Biomedical subjects

F Pellestor

Publications and source records attributed to F Pellestor.

At least 55 records · Page 3Linked to original sources

PubMed1996

The PRINS technique: potential use for rapid preimplantation embryo chromosome screening.

The primed in-situ labelling (PRINS) method is an alternative to in-situ hybridization for chromosomal detection based on the use of chromosome-specific oligonucleotide primers. Using this process, we have developed a simple and semi-automatic method for rapid in-situ detection of human chromosomes. The reaction was performed on a programmable temperature cycler. Specific labelling was obtained in < 2 h reaction. Double PRINS techniques were performed on six morphologically abnormal preimplantation embryos using primers specific for chromosomes 9, 16, 18, 21, X and Y. The majority of these embryos displayed chromosomal abnormalities. The present results demonstrate that PRINS may be a simple and reliable technique applicable in human preimplantation diagnosis.

Blastocyst↗

PubMed1996

FISH and PRINS, a strategy for rapid chromosome screening: application to the assessment of aneuploidy in human sperm.

The co-utilization of FISH and PRINS techniques for in situ chromosome screening was tested on human sperm nuclei. We used a centromeric repeat probe specific for chromosome 4 for FISH. PRINS reactions were performed with alpha-satellite primers specific for either chromosomes 9 or chromosome 18. Double labeling was obtained and estimates of disomy rates were carried out for the three chromosomes.

Adult↗

PubMed1996

Double and triple in situ chromosomal labeling of human spermatozoa by PRINS.

The PRimed IN Situ (PRINS) labeling method allows rapid, specific detection of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for simultaneous in situ decondensation and denaturation of sperm nuclei. Using fluorochrome-labeled dNTPs in a sequential PRINS reaction, the direct detection of two or three distinct chromosomes must be performed within a timespan of 3 h. The method was tested with primers specific for chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11% to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that are indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rapid and efficient alternative to FISH for detecting nondisjunction in human sperm.

Aneuploidy↗

PubMed1996

Assessment of aneuploidy for chromosomes 8, 9, 13, 16, and 21 in human sperm by using primed in situ labeling technique.

The incidence of aneuploidy was estimated for chromosomes 8, 9, 13, 16, and 21 in mature human spermatozoa by primed in situ (PRINS) labeling technique. This method allows us to perform a chromosome-specific detection by in situ annealing of a centromeric specific primer. A dual color PRINS protocol was adapted to human sperm. The decondensation and the denaturation of sperm nuclei were simultaneously performed by 3-M NaOH treatment. Double labeling of spermatozoa was obtained in <2 h. A total of 96,292 sperm nuclei were analyzed by two independent observers. The estimates of disomy were 0.31% for chromosome 8, 0.28% for chromosome 9, 0.28% for chromosome 13, 0.26% for chromosome 16, and 0.32% for chromosome 21. These homogeneous findings suggest an equal distribution of aneuploidies among autosomal chromosomes in males.

Adult↗

PubMed1996

[Rapid identification of chromosomes by in situ hybridization of labelled oligonucleotides and comparison with the PRINS method].

We propose a simple, fast and inexpensive method of identification of human centromeres on metaphasic chromosomes and interphasic nuclei. This is based on in situ hybridization of labelled oligonucleotides. The efficiency of the methodology was demonstrated on cytogenetic preparations from human heteroploid and human x hamster hybrid cell lines and also on frozen tissue sections using an oligonucleotide specific for the alpha-satellite DNA of chromosome 1. Three versions of this oligonucleotide respectively labelled with 1, 4 and 10 fluorescein molecules were synthesized. The signal intensity provided by the oligonucleotide coupled with 4 fluoresceins allowed unambiguously the detection of the chromosome and the establishment of its ploidy using a classical cytogenetic microscope without the need for an amplification procedure. The use of different fluorochromes and possibly combination with an unlabelled elongation in 3' of the oligonucleotides which stabilize its hybridization, lead to a simple multicolour method. Preliminary quantification of the signals obtained by in situ hybridization of labelled oligonucleotides and comparison with those obtained by primed in situ labelling (PRINS) using the same nucleotides as primers, suggest that the elongation generated by PRINS may be very short compared with a PCR in solution. This limited efficiency of the in situ elongation may reflect the present difficulties of PRINS and DISC PCR (direct in situ single copy polymerase chain reaction) with primers specific for non-repetitive sequencies.

Animals↗

PubMed1995

Rapid in situ detection of chromosome 21 by PRINS technique.

The "PRimed IN Situ labeling" (PRINS) method is an interesting alternative to in situ hybridization for chromosomal detection. In this procedure, chromosome labeling is performed by in situ annealing of specific oligonucleotide primers, followed by primer elongation by a Taq polymerase in the presence of labeled nucleotides. Using this process, we have developed a simple and semi-automatic method for rapid in situ detection of human chromosome 21. The reaction was performed on a programmable temperature cycler, with a chromosome 21 specific oligonucleotide primer. Different samples of normal and trisomic lymphocytes and amniotic fluid cells were used for testing the method. Specific labeling of chromosome 21 was obtained in both metaphases and interphase nuclei in a 1 hour reaction. The use of oligonucleotide primer for in situ labeling overcomes the need for complex preparations of specific DNA probes. The present results demonstrate that PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

Base Sequence↗

PubMed1995

PRINS as a method for rapid chromosomal labeling on human spermatozoa.

Direct in situ labeling of human spermatozoa was performed using the PRINS method. This technique is based on annealing of specific oligonucleotide primers, and subsequent primer extension by a Taq DNA polymerase. The reaction was carried out on a programmable temperature cycler, and labeling was obtained in a 1-hr reaction. The method was successfully tested with specific primers for chromosomes 13, 16, and 21. This suggests that PRINS may be a fast and reliable technique for detecting aneuploidies.

Aneuploidy↗

PubMed1995

Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y.

The primed in situ labelling (PRINS) technique is an alternative to in situ hybridization for chromosomal screening. We have developed a semi-automatic PRINS protocol, using a programmable thermocycler. The method has been successfully tested with specific primers for chromosomes, 13, 16, 18, 21, X and Y. Specific chromosome detection has been obtained on both metaphases and interphase nuclei. This suggests that PRINS may be a reliable technique for detecting aneuploidies and some chromosomal aberrations.

Base Sequence↗

PubMed1995

The cytogenetic analysis of human zygotes and preimplantation embryos.

A review of cytogenetic studies on human zygotes and preimplantation embryos is presented. This survey documents the high incidence of chromosomal abnormalities in embryos in vitro. Up to date, 914 zygotes and embryos have been karyotyped. The rate of abnormalities is significantly higher in morphologically poor-quality embryos than in good-quality embryos (86.6 and 36.6% respectively). In both groups, aneuploidy is the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism or fragmentation are also found. Among tripronucleated zygotes, 81.9% display chromosomal abnormalities. Data suggest that some parameters of IVF procedures might be responsible for the occurrence of some abnormalities.

Aneuploidy↗

PubMed1995

Selection of chromosome-specific primers and their use in simple and double PRINS techniques for rapid in situ identification of human chromosomes.

The PRimed IN Situ labeling (PRINS) technique is an alternative to in situ hybridization for rapid chromosome screening. We have defined and tested new specific oligonucleotide primers for alpha-satellite DNA of several chromosomes. When using a semiautomatic PRINS protocol, specific labeling was obtained in both metaphase cells and interphase nuclei in a 1-h reaction. PRINS may be a simple and reliable technique for rapidly detecting aneuploidies.

Base Sequence↗

PubMed1995

[Direct analysis of the frequency of disomy in human sperm using the PRINS technique].

The PRINS method allows a rapid and specific detection of human chromosomes in situ. We have adapted the PRINS protocol to human sperm. Estimates of disomy have thus been performed for chromosomes 9, 12, 16 and 21 by using alpha-satellite DNA specific primers. The frequencies of disomy ranged from 0.27% to 0.31%. No significant difference was found. These data agree with the hypothesis of an equal distribution of non-disjunctions among chromosomes in male meiosis.

Adult↗

PubMed1994

Relationship between morphology and chromosomal constitution in human preimplantation embryo.

In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities.

Aneuploidy↗

PubMed1994

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS).

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.

Base Sequence↗

PubMed1994

Direct assessment of the rate of chromosomal abnormalities in grade IV human embryos produced by in-vitro fertilization procedure.

During in-vitro fertilization (IVF) procedures, human preimplantation embryos were classified into four grades according to their morphological appearance under light microscopy. The grade IV group included poor quality embryos. In our IVF programme, these embryos were never transferred or frozen, and were thus available for cytogenetic analysis. Cytogenetic analysis was performed on 411 grade IV embryos from 327 couples participating in the IVF programme. A total of 118 embryos were successfully karyotyped using at least one metaphase. Normal diploid chromosomes were found in only 12 embryos, containing a total of 19 metaphases. All others (90%) showed abnormal or aberrant chromosome complements; 48 were aneuploid and six cases of single chromatids were noted; 14 embryos (11.8%) contained haploid complements, while the remaining 44 exhibited mosaics (2n/3n, n/2n, n/3n) or fragmented chromosome sets. Also, several structural aberrations and rearrangements were observed. These results indicate that the large majority of grade IV human embryos are chromosomally abnormal. This confirms the morphological assessment of the poor quality of these embryos and demonstrates the uselessness of both the transfer and the cryopreservation of grade IV embryos.

Blastocyst↗

PubMed1994

[Rapid in situ detection of chromosome 21 by the PRINS technique].

The PRINS technique has been used to rapidly detect chromosomes 21 in both metaphases and interphase nuclei. A specific labeling was obtained in preparations from lymphocytes and amniotic fluid cells. The method is based on annealing of specific oligonucleotide primers and subsequent primer extension by a Taq DNA polymerase. PRINS is an interesting alternative to in situ hybridization for cytogenetic diagnosis and physical mapping.

Amniocentesis↗

PubMed1993

A simplified method for R banding of human oocyte chromosomes.

A simple and reliable R banding technique was developed for karyotyping mature human oocytes. The banding quality obtained is sufficient for the diagnosis of specific aneuploidies and the discrimination between whole chromosomes and separated chromatids. The ability to karyotype human oocytes accurately will facilitate study of the aetiology of chromosomal abnormalities in human concepti.

Chromosome Banding↗

PubMed1993

[Cytogenetic study of fragmented embryos not transferred in in vitro fertilization].

A cytogenetic analysis was performed on a sample of 411 human grade IV embryos (i.e. poor morphological quality embryos, never transferred in our in vitro fertilization (IVF) pro Gram) in order to investigate the chromosomal status of these embryos. One hundred eighteen were successfully karyotyped from at least one metaphase. Only 10% displayed normal diploid metaphases. Aneuploidy was the most frequently observed abnormality, with a rate of 36.4%. Six cases of single chromatids were noted and 9 embryos showed structural aberrations. Polyploidy (from 3n to 7n) and haploidy were also observed, suggesting parthenogenetic activation, polyspermy or chromosomal duplication. Mosaicism constituted 6% of the abnormalities. Thirty embryos exhibited fragmented chromosome sets which might result from in vitro delayed fertilization.

Chromosome Aberrations↗

PubMed1991

Frequency and distribution of aneuploidy in human female gametes.

During the past 6 years, 14 cytogenetic studies on human oocytes recovered during in vitro fertilization procedures have been published; they report contradictory results. The present survey has pooled the more than 1500 oocyte chromosome complements examined to date, in order to determine generalized trends in chromosomal abnormalities of female gametes. The overall frequency of abnormalities in mature oocytes is 24.0% with a large majority of aneuploidies (22.8%) over structural aberrations (1.2%), which could be explained by the difficulty in the detection of structural abnormalities in oocyte chromosome sets. An analysis of the distribution of non-disjunction among all chromosomes was also performed. In the A, C, D, and especially in the G groups, there is a significant difference between the observed non-disjunction and the frequencies expected from an equal partitioning of non-disjunction among all chromosomes. These data are discussed with reference to the differences obtained from cytogenetic studies on human sperm and from investigations on spontaneous abortion.

Adult↗