Evolution of a culture protocol for successful blastocyst development and pregnancy

Jones, Gayle M; Trounson, Alan; Gardner, David K.; Kausche, Annette; Lolatgis, Nicholas; Wood, E. C.

doi:10.1093/humrep/13.1.169

Cited by 211 publications

(95 citation statements)

References 46 publications

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“…fragmentation [35,36]. Examination of mtDNA sequence from the HVII region showed that one SCNT blastocyst (coded reference K8) was matched to egg donor 3 (coded references F1 and F2) mtDNA and was identical to the parthenogenetic embryos (coded references K1 and K6) obtained in the same experimental series.…”

Section: Discussionmentioning

confidence: 93%

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

French¹,

et al. 2008

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Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20 -24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer. STEM CELLS 2008;26:485-493 Disclosure of potential conflicts of interest is found at the end of this article.

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Section: Discussionmentioning

confidence: 93%

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

French¹,

et al. 2008

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“…Embryo culture at 5% O2 has shown to produce more blastocysts per cycle with higher mean cell numbers compared to 20% O2, where significantly more blastocysts had abnormally lower cell number (<25) [30]. An efficient development to the blastocyst stage has been achieved for human [31] and bovine [32] embryos using a culture protocol of reduced O2 (5%). Subsequent studies [33,34] have confirmed that blastocysts consume two to four times more O2 than cleavage stage embryos.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

Farouk

Vlad

2008

Journal of Reproduction and Development

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Abstract. The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects. Key words: Blastocyst, Co-culture, Mouse, Pronuclear embryo, Sequential culture medium (J. Reprod. Dev. 54: [385][386][387][388][389][390] 2008) t has been suggested that the co-culture system exerts its beneficial effects by negative [1][2][3][4][5] or positive conditioning [4,[6][7][8][9][10][11] of the culture media, and by embryo-to-cell contact. Negative conditioning or detoxification is the ability of the feeder cells to remove deleterious components from the culture medium whereas positive conditioning is the ability of the feeder cells to modify it by releasing some components (embryotrophic factors) into the culture medium [12,13].There have been studies proposing that embryo-to-cell contact is necessary to allow cellular connections between the zona pellucida and feeder cells for cross-transfer of glycoproteins and other important metabolites [12]. In the cumulus oocyte complex, gap junctions have been shown to be present in the regions of contact between the cumulus cell projections and the oolemma, as well as between adjacent cumulus and granulosa cells [14,15]. Nevertheless, whether gap junctions are present ...

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“…As a routine method in clinical IVF, blastocyst culture and transfer has proved to bring in a significantly increase in implantation [10], which helps to select embryos with high developmental potential and chromosomally normal embryos [11]. Although there have been several case reports about transferring 1PN blastocysts and achieving normal live-birth, the chromosomal constitution of 1PN blastocyst still remains uncertain.…”

Section: Introductionmentioning

confidence: 99%

Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes

Liao

Zhang

Cheng

et al. 2009

J Assist Reprod Genet

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Purpose By comparing the chromosomal constitution among the arrested cleavage-stage embryos, blastocysts and human embryonic stem cells (hESCs) which are all derived from monopronuclear (1PN) zygotes, it is aimed to determine whether chromosomally normal embryos can be reliably selected by blastocyst culture. Methods After 1PN zygotes are sequentially cultured for 5 days, the blastocysts and arrested cleavage-stage embryos were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosomes 18, X and Y; G-banding analysis was adopted to analyze the karyotype of 1PN hESCs.Results The diploid rate of blastocysts was 74.6%, which was significantly (P<0.001) higher than that of arrested cleavage-stage embryos (31.6%), and the diploid rate of hESCs was 97.0%, which was significantly (P < 0.01) higher than that of blastocysts; the haploid embryos were excluded by blastocyst culture; nevertheless, there still existed such chromosomal abnormalities as mosaic and monosomic in blastocysts and trisomy in hESCs. Conclusions Blastocyst culture is an effective method to select against chromosomal abnormalities, especially the haploids in 1PN embryos; however, development to the blastocyst stage is not a reliable marker for mosaicism or aneuploidy.

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Evolution of a culture protocol for successful blastocyst development and pregnancy

Cited by 211 publications

References 46 publications

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes

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