Preimplantation genetic diagnosis of X-linked diseases examined by indirect linkage analysis.

BACKGROUND: Many centers of assisted reproduction in the Czech Republic offer preimplantation genetic diagnosis with fluorescent in situ hybridization (FISH) to couples requiring preimplantation genetic diagnosis (PGD) of X-linked diseases. However, this process results in discarding all male embryos and is not able to distinguish a carrier or healthy female embryo in X-linked recessive disorders. OBJECTIVES: The main aim of this study was to summarize a six-year period of PGD of X-linked monogenic diseases using indirect linkage analysis. METHODS AND RESULTS: We wanted to accentuate the advantage indirect analysis of PGD using multiple displacement amplification (MDA) followed by short tandem repeat (STR) analysis. We present forty-six PGD cycles, including pre-case haplotyping (PGH) panel, for fifteen X-linked diseases. Embryo transfer was made thirty-eight times and gravidity was confirmed in thirteen female probands with a success rate of pregnancy calculated at 42 %. CONCLUSIONS: PGD procedure using MDA amplification followed by STR analysis provides help in identifying genetic defects within embryos prior to implantation. The reliability of the method was also supported by high pregnancy rate compared to other publications, which commonly achieved a 30-35 % success rate (Tab. 2, Fig. 1, Ref. 33).

Effect of protein supplement source on porcine oocyte maturation and subsequent embryonic development after parthenogenetic activation.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

Nucleus replacement in Mammalian oocytes.

Our contribution discusses the potential use of cell therapies (nucleus replacement) in mammalian oocytes. It is assumed that these approaches may be used, for example, for the elimination of mutated maternally transmitted mitochondrial DNA (mtDNA) as well as for the reconstruction of normal oocytes from oocytes that are developmentally compromised. Moreover, it is speculated that the replacement of germinal vesicles by somatic cells may result in cells of the haploid genome: the production of germ cells from somatic cells. The preliminary results obtained in our laboratories are discussed in this article.