Cell death and lung cell histology in meconium aspirated newborn rabbit lung.

UNLABELLED: Meconium aspiration syndrome (MAS) is a major cause of newborn mortality and morbidity. In this study we investigated the inflammatory responses and morphological changes in the newborn lung to debris-free meconium instillation. We developed a model for studies of MAS using 2-week-old rabbit pups. Cell death was assessed by DNA staining and detection of DNA fragmentation by in situ end labeling. Cell death was seen in association with an increase of inflammatory cytokines levels, studied by ELISA. Necrotic cells were detected by staining of lavage cells with ethidium bromide and 4',6'-diamino-2'-phenylidon. Meconium instillation resulted selectively in loss of airway and alveolar epithelial cells followed by cell death, which increased with time. Necrotic cells looked smaller and damaged with maximal counts at 24 h after instillation. CONCLUSION: Meconium instillation into lungs caused massive cell death, possibly by apoptosis, and necrosis that may have been activated by the inflammatory cytokine production.

A cytogenetic study of G1-chromosomes in one-cell stage mouse embryo and in corresponding second polar body. Evaluation of aneuploidy originated in females heterozygous for translocation T[14;15]6Ca.

Meiotic chromosomes in ovulated oocytes and G1-chromosome sets visualized in the 2nd PB and in the pronuclei of one-cell stage embryos treated with okadaic acid were studied in female mouse heterozygous for reciprocal translocation T[14;15]6Ca. It was found that 61.5% of oocytes were haploid, 14.9% hyperhaploid and 23.6% hypohaploid. Unpaired chromatid (a half-dyad), in addition to (or replacing) a whole chromosome (a dyad), was detected in 20% of oocytes. G1-chromosome complements in the 2nd PB and in the MPN expected in the case of aneuploidy due to chromosome non-disjunction or to chromatid abnormal segregation at the first and second meiotic division were detected in one-cell stage embryos. The hypo- and hyperhaploidy caused by non-disjunction was revealed in 17.6% of embryos. Aneuploidy due to abnormal segregation of a chromatid in the first and the second meiotic division was in 20% and 4.4% of all cases respectively. The incidence of different classes of aneuploid oocytes were almost fully conformable to that of corresponding types of aneuploidy detected in one-cell stage embryos. The main advantage of the proposed new approach based on cytogenetic analysis of G1-chromosomes in the 2nd PB and in the corresponding MPN is that it allows to study not only the chromosome non-disjunction, but also abnormal segregation of chromatids in the first and in the second meiotic divisions, and to estimate accurately the incidence of these maternal meiotic errors.

Birth of a healthy girl after preimplantation gender determination using a combination of polymerase chain reaction and fluorescent in situ hybridization analysis. Preimplantation Genetics Group.

OBJECTIVE: To perform preimplantation gender determination by a combination of polymerase chain reaction (PCR) sexing and fluorescent in situ hybridization technique using the directly labeled fluorescent alpha-satellite centromeric DNA probes for X and Y chromosomes. SETTING: The IVF program of Illinois Masonic Medical Center. PATIENTS: A couple requested preimplantation diagnosis because the mother is a carrier for hemophilia A. RESULTS: Two blastomeres were aspirated from each of the four- to eight-cell embryos, and only the embryos with both fluorescent in situ hybridization and PCR results indicating female sex chromosomal complement were transferred, resulting in a singleton pregnancy and delivery of a healthy female infant, after prenatal confirmation of the diagnosis as female. The male embryos or embryos diagnosed as females only by PCR were followed up by confirmatory fluorescent in situ hybridization analysis demonstrating a discrepancy of PCR and fluorescent in situ hybridization results in four embryos, presumably because of a possible sperm contamination of the PCR reaction or chromosomal mosaicism. CONCLUSION: The analysis of two blastomeres from the same embryo by a combination of PCR sexing and fluorescent in situ hybridization increases the reliability of preimplantation gender identification at the cleavage stage.

Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling).

PURPOSE: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. METHODS: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. RESULTS: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91-95%) than for PCR (85-87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. CONCLUSIONS: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.

Polar body diagnosis of common aneuploidies by FISH.

PURPOSE: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. DESIGN: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. RESULTS: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. CONCLUSIONS: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.

Expression of homebox-containing genes in human preimplantation development and in embryos with chromosomal aneuploidies.

PURPOSE: The purpose of the study was to investigate homeobox gene expression in human oocytes and preembryos and in postimplantation embryos with impaired embryonic development determined by chromosomal abnormalities. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with intron spanning primer sets for Homeobox gene sequences was used. RESULTS: The homeobox genes HoxA4, HoxA7, HoxB4, and HoxB5 were present in human oocytes and cleaving normal and triploid embryos. The expression pattern was different between chromosomally abnormal and normal first-trimester embryos. Of four homeobox transcripts (HoxA7, HoxB4+ ++, HoxB5, and HoxC6) that are expressed in diploid embryos, only HoxA7, HoxB4 and HoxC6 were present in a trisomy 7 embryo, and only HoxB4 and HoxB 5 in triploid embryos and an embryo with trisomy 9. Cloning experiments revealed differences in the number of homeobox clones obtained from trisomy 7 and control embryos. CONCLUSIONS: The transcripts of homeobox genes, HoxA4, HoxA7, HoxB4, and HoxB5, were present in oocytes and cleaving embryos. The pattern of expression of homeobox genes in cultured fibroblasts derived from spontaneously aborted embryos with aneuploidies was different from that in control diploid cells.

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization.

PURPOSE: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies. DESIGN: Human unfertilized oocytes and their extruded IPB were analyzed using the directly labeled fluorescence alpha-satellite DNA probes to chromosomes X and 18. RESULTS: Paired signals for chromosomes X and 18 were observed in the second meiotic prophase (MII) of unfertilized oocytes and their extruded IPB. In the series of 156 unfertilized oocytes in which the number of X chromosome- and chromosome 18-specific signals were analyzed in both MII and IPB, five nondisjunction events have been detected, with corresponding signals in MII and their IPB: missing signals in MII corresponded to extra signals in their IPB and extra signals in MII corresponded to missing signals in IPB. In one oocyte chromosome 18 nondisjunction was detected, with both chromosome 18 signals in MII and no chromosome 18 signal in IPB. In four oocytes chromatid malsegregations for chromosome X or chromosome 18 were detected: in two oocytes, three of four chromosome 18 signals were present in MII, with only one in IPB, and in the other two oocytes, three of four chromosome signals were present in MII, with only one left in IPB. CONCLUSIONS: The data suggest the possibility of detecting chromosomal aneuploidy in oocytes through cytogenetic analysis of their corresponding IPB by FISH as a possible approach for preimplantation diagnosis of major chromosomal trisomies.

Cross-hybridization of the chromosome 13/21 alpha satellite DNA probe to chromosome 22 in the prenatal screening of common chromosomal aneuploidies by FISH.

In a routine application of commercially available centromeric DNA probes for the prenatal screening of common trisomies involving the autosomes 13, 18, and 21, and sex chromosomes, four cases of discrepancy between fluorescence in situ hybridization (FISH) results and follow-up cytogenetic analysis were observed from a total of 516 cases of amniocentesis. In three of these cases, the results were false negative, and in one false positive. In this case, amniocentesis was performed because of a positive triple test in a 34-year-old woman with previous infertility treatment. The alpha satellite DNA probe for chromosomes 13/21 revealed five signals in 50 per cent of uncultured amniocytes, while standard cytogenetic analysis showed a normal karyotype. FISH analysis on metaphase chromosomes demonstrated the location of the additional signal in the centromeric region of chromosome 22. This additional signal was also present in the centromeric region of chromosome 22 of the mother, providing evidence for a possible inherited polymorphism in chromosome 22 responsible for unspecific hybridization with the alpha satellite probe for chromosomes 13/21 in this case. The observed polymorphism in centromeric regions may contribute to unreliability of the use of the 13/21 alpha satellite probe for prenatal screening by FISH.

Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization.

Chromosomal aneuploidies contribute considerably to the low pregnancy rate in in-vitro fertilization (IVF). The objective of this experimental work was to explore the possibility of detecting common aneuploidies in oocytes by polar body sampling. The study included 45 infertile patients of advanced maternal age participating in an IVF programme. The first polar body was removed prior to fertilization or both the first and second polar bodies were removed after fertilization and studied by fluorescent in-situ hybridization (FISH) using chromosome-specific probes for chromosomes X, 18 and/or 13/21. Of 155 oocytes with FISH results, 36 demonstrated chromosomal abnormalities. Of 119 oocytes predicted to be free from aneuploidy of chromosomes X, 18 and/or 13/21, 72 were normally fertilized, cleaved and transferred in 23 treatment cycles, which resulted in two healthy deliveries and three ongoing pregnancies confirmed to be unaffected by chorionic villous sampling. The method may appear useful for the detection of oocytes with common chromosomal aneuploidies in IVF patients of advanced maternal age.