Carmen Márquez

Chromosome abnormalities in 1255 cleavage-stage human embryos

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.

Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect

The present study evaluated mosaicism in a large series of cleavage-stage human embryos analysed by fluorescence in-situ hybridization. Only embryos with at least three cells analysed were included (n = 1235), of which 556 were mosaics. The most common types of mosaicism were chaotic (48%), diploid/polyploid (26%), and those caused by mitotic non-disjunction (25%). The number of abnormal cells per embryo ranged from 44% in diploid/polyploid to 84% in chaotic mosaics. Chromosome 16 was most commonly involved in mitotic non-disjunction mosaics. While overall mosaicism did not increase with maternal age, the average maternal age of the embryos that had mosaics caused by mitotic non-disjunction was significantly higher than that for normal or other mosaic embryos (P < 0.001). During the cleavage stage, the embryonic genome is not yet fully activated and consequently the mRNA and protein pools are still similar to those found in the oocyte. We therefore propose that the malfunctioning of the meiosis apparatus, which is similar to the mitotic one, may cause either meiotic errors or mitotic non-disjunction at cleavage-stage embryo development.

Differences in chromosome susceptibility to aneuploidy and survival to first trimester.

The purpose of this study was to find specific rates of aneuploidy in cleavage-stage embryos compared with first trimester data and to evaluate post-zygotic selection against aneuploidy. A total of 2058 embryos were analysed by flurorescence in-situ hybridization (FISH), and specific aneuploidy rates were obtained for 14 chromosomes. Data from morphologically abnormal embryos could be pooled with data from preimplantation genetic diagnosis (PGD) cycles because it was observed that they had similar rates of aneuploidy; thus, for the purpose of studying aneuploidy they could be, and were, pooled. Specific chromosome aneuploidy rates were not related to morphology or development of the embryos. The average maternal age of patients with aneuploid embryos was significantly higher than the overall analysed population. Monosomy appeared more commonly than trisomy. The chromosomes most frequently involved in aneuploidy were (in order) 22, 16, 21 and 15. When compared with first trimester pregnancy data, aneuploidies detected at cleavage stage seem to die in excess of 90% before reaching first trimester, with the exception of chromosome 16 and gonosomes (76% and 14% respectively). Differences in chromosome-specific aneuploidy rates at first trimester conceptions are probably produced by different chromosome-specific aneuploidy rates at cleavage stage and different survival rates to first trimester.

Spontaneous Abortions Are Reduced After Preconception Diagnosis of Translocations

Purpose: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).Methods: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.Results: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%).Conclusions: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.

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

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)(p13.1;q13.3), 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.

Chromosome abnormalities in embryos obtained after conventional in vitro fertilization and intracytoplasmic sperm injection

OBJECTIVE To compare the rate of numerical chromosome abnormalities in embryos derived from bipronucleated zygotes produced by intracytoplasmic sperm injection (ICSI) and conventional IVF. DESIGN Embryos were classified by maternal age and morphological and developmental characteristics to avoid bias when comparing chromosome abnormalities in ICSI and IVF embryos. SETTING The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, West Orange, New Jersey. PATIENT(S) Seventy-nine couples undergoing IVF and 53 couples undergoing ICSI. INTERVENTION(S) 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. MAIN OUTCOME MEASURE(S) Embryo chromosome abnormalities. RESULT(S) 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%). CONCLUSION(S) 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.

Chromosome identification in human oocytes and polar bodies by spectral karyotyping

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.

High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in the nucleus. To facilitate the detection, DNA probes for breakpoints on different chromosomes are labeled in different colors, so the translocation event can be detected as a fusion of red and green hybridization domains. We applied this scheme successfully for the analysis of somatic and germ cells from more than 20 translocation patients, each with individual breakpoints, and provide summaries of our experience as well as strategies, cost and time frames to prepare case-specific translocation probes.