Microsurgical bisection of porcine morulae and blastocysts to produce monozygotic twin pregnancy

Nagashima, Hiroshi; Kato, Yukio; Ogawa, Shyoso

doi:10.1002/mrd.1120230102

Cited by 26 publications

(7 citation statements)

References 22 publications

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“…Porcine morulae and blastocysts having half the normal cell number after embryo-splitting have been shown to be competent to develop to normal offspring [42,43]. On the basis of this observation, porcine morulae and blastocysts cryopreserved after centrifugation are likely to maintain their viability, provided that the proportion of damaged cells does not exceed the half.…”

Section: Discussionmentioning

confidence: 99%

Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification.

Nagashima

Cameron

Kuwayama

et al. 1999

Journal of Reproduction and Development

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Abstract. The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided.

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Section: Discussionmentioning

confidence: 99%

Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification.

Nagashima

Cameron

Kuwayama

et al. 1999

Journal of Reproduction and Development

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“…However different mammalian embryos have demonstrated a high ability for embryonic regulation at this stage. For example, microsurgical splitting of bovine, goat, porcine, and murine morulae does not adversely affect embryonic development or birth rate [35], [36], [37], [38], [39].…”

Section: Discussionmentioning

confidence: 99%

Biopsy of Human Morula-Stage Embryos: Outcome of 215 IVF/ICSI Cycles with PGS

Zakharova¹,

Zaletova²,

Krivokharchenko³

2014

PLoS ONE

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Preimplantation genetic diagnosis (PGD) is commonly performed on biopsies from 6–8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS), and 3–7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21) were screened by fluorescence in situ hybridization (FISH). No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5–6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5–6 in the current IVF cycle.

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“…In the present study, we evaluated the effectiveness of the HFV method using IVM-derived porcine morulae. Porcine embryos are known to be highly cryosensitive [37,38], and IVM-derived porcine morulae are even more cryosensitive than in vivo derived embryos. To date, successful application of cryopreservation with IVM-derived porcine morulae has required treating the embryos to increase their cryotolerance by removing cytoplasmic lipid droplets, i.e., delipation [30,39].…”

Section: Discussionmentioning

confidence: 99%

Hollow Fiber Vitrification: A Novel Method for Vitrifying Multiple Embryos in a Single Device

Matsunari

Maehara

Nakano

et al. 2012

Journal of Reproduction and Development

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Abstract. Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 µl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/ group). The HFV method was also proven to be effective for vitrifying porcine in vitro-and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs. Key words: Cryopreservation, Hollow fiber, Mouse embryos, Pig embryos, Vitrification (J. Reprod. Dev. 58: [599][600][601][602][603][604][605][606][607][608] 2012) E mbryo cryopreservation is widely and routinely used both for research in reproductive biology and for the development of practical applications in animal industries and human reproductive medicine. The cryopreservation of oocytes and embryos is essential for the long-term preservation of valuable genetic resources in experimental and livestock animals [1][2][3][4][5], and the cryopreservation of embryos improves pregnancy rates in assisted reproductive technology applications [6][7][8]. Various protocols have been developed for the cryopreservation of animal embryos, and many are used in research and clinical applications [1,5,9].In recent years, embryo vitrification has been applied to an increasing number of animal species, and high post-cryopreservation embryo viability has been achieved [10,11]. The basic concept of vitrification is as follows: a solution containing a high concentration of a cryoprotective agent (CPA) is rapidly cooled, causing it to transform from the liquid phase to the solid phase without forming ice crystals. As a result, cells suspended in the solution are preserved in an ultra-low temperature glassy (amorphous) material [10,12]. To create this glassy state, a solution containing a high concentration of CPA (normally 4-6 M) needs to be cooled ultrarapidly. Howeve...

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Microsurgical bisection of porcine morulae and blastocysts to produce monozygotic twin pregnancy

Cited by 26 publications

References 22 publications

Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification.

Survival of Porcine Delipated Oocytes and Embryos after Cryopreservation by Freezing or Vitrification.

Biopsy of Human Morula-Stage Embryos: Outcome of 215 IVF/ICSI Cycles with PGS

Hollow Fiber Vitrification: A Novel Method for Vitrifying Multiple Embryos in a Single Device

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