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ABSTRACT: Ultrastructural and morphometric analyses of centromeric regions by scanning and transmission electron microscopy have been performed in chromosomes from male pronuclei obtained by heterologous fertilisation of hamster oocytes with human spermatozoa. In 1308 of 1323 chromosomes analysed, the primary constriction showed a defined biconcave constriction of variable length (0.56-1.34 microns) and constant width (0.64-0.7 micron). A positive correlation was observed between centromeric length and chromosome length. In some chromosomes, the primary constriction appears as decondensed regions of variable length (1.6-2.51 microns) composed of chromatin fibres with a minimum diameter of 30 nm.
Zygote 03/2000; 8(1):79-85. · 1.17 Impact Factor
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ABSTRACT: The relationship was examined between chromosome abnormalities in cleavage stage human embryos and maternal age, embryo morphology and development rate. Embryos that were classified as suboptimal for transfer from patients undergoing IVF treatment were disaggregated, and all or most of their cells were fixed for analysis by fluorescence in-situ hybridization. Chromosomes X, Y, 13, 18 and 21, and in some instances 16 were examined. A total of 731 non-viable embryos was analysed. An increase in chromosome abnormalities with decreasing embryo competence and increasing maternal age was shown. Compared with an earlier study, the major difference was that polyploidy (P<00.01) and aneuploidy were previously more common. After pooling results, it was found that aneuploidy increased with maternal age, from 3.1% in embryos from 20-34 years old patients to 17% in patients 40 years or older. Also, aneuploidy occurred more frequently in embryos with good morphology and development rate than in embryos developing poorly. In contrast, dysmorphic and slowly developing or arrested embryos had significantly more polyploidy and mosaicism than normally developing embryos. Clear associations between maternal age and aneuploidy, and between cleavage anomalies and mosaicism have been established in non-viable embryos. Arrested embryos were mostly polyploid. Moreover, polyploidy was found more frequently in embryos analysed on day 4, suggesting that developmentally compromised embryos became arrested in extended culture. A slightly higher aneuploidy rate in the earlier study may be attributed to differences in hormonal stimulation, which also resulted in different numbers of oocytes recruited and matured.
Reproductive biomedicine online 01/2000; 1(1):17-26. · 2.04 Impact Factor
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S Munné,
C Magli,
J Cohen,
P Morton,
S Sadowy,
L Gianaroli,
M Tucker, C Márquez,
D Sable,
A P Ferraretti,
J B Massey,
R Scott
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ABSTRACT: usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.
Human Reproduction 09/1999; 14(9):2191-9. · 4.47 Impact Factor
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ABSTRACT: The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient's embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources.
Human Reproduction 03/1999; 14(2):470-5. · 4.47 Impact Factor
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ABSTRACT: Fluorescence in-situ hybridization (FISH) for application in preimplantation genetic diagnosis (PGD) of aneuploidy has been used successfully, but stringent scoring criteria to score FISH signals have not been developed. In the present study a FISH protocol to simultaneously enumerate chromosomes X, Y, 13, 16, 18, and 21 was used to evaluate two different scoring criteria. The criteria consider hybridization signal size, shape, and vicinity to other signals and nuclear diameter. For this purpose, 74 embryos (412 blastomeres) donated for research had most or all of their cells analysed. The least error-prone criterion (9%) was selected for use in PGD cases. Some probes produced more errors than others, and these criteria may provide clues to improve these probes. The same probe solution was applied to 55 PGD cases and a total of 307 embryos. Of the non-transferred embryos, 67 were fully reanalysed and 1.5% (1/67) of them were falsely diagnosed as normal, while 19% (13/67) were falsely diagnosed as abnormal. Twelve of the patients became pregnant after PGD.
Molecular Human Reproduction 10/1998; 4(9):863-70. · 3.85 Impact Factor
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ABSTRACT: Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother's womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p 13.1 ;q 13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring.
Human Genetics 07/1998; 102(6):663-74. · 5.07 Impact Factor
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S Munné,
L Morrison,
J Fung, C Márquez,
U Weier,
M Bahçe,
D Sable,
L Grundfeld,
B Schoolcraft,
R Scott,
J Cohen
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ABSTRACT: Preimplantation genetic diagnosis of translocations has seldom been attempted. Recently, a genetic test based on analyzing polar bodies at the methaphase stage, following fluorescent in situ hybridization with commercially available whole-chromosome painting DNA probes has been presented. Here we report the use of this method in seven couples in whom the female was a carrier of one of these balanced translocations: 45,XX,der (13q;14q)(q10;q10) (two cases), 46,XX,t(4;14)(p15.3;q24), 45,XX,der(14q;21q) (q10;q10), 46,XX,t(7;20)(q22;q11.2), 46,XX,t(9,11)(p24;q12), 46,XX,t(14;18)(q22;q11), and 46,XX,t(3;8)(q11;q11).
The original method was improved in two ways. First, centromeric probes for one or both chromosomes involved in the translocation were added to avoid misdiagnosis caused by possible confusion of first polar body monovalent chromosomes (with two chromatids each) with single chromatids. Second, for cases with terminal translocations where commercially available probes do not cover telomere sequences, a telomere probe labeling the translocated fragment was added.
A total of 26 abnormal, 18 balanced, and 22 normal eggs was detected. Nine normal and seven balanced embryos were transferred, resulting in eight (50%) implanting, of which one spontaneously aborted. To date, the remainder have produced karyotypically normal or balanced babies and ongoing pregnancies. The rate of spontaneous abortions after preimplantation genetic diagnosis (12.5%) was significantly reduced (P < 0.001) compared to natural cycles in the same patients (95%).
With the above improvements, the test can characterize any translocation of maternal origin and produce a high pregnancy rate and an apparently low frequency of spontaneous abortion.
Journal of Assisted Reproduction and Genetics 06/1998; 15(5):290-6. · 1.84 Impact Factor
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ABSTRACT: To compare the rate of numerical chromosome abnormalities in embryos derived from bipronucleated zygotes produced by intracytoplasmic sperm injection (ICSI) and conventional IVF.
Embryos were classified by maternal age and morphological and developmental characteristics to avoid bias when comparing chromosome abnormalities in ICSI and IVF embryos.
The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, West Orange, New Jersey.
Seventy-nine couples undergoing IVF and 53 couples undergoing ICSI.
Embryos donated for research were fully biopsied, and their cells were analyzed by fluorescence in situ hybridization with specific probes for chromosomes X, Y, 13, 18, and 21 and some with also a probe for chromosome 16.
Embryo chromosome abnormalities.
A total of 245 embryos obtained through conventional IVF and 136 embryos obtained through ICSI were analyzed. There were no statistical differences between the rates of numerical chromosomal abnormalities detected in the IVF (61%) and ICSI (52%) embryos analyzed. Regarding gonosomal aneuploidy, the same rate was found in both ICSI (1%) and IVF groups (2%).
If the parents are chromosomally normal, the results indicate that, at the embryo level and before any embryo selection has occurred in utero, ICSI does not produce more numerical chromosomal abnormalities than conventional IVF.
Fertility and Sterility 06/1998; 69(5):904-8. · 3.56 Impact Factor
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ABSTRACT: Sixty unfertilized human oocytes and two fresh polar bodies were karyotyped by spectral karyotyping (SKY). The oocytes were provided by 29 women ranging from 30 to 42 yr of age. The mean hybridization efficiency for oocytes was 95.2% (60/63). Nondisjunction of bivalent chromosomes (13.3%) and predivision of sister chromatids at meiosis I (3.3%) were unequivocally determined by analysis first with SKY and then fluorescence in situ hybridization. Four oocytes (6.7%) were hyperhaploid, six (10.0%) were hypohaploid, one (1.7%) showed balanced predivision, and another (1.7%) was diploid. No specific structural rearrangements were detected. This study demonstrates that the SKY technique can be used successfully as an alternative method of karyotyping second meiotic metaphase chromosomes from human oocytes and polar bodies in appropriate spreads.
Cytogenetics and cell genetics 02/1998; 81(3-4):254-8.
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ABSTRACT: Analysis of sperm karyotypes and two-color fluorescent in situ hybridization (FISH) on sperm nuclei were carried out in a man heterozygous for the pericentric inversion inv(9)(p11q13). Sperm chromosome complements were obtained after in vitro fusion of zona-free hamster oocytes and donor sperm. A total of 314 sperm complements was analyzed: 153 (48.7%) carried the inverted chromosome 9 and 161 (51.3%) carried the normal one. None of the sperm complements contained a recombinant chromosome 9, suggesting that no chiasmata were formed in the heterochromatic region. The frequency of structural chromosome aberrations unrelated to the inversion (8.3%) and the frequency of conservative aneuploidy (3.2%) were within the limits observed in our control donors. The proportions of X-bearing (47.3%) and Y-bearing sperm (52.7%) were not significantly different from the expected 1:1 ratio. The percentage of disomy for chromosome 21 was analyzed by two-color FISH in 10336 sperm nuclei. The disomy rate for chromosome 21 (0.30%) was not significantly different from that found in our controls. These results suggest that the risk for this man of producing chromosomally abnormal offspring or spontaneous abortions was not increased, and do not support the existence of an interchromosomal effect for chromosome 21.
Human Genetics 07/1997; 99(6):761-5. · 5.07 Impact Factor
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ABSTRACT: We describe a modification of the human sperm-zona-free hamster egg fusion method that permits the study of aneuploidy in sperm-derived pronuclei by multicolour fluorescent in-situ hybridization (FISH). Zona-free hamster eggs and human spermatozoa were fused and cultured for 15 h in the presence of colcemid (1 microg/ml of medium) to obtain hamster oocytes arrested at metaphase II and human spermatozoa at the pronuclear stage. By applying a whole human genomic DNA probe we confirmed that 100% of pronuclei tested (372/372) were of human origin. One-colour fluorescent in-situ hybridization using a centromeric 18 probe was applied to 919 pronuclei with different dithiothreitol (DTT) pretreatments: 50 mM (10 min) or 25 mM (20 and 25 min). The highest hybridization efficiency was obtained with treatment with 25 mM DTT for 20 min (90.3%). Sex chromosome aneuploidy was analysed by three-colour FISH in a total of 2596 pronuclei from a normal donor. Hybridization efficiency was 98.6%. The disomy rates for X, Y and XY chromosomes (0.11, 0.04 and 0.08% respectively) were similar to data reported for sperm nuclei by three-colour FISH and to those obtained in sperm chromosomes. These results suggest that selection of potentially fertile spermatozoa (spermatozoa able to fertilize zona-free hamster eggs and produce a pronucleus) does not imply chromosomal selection.
Human Reproduction 05/1997; 12(4):641-5. · 4.47 Impact Factor
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ABSTRACT: Using the methylase SssI enzyme, we have analyzed the degree of in situ methylation of human sperm pronuclear chromosomes obtained by fertilizing hamster oocytes with human sperm. Untreated (control) sperm chromosome complements showed a higher degree of in situ methylation, compared to sperm complements previously treated with 5-azadeoxycytidine or lymphocyte chromosomes. This indicates that human sperm pronuclear chromosomes have a lower degree of genomic methylation compared to that of other somatic cells. The similarity in the degree of in situ methylation of the euchromatic and heterochromatic regions of chromosomes 1, 9, 15, and 16 and the Y chromosome in human sperm does not support the existence of a possible correlation between hypomethylation and heterochromatin decondensation.
Cytogenetics and cell genetics 02/1997; 76(3-4):123-7.
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ABSTRACT: The effect of Colcemid on the frequency and type of chromosomal abnormalities in human sperm was investigated. Using the human sperm and zona-free hamster egg fusion technique, penetrated eggs were cultured in the presence or absence of Colcemid. We used two different times of Colcemid treatment: standard Colcemid treatment (Colcemid-5 h) or long Colcemid treatment (Colcemid-17 h). Each Colcemid series had its own control series without Colcemid, thus ensuring that Colcemid was the only significant variable. A total of 771 sperm karyotypes from one normal donor was analyzed: 286 in the Colcemid-5 h series, 262 in the Colcemid-17 h series, and 223 in the two control series. In both Colcemid series there was a significant increase in the frequencies of hypohaploidy vs. hyperhaploidy (9.4% and 7.3% vs. 2.4% and 1.1%, for the Colcemid-5 h and Colcemid-17 h series, respectively), in contrast to those obtained in the control series, in which the frequencies of hypohaploidy and hyperhaploidy were close to the 1:1 relationship (4.9% vs. 4.0%) expected from nondisjunction. There was a significant increase in the frequency of structural abnormalities in both Colcemid series (16.1% and 14.5% for the Colcemid-5 h and Colcemid-17 h series, respectively) compared to the control series (6.3%). These results suggest that Colcemid significantly increases the frequency of hypohaploidy and unstable structural aberrations in human sperm.
Cytogenetics and cell genetics 02/1996; 72(2-3):164-70.
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ABSTRACT: A sperm chromosome analysis of 24 men with normal or balanced karyotypes was carried out to study the frequency of sperm chromosome aneuploidy. A total of 3,446 human sperm complements (36-315 per donor) was analyzed after in vitro penetration of hamster eggs. Two sets of donors were studied at two different centers in the United States (center 1) and Spain (center 2). The frequencies of hyperhaploidy and hypohaploidy in control donors were similar between center 1 (1.9% vs. 7.7%) and center 2 (1.8% vs. 10.3%). In carrier donors there were no significant differences between the two centers in the frequency of hyperhaploidy (0.8% vs. 1.9%), but that of hypohaploidy was significantly higher in center 2 (11.0%) than in center 1 (4.6%). A significant excess of hypohaploid complements, as compared to hyperhaploid complements, was found in both centers in both control and carrier donors. The sex ratio was similar in both centers and did not differ significantly from a 1:1 sex ratio. The larger chromosomes in the complement (1, 2, 3, 4, 5, 7, and 10) presented a significantly lower frequency of hypohaploidy, while some of the smaller chromosomes (13, 19, and 21) showed a higher frequency of hypohaploidy than expected. Chromosome 21 and the sex chromosomes showed an increase in the percentage of hyperhaploidy, as compared to other chromosomes, that was close to statistical significance (P = 0.08). Our results reflect a preferential loss of small chromosomes during slide preparation and suggest that chromosome 21 and the sex chromosomes could be more frequently involved in aneuploidy.
Cytogenetics and cell genetics 02/1996; 74(3):194-200.
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ABSTRACT: Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).
The American Journal of Human Genetics 03/1995; 56(2):452-60. · 10.60 Impact Factor
