New micromanipulative techniques in reproductive biology

New micro manipulative techniques have facilitated development to help patients who suffer from infertility and spontaneous abortion, as well as those who carry severe genetic diseases and have risks of delivering an affected child. Preimplantation genetic diagnosis (PGD), which is based on obtaining blastomere biopsy from an eight-cell embryo, has made it possible to diagnose severe defects caused by a genetic factor. In the animal laboratory, ovarian transplantation is a new technique that provides a tool in research to save valuable mutants and transgenic animals that have an infertility not caused by germ cells. The infertility can be due to prepubertal lethality, and inability to mate or incapacity to complete a successful fertilization, gestation or parturition. The creation of a technique for transfer of mouse embryos into germ free recipients to produce gnotobiotic mice will facilitate researchers to study the micro flora of transgenic and special mutants more specifically. In this thesis we improved three micro manipulative techniques and the possibilities of applying them in research laboratories and in in-vitro fertilization units. 1) We created an improvement in the embryo biopsy micro technique that is used to allow the preimplantation genetic diagnosis (PGD) using a single-needle to perform the blastomere biopsy. 2) We used the ovarian transplantation micro-surgical technique to save valorous mutants and transgenic animals when the reason for infertility was other than non-functional germ cells. 3) We created a new micro technique to allow transfer of transgenic animals to a germ free state, through the embryo transfer into a germ-free or gnotobiotic recipient. We tested and successfully applied a single-needle approach to obtain blastomere biopsies from human preimplantation embryos for preimplantation genetic diagnosis (PGD). The method was first evaluated in a mouse system and shown to be compatible with a high degree of in-vitro and in-vivo development of biopsied mouse embryos. Biopsied mouse embryos after transfer to recipient mice underwent implantation, normal development and delivery. Successful human preimplantation diagnosis, followed by pregnancies and birth of healthy babies, was established with two out of three couples carrying a risk to transmit chromosomal abnormalities leading to severe diseases. This is the first report of the use of a single-needle approach in human PGD. We evaluated an ovary transplantation method by transplanting ovaries from females belonging to a non-reproductive BALB/cByJ mutant mouse strain. All transplanted mice, BALB/c.C57BL/6By, produced offspring and 94 % of the progeny originated from the transplanted ovaries. The mean litter size and the mating period needed for productive mating to occur were similar to what is observed for corresponding control mice. Subsequently we also performed ovary transplantation of estrogen beta-receptor knockout (ERbeta-/-) ovaries into wild type and normal C57B1/6 recipient mice. The litters' sizes were similar after transplantation of ERbeta-/- and WT ovaries to normal recipients, hence referring to an extra-ovarian cause of subfertility. Lastly we demonstrated that it is possible to obtain germ-free animals using embryo transfer of conventional or transgenic mice to germ-free recipients inside a steel isolator. We have developed a unique embryo transfer technique that will facilitate researchers to use this procedure as a tool in changing the status of different mutants into germ-free mice. All these three techniques are powerful tools in the field of reproductive medicine and biology