estrous cycle). Ten days later the recipients were slaughtered, the uteri were recovered, and pregnancy was verified by the presence of at least one normally developed embryo. Endometrium samples of 9 SCNT and 10 IVF pregnancies were used for transcriptome profiling with a custom cDNA microarray (BOE array; Bauersachs S et al. 2007 J. Dairy Sci. 90, 4420–4423). In total, 58 transcripts were found to be differently abundant between endometrium samples from SCNT v. IVF pregnancies (SAM, FDR 5.24%). Interestingly, for some of them an important role in implantation and/or placentation has already been shown. NR2F2, encoding the orphan nuclear receptor superfamily member NR2F2 (COUP-TFII), was downregulated in endometrium from SCNT pregnancies (1.5-fold; P < 0.01). Uterine-specific Nr2f2 mutant mice are infertile due to implantation failure (Kurihara I et al. 2007 PLoS Genetics 3, e102). Another interesting candidate is GJA1, encoding connexin 43 (Cx43), with 1.8-fold (P < 0.001) downregulated transcript levels in SCNT pregnancies. A striking increase of stromal Cx43 has been observed in the ovine intercaruncular and caruncular endometrium during intensification of the feto-maternal contact (Gabriel et al. 2005 Placenta 25, 287–296), suggesting that reduced GJA1 mRNA expression in bovine clone pregnancies may negatively affect placentation. In view of the well-orchestrated spectrum of transcriptome changes in endometrium during the peri-attachment period (Bauersachs S et al. 2006 Reproduction 132, 319–331), these findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period. This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478).
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Kwong, W. Y., S. J. Adamiak, A. Gwynn, R. Singh, and K. D. Sinclair. "Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre-implantation embryo." REPRODUCTION 139, no. 4 (April 2010): 705–15. http://dx.doi.org/10.1530/rep-09-0517.
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Maternal B-vitamin status at conception can affect fertility and the health of offspring. This study details transcript expression for genes encoding key enzymes in the linked methionine/folate cycles in the bovine oocyte, somatic cells of the ovarian follicle and pre-implantation embryo. Transcripts for all 12 enzymes that were studied and for the two folate receptors (FOLR1andFOLR2) and reduced folate carrier (SLC19A1) were expressed in liver cells, but transcripts for betaine-homocysteine methyltransferase and methionine adenosyl transferase 1A were absent in all ovarian cells, and transcripts forFOLR2were absent in embryonic cells. Transcripts for glycine methyltransferase were also absent/weak in cumulus and granulosa cells. The absence of these enzymes could have a profound effect on single-carbon metabolism within the ovary and pre-implantation embryo. Immunocytochemical analysis revealed SLC19A1 protein expression on the plasma and basal-lateral membranes of the pre-implantation embryo. The folate antagonist methotrexate (MTX) enters the cell via SLC19A1, and in the current study, MTX inclusion in bovine/ovine culture media at either 1 or 10 μM from the 1-cell stage inhibited embryo development beyond the 8-cell stage. Hypoxanthine and thymidine (100 μM) increased the proportion of embryos that developed to blastocysts, but the cell number was reduced by 20%. The reduced uptake of [35S] methionine into intra-cellularS-adenosylmethionine andS-adenosylhomocysteine pools, together with reduced uptake of glutamate and tryptophan, was consistent with depleted intra-cellular pools of reduced folates. These data provide an insight into the importance of maternal dietary folate/B-vitamin status during the peri-conceptional period.
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Sayed, Shabana, Marte Myhre Reigstad, Bjørn Molt Petersen, Arne Schwennicke, Jon Wegner Hausken, and Ritsa Storeng. "Time-lapse imaging derived morphokinetic variables reveal association with implantation and live birth following in vitro fertilization: A retrospective study using data from transferred human embryos." PLOS ONE 15, no. 11 (November 19, 2020): e0242377. http://dx.doi.org/10.1371/journal.pone.0242377.
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The purpose of this retrospective time-lapse data analysis from transferred preimplantation human embryos was to identify early morphokinetic cleavage variables that are related to implantation and live birth following in vitro fertilization (IVF). All embryos were monitored from fertilization check until embryo transfer for a minimum of 44 hours. The study was designed to assess the association between day 2 embryo morphokinetic variables with implantation and live birth based on Known Implantation Data (KID). The kinetic variables were subjected to quartile-based analysis. The predictive ability for implantation and live birth was studied using receiver operator characteristic (ROC) curves. Three morphokinetic variables, time to 2-cells (t2), duration of second cell cycle (cc2) below one threshold and cc2 above another threshold had the highest predictive value with regards to implantation and live birth following IVF treatment. The predictive pre-transfer information has little divergence between fetal heartbeat and live birth data and therefore, at least for early morphokinetic variables up to the four-cell stage (t4), conclusions and models based on fetal heartbeat data can be expected to be valid for live birth datasets as well. The three above mentioned variables (t2, cc2 below one threshold and cc2 above another threshold) may supplement morphological evaluation in embryo selection and thereby improve the outcome of in vitro fertilization treatments.