The Well-of-the-Well system: an efficient approach to improve embryo development.

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.

[1]  G. Vajta,et al.  A comparison of established and new approaches in ovine and bovine nuclear transfer. , 2003, Cloning and stem cells.

[2]  D. Gardner,et al.  Culture and transfer of viable blastocysts: a feasible proposition for human IVF. , 2000, Human reproduction.

[3]  P. Holm,et al.  Application of the zona-free manipulation technique to porcine somatic nuclear transfer. , 2001, Cloning and Stem Cells.

[4]  T. Peura.  Improved in vitro development rates of sheep somatic nuclear transfer embryos by using a reverse-order zona-free cloning method. , 2003, Cloning and stem cells.

[5]  P. Devroey,et al.  The relationship between embryo quality and the occurrence of multiple pregnancies , 1992, Fertility and sterility.

[6]  L. Gianaroli,et al.  Human embryo co-culture: results of a randomized prospective study. , 1995, International journal of fertility and menopausal studies.

[7]  E. C. Wood,et al.  Evolution of a culture protocol for successful blastocyst development and pregnancy. , 1998, Human reproduction.

[8]  B. Bavister,et al.  Culture of preimplantation embryos: facts and artifacts. , 1995, Human reproduction update.

[9]  J. Rossant,et al.  Non-injection methods for the production of embryonic stem cell-embryo chimaeras , 1993, Nature.

[10]  Zsolt Peter Nagy,et al.  High fertilization and implantation rates after intracytoplasmic sperm injection , 1993 .

[11]  P. Mermillod,et al.  In vitro production of bovine embryos using individual oocytes , 1996, Molecular reproduction and development.

[12]  C. Feltrin,et al.  35 In vitro bovine embryo development after nuclear transfer by handmade cloning using a modified WOW culture system. , 2006 .

[13]  Thomas B Pool.  Recent advances in the production of viable human embryos in vitro. , 2002, Reproductive biomedicine online.

[14]  L. Bolund,et al.  Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation. , 2005, Theriogenology.

[15]  B. Kovačič,et al.  Comparable effectiveness using flexible single-dose GnRH antagonist (cetrorelix) and single-dose long GnRH agonist (goserelin) protocol for IVF cycles--a prospective, randomized study. , 2003, Reproductive biomedicine online.

[16]  G. Vajta,et al.  Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the Handmade Cloning (HMC) and the Submarine Incubation System (SIS). , 2004, Theriogenology.

[17]  D. Gardner,et al.  Culture and selection of viable blastocysts: a feasible proposition for human IVF? , 1997, Human reproduction update.

[18]  K. Yoshioka,et al.  Birth of Piglets Derived from Porcine Zygotes Cultured in a Chemically Defined Medium1 , 2002, Biology of reproduction.

[19]  A. Trounson,et al.  The 'GO' system--a novel method of microculture for in vitro development of mouse zygotes to the blastocyst stage. , 2003, Reproduction.

[20]  R C Burghardt,et al.  Viability and ultrastructure of equine embryos following culture in a static or dynamic system. , 1991, Journal of reproduction and fertility. Supplement.

[21]  D. Gardner,et al.  Amino acids and ammonium regulate mouse embryo development in culture. , 1993, Biology of reproduction.

[22]  J. M. Wilson,et al.  Hormone secretion by preimplantation embryos in a dynamic in vitro culture system. , 1992, Biology of reproduction.

[23]  L. Bolund,et al.  Production of transgenic porcine blastocysts by hand-made cloning , 2004 .

[24]  G. Vajta,et al.  Hand-made cloning approach: potentials and limitations. , 2005, Reproduction, fertility, and development.

[25]  G. Vajta,et al.  Somatic cell cloning without micromanipulators. , 2001, Cloning.

[26]  Velimir Šimunić,et al.  Izvantjelesna oplodnja i ostale metode pomognute oplodnje , 2001 .

[27]  L. Bolund,et al.  High overall in vitro efficiency of porcine handmade cloning (HMC) combining partial zona digestion and oocyte trisection with sequential culture. , 2005, Cloning and stem cells.

[28]  D. Gardner,et al.  Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. , 1992, Human reproduction.

[29]  S. McKiernan,et al.  Regulation of Hamster Embryo Development In Vitro by Amino Acids , 1993 .

[30]  P. Holm,et al.  High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. , 1999, Theriogenology.

[31]  Z. Nagy,et al.  The influence of the site of sperm deposition and mode of oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates. , 1995, Human reproduction.

[32]  J. Lewis,et al.  An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. , 1989, Journal of reproduction and fertility.

[33]  G. Vajta,et al.  New method for culture of zona‐included or zona‐free embryos: The Well of the Well (WOW) system , 2000, Molecular reproduction and development.

[34]  C. Galli,et al.  Birth of cloned pigs from zona-free nuclear transfer blastocysts developed in vitro before transfer. , 2006, Cloning and stem cells.

[35]  A. Gutiérrez-Adán,et al.  Effect of culture environment on embryo quality and gene expression - experience from animal studies. , 2003, Reproductive biomedicine online.

[36]  P. Holm,et al.  In vivo versus in vitro produced bovine ova: similarities and differences relevant for practical application. , 1998, Reproduction, nutrition, development.

[37]  G. Vajta,et al.  Birth of a cloned calf derived from a vitrified hand-made cloned embryo. , 2003, Reproduction, fertility, and development.

[38]  Usa Serono Symposia,et al.  Preimplantation Embryo Development , 1993, Serono Symposia, USA Norwell, Massachusetts.

[39]  Dk Gardner,et al.  In-vitro culture of human blastocysts , 1999 .

[40]  S. Stice,et al.  In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors. , 1994, Theriogenology.

[41]  M. O'connor,et al.  Preimplantation stages of pregnancy. , 1965 .

[42]  Mohammad NasrEsfahani,et al.  Radical solutions and cultural problems: Could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[43]  D. Gardner,et al.  Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. , 1998, Fertility and sterility.

[44]  D. Gardner,et al.  Culture of viable human blastocysts in defined sequential serum-free media. , 1998, Human reproduction.

[45]  J. J. Robinson,et al.  Culture of sheep zygotes can alter fetal growth and development , 1997 .

[46]  I. Wilmut,et al.  Modifications to improve the efficiency of zona-free mouse nuclear transfer. , 2006, Cloning and stem cells.