The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells.

BACKGROUND: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. MATERIALS AND METHODS: In this cohort study, we used fluorescence in situ hybridi- zation (FISH) to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD) on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. RESULTS: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%), while 2(6.7%) were diploid, and 2(6.7%) were triploid. Of those with mosaicism, 23(88.5%) were determined to be diploid-aneuploid and 3(11.5%) were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05); however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. CONCLUSION: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

DNA repair signalling pathway genes are overexpressed in poor-quality pre-implantation human embryos with complex aneuploidy.

OBJECTIVE: Chromosomal abnormalities and poor quality are correlated with DNA damage in the pre-implantation stage in humans. This study aimed to explore the altered expression of DNA damage signalling pathways - including apoptosis, cell cycle and DNA repair pathways - in poor-quality pre-implantation human embryos with complex aneuploidy. STUDY DESIGN: Surplus Day 4 embryos from candidates undergoing pre-implantation genetic screening were pooled into two groups. Group 1 included good-quality embryos that had simple aneuploidy, a single chromosome [according to fluorescence in situ hybridization-based pre-implantation genetic diagnosis (PGD) on Day 3], a normal rate of cell division, and graded as A or B (excellent to good). Group 2 included embryos with more than one aneuploid chromosome on PGD on Day 3, an abnormal rate of cell division, and graded as C or D (fair to poor). Gene expression of DNA damage signalling pathways was analysed using a real-time polymerase chain reaction-based array, which included 84 genes after specific pre-amplification of cDNA by a primer mix, including all array genes. RESULTS: In Group 2, five of the 84 genes studied showed significant overexpression (p<0.05): MSH3, XRCC1, RAD50, LIG1 and CDK7. Alterations were in agreement with genetic relationships in pathway analyses on DAVID. CONCLUSIONS: The five overexpressed genes are involved in DNA repair. Therefore, in comparison with cell cycle control and apoptotic pathways, DNA repair pathways are more activated in poor-quality pre-implantation human embryos with complex aneuploidy. This suggests that the dominant response to DNA damage in such embryos is DNA repair rather than cell division or apoptosis.

Delineating the association between isodicentric chromosome Y and infertility: a retrospective study.

OBJECTIVE: To report on 14 infertile patients who had a de novo form of the same isodicentric (idic)(Yq) karyotype with variable degrees of mosaicism. DESIGN: Retrospective study and review of the literature. SETTING: Medical genetics laboratory in a research institute for reproductive biomedicine. PATIENT(S): Fourteen infertile patients, including 13 male patients and 1 female patient who had infertility with the same idic(Y) karyotype. INTERVENTION(S): Conventional cytogenetic methods, fluorescence in situ hybridization (FISH) on seminal germ cells and blood, and polymerase chain reaction (PCR)-based molecular approaches. MAIN OUTCOME MEASURE(S): Karyotype, FISH, and PCR results. RESULT(S): Cytogenetic results revealed abnormal Y chromosome: 45,X/46,X,idic(Y)(q11.22). The FISH technique on blood lymphocytes confirmed a rearranged Y chromosome, with two centromeres and two SRY signals, and marker chromosome with various levels of mosaicism. Moreover, aneuploidy of sex chromosomes was also detected in haploid seminal germ cells. Multiplex PCR analysis of blood samples demonstrated microdeletion in AZFb and AZFc loci. CONCLUSION(S): Because of the resemblance between inversion of chromosome Y and idics(Y), use of confirmatory techniques (e.g., FISH or PCR-based methods) could help prevent medical errors in healthcare systems and precisely delineate chromosomal aberrations in infertile patients when clinical data fail to clarify the cause of infertility.

Optimization of The Electroporation Conditions for Transfection of Human Factor IX into The Goat Fetal Fibroblasts.

OBJECTIVE: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk. MATERIALS AND METHODS: In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 µg DNA per pulse in the first experiment and increments of 10 µg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR)-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05. RESULTS: The results showed no significant difference among three first concentrations except for group 1 (10 µg/pulse) and group 3 (30 µg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 µg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells. CONCLUSION: Increasing amount of using the vector 40µg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ß-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats.

Generation of human induced pluripotent stem cells from a Bombay individual: moving towards "universal-donor" red blood cells.

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, alpha-globulin, and gamma-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.