好大夫在线> 陈智勤 > Aneuploidy screening in early human embryos上海市第一妇婴保健院辅助生殖医学科陈智勤 Introduction Research suggests that fewer than 50% of naturally conceived human embryos reach full term. They are lost mostly before or shortly after implantation.(Short et al, 1979; Edwards et al, 1976) A similar pattern of early embryonic mortality is seen in in vitro fertilization (IVF), where a significant proportion of embryos arrest in development during the first few days following fertilization. Those embryos that survive are transferred to the mother, only a minority result in a live birth.Although multiple factors may adversely influence embryo survival, chromosomal aneuploidy is one of the most significant. It is a major pattern of numerical abnormities and usually has a catastrophic effect on the developing embryo by altering the dosage of hundreds of expressed genes, therefore, all autosomal monosomies are lethal and most trisomies lead to early fetal loss. The studies of material from spontaneous abortions reveal that the majority of these failed pregnancies are aneuploidy. The impact of aneuploidy on in vitro fertilization Up to now, the probability of achieving a pregnancy using IVF is generally less than 30% per embryo transferred. In most cases, multiple embryos are generated in treatment cycle and IVF clinics decide which of these to transfer based on morphological characteristics. However, as many aneuploid embryos display normal morphology during preimplantation phase, morphological assessment is insufficient for the exclusion of chromosome abnormality. It has been suggested that the efficiency of assisted reproductive techniques could be improved if chromosomally normal embryos can be detected and preferentially selected for transfer to the mother. In an effort to identify the chromosomally normal embryos, some infertility centres have now introduced tests specifically designed for cytogenetic screening.(Munne et al, 1998;Gianaroli et al, 1997) Tests of this type focus on the use of technologies developed for preimplantation genetic diagnosis (PGD-AS). Indication of aneuploidy screening PGD-AS has been applied primarily to women with one of three indications for infertility. These are advanced maternal age (AMA), recurrent implantation failure (RIF) and recurrent miscarriage (RM). Many of the chromosome abnormalities in human embryos would have caused the embryos to die in early development, or even before implantation. There has the positive correlation of advanced maternal age (AMA) with embryonic aneuploidy, means that the older women would be at increased risk of producing aneuploid embryos which would die around the time of implantation or cause early period of miscarriage. There are many infertile couples that fail to become pregnant even after repeated transfers of apparently morphologically normal embryos. This led to the expansion of PGD techniques to patients who had recurrent implantation failure or recurrent miscarriage after IVF treatment. Methods of cytaogenetic testing Complete karyotyping of metaphase chromosomes in early human embryos using traditional cytogenetic techniques is a problem a few years ago, primarily because the nuclei are in metaphase for only a fraction of the entire cell cycle. Cells can be arrested in metaphase by the use of mitotic inhibitors but these results in shortened chromosomes that are difficult to classify. Attempts have been made to karyotype every blastomere from individual embryos but the efficiency is very low (Wilton, 1993; Jamieson et al., 1994; Santalo et al., 1995, Clouston et al., 1997). An alternative to karyotyping is fluorescent in situ hybridization (FISH) using fluorochrome-labelled DNA probes that are complementary to DNA sequences specific to individual chromosomes. FISH enables enumeration of individual chromosomes even in interphase cells. FISH was first used on human blastomeres to detect the sex chromosomes (Griffin et al., 1992; Delhanty et al., 1993; Harper et al., 1994a) and was then applied to single cells biopsied from 3-day old human embryos from patients who carried an X-linked recessive disease (Griffin et al., 1993, 1994). This form of preimplantation genetic diagnosis (PGD) enables to select and transfer unaffected embryos to the mother. Several FISH protocols for simultaneous detection of multiple chromosomes with specific probes are being used for PGD. These protocols are based on the use of ratios of fluorochromes to labeling five or more chromosomes with only three fluororchromes 35. For the blastomere studies the most recently published protocols include X,Y, 13,16,18,and 21chromosomes have been used. These probes are labeled with different spectrums: Chromosome X was labeled with Aqua; 16 with green; 21 with orange. The other chromosomes were labeled with mixtures of fluorochromes. For instance: Chromosome 13 in orange and green; Chromosome 18 with orange and Aqua chromosome Y with combination of the 3 fluorochromes. Thus, chromosomes 13 appear as yellow; chromosome 18 as pink and chromosome Y as white. However, the use of mixtures of colors has the disadvantage that overlapping signals produce a misdiagnosis. For that reason, new colors have been developed, such gold and blue, and are currently being tested in some IVF center (Vysis et al) Misdiagnosis after aneuploidy screening Like many techniques, misdiagnoses of FISH have been reported. Blastomeres from untransferred embryos that were diagnosed as abnormal by FISH have been analyzed with one report of error rates as high as 15% (Munne et al., 1998b); Others have reported much lower, only 3% of embryos possibly being misdiagnosed (Wilton et al., 2000) About two-thirds of these errors have no impact on the outcome, because the embryo was still abnormal but had a different abnormality from that predicted by the initial analysis of a single cell. Misdiagnosis can occur because of chromosomal mosaicism in human embryos. One way to minimize the problem of mosaicism would be to biopsy two cells instead of one cell and only transfer the embryo if both were normal. However, errors could still occur as in some embryos there is a mixture of euploid and aneuploid cells (Voullaire et al., 2000; Wilton et al., 2000) and it would still be possible to biopsy two euploid cells from an embryo that was predominantly aneuploid. Misdiagnoses can also be caused by technical errors related to FISH. Reasons for this include signal failure and this is known to increase after successive rounds of FISH (Liu et al., 1998) and signal overlap. Many workers increase the number of FISH probes used in each analysis by using combination labeling, where some chromosomes will be detected by probes labeled with a single fluorochrome and others with a combination of two fluorochromes. The relatively low error rates reported by Wilton et al.(2000) were after only one round of FISH and using monocolour labeling of probes. Another approach to minimizing technical FISH errors is to include a second probe for some chromosomes. This has been reported for chromosome 21 (Magli et al., 2001), where misdiagnoses could potentially result in the birth of abnormal babies. Clinical application of CGH FISH technology has proved to be an invaluable tool in the analysis of chromosome errors and the diagnosis of aneuploidy in human embryos but its obvious limitation is that only a restricted number of chromosomes can be tested in a single cell. There is no doubt that some embryos that are diagnosed as ‘normal’ by FISH are actually aneuploid for chromosomes that have not been analyzed. It is likely that the benefit of PGD-AS could be increased if more chromosomes could be analyzed, hence maximizing the chances to select high quality embryos. The most successful technique for complete karyotyping of interphase cells is comparative genomic hybridization (CGH) (Kallionemi et al., 1993). In a CGH, test DNA and known normal reference DNA are differentially labeled (usually with green and red fluorochromes, respectively) and hybridized simultaneously to normal metaphase chromosomes that act as a template. The green: red fluorescence ratio along the length of the template chromosomes is indicative of the relative chromosome copy number of the test DNA compared to the reference DNA. CGH has been successfully applied to single cells (Voullaire et al., 1999; Wells et al., 1999) and interphase blastomeres with very high efficiency (Voullaire et al., 2000; Wells and Delhanty, 2000). CGH has now been applied to single blastomeres biopsied from human embryos resulting in the first birth from an embryo that has been fully karyotyped prior to transfer (Wilton et al.2001). The major limitation of CGH for PGD is that the analysis takes at least 5 days to complete because of the long hybridization time required and the laborious analysis of template chromosomes. This is longer than human embryos can be maintained in culture after a blastomere biopsy on Day 3. These difficulties will be overcome when the template chromosomes are replaced by microarrays where hundreds or thousands of chromosome specific probes are spotted onto a glass slide. Polar body and blastomere and blastocyst analysis, which is better? People use to analysis of polar bodies and infer aneuploidy in embryos that result from the oocytes. It has significant limitations. First, because the diagnosis is performed prior to fertilization, thus chromosome abnormalities derived from aneuploid sperm cannot be accounted for. Although the paternal contribution to aneuploidy is probably much lower than the maternal contribution, it is not unreasonable that patients will have an expectation that all errors of the chromosomes being analyzed will be determined. Also, it is now clear that many human embryos are chromosomally mosaic, i.e. that the chromosomal constitution is not the same in every cell. These errors must arise during early cleavage divisions of the embryo and so will not be present in the first or second polar body and will remain undetected if only polar body diagnosis is performed. Most laboratories now offering PGD-AS perform cleavage stage embryo biopsy, which will identify meiotic and post-zygotic chromosome abnormalities. Oocyte retrieval and fertilization are followed by the biopsy of 1–2 cells (blastomeres) derived from 6–10 cell embryos. These biopsied cells then provide for subsequent FISH test. If biopsied cell is found to be free of the chromosomal errors, then it is inferred that the rest of the embryo is also unaffected by the abnormalities. Only ‘‘unaffected’’ embryos are transferred to the mother’s uterus. However, embryo biopsy is invasive and PGD-AS using FISH is time-consuming and expensive. As described earlier, poor embryo development has been associated with an increased frequency of chromosomal abnormalities. It would appear that embryos that are chromosomally abnormal on Day 3 are less likely to develop to the blastocyst stage than euploid embryos (Magli et al., 2000; Sandalinaset al., 2001). This raises the possibility that extended in vitro culture of human embryos to the blastocyst stage could be a very simple way of eliminating aneuploid embryos and facilitating the selection of euploid embryos for transfer (Janny and Me ne zo 1996; Jones et al., 1998) FISH analysis of the ICM of human blastocysts showed that many were aneuploid (Evsikov and Verlinsky, 1998; Magli et al., 2000) or mosaic(Ruangvutilert et al., 2000) and that the incidence of aneuploidy was the same in the ICM as in the entire blastocyst (Evsikov and Verlinsky, 1998). The information got from the trophectoderm can represent the entire blastocyst, so biopsy from trophectoderm which develop to placenta tissue later on is less invasive to the embryo and will have a prosperous future in the PGD-AS. Conclusions Methods for aneuploidy screening of early embryos have become increasingly powerful, benefiting enormously from the fusion of traditional cytogenetic techniques and molecular genetics. It provides less invasive and more confident prediction that an individual embryo will implant and result in a healthy fetus which will complete development to term. PGD- AS will soon become a standard IVF practice especially to the woman with high maternal age and low success rates after conventional IVF. References Short RV. When a conception fails to become a pregnancy. Ciba Found Symp 1979;64:377–394. Edwards RG, Gardner RL. Sexing of live rabbit blastocysts. Nature 1967;214:576–577. Munne S, Magli C, Bahce M, Fung J, Legator M, Morrison L, Cohert J, Gianaroli L. Preimplantation diagnosis of the aneuploidies most commonlyfound in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn 1998;18:1459–1466. 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FISH analysis on day 5 post insemination of human arrested and blastocyst stage embryos. Prenat Diagn 20: 552–560. In early human embryonic aneuploidy screening, very profound, is not easy to understand!