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The First World Congress On: Controversies in Obstetrics, Gynecology & InfertilityPrague, Czech Republic - 1999
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The relatively low viability of human embryos is thought to be largely determined by the large proportion of chromosomal abnormalities that have been frequently and consistently reported in embryos examined by karyotype analyses (1). The use of fluorescent in situ hybridization (FISH) to identify the number of chromosomes in interphase nuclei of embryos, has confirmed the high level of aneuploidy. Furthermore, the embryo aneuploidies increase with increasing maternal age (2). For example, the number of aneuploid embryos in three groups of IVF patients with poor prognosis for pregnancy in IVF (maternal age ³ 38 yrs; IVF failure on 2 or more attempts; mosaic karyotype - 46 XX / 45 XO in blood of patients) ranged from 57 to 63% (3). In this study only five chromosomes were examined (X, Y, 13, 18 and 21) and raised the concern that examination of all chromosomes would leave very few chromosomally normal embryos in these patients. The use of six chromosome probes (X, Y, 13, 16, 18 and 21) for the same poor prognosis patients (except IVF failures were after 3 or more attempts), didn’t appear to increase the proportion of aneuploid embryos (60%) in further data from this research group (4).
A detailed examination of 247 embryos for nine chromosomal probes (X, Y, 13, 14, 15, 16, 18, 21, 22) by Munn et al. (5) from a mixed group of patients (advanced maternal age - 14 patients; habitual pregnancy loss - 3 patients; poor embryonic development or failed more than 3 IVF attempts - 4 patients; gonosomal mosaicism - 4 patients), showed 42% normal embryos. Of the abnormal embryos, 13% were monosomic, 10% were trisomic, 22% were complex (2 or more numerical chromosomal errors), 6% were polyploid and 3% were haploid. It is probable that the multiple chromosome errors account for the relatively constant number of aneu ploid embryos found despite the increased number of FISH probes being used. Given the present information, it is apparent that around 55 to 75% of 8-cell human embryos are aneuploid in the group of patients considered difficult to succeed with establishing viable IVF pregnancies. The upper limit would allow for aneuploidies which may occur for chromosomes that are not presently examined. Diagnosis of these aneuploidies enables the transfer of an increased number of chromosomally normal embryos and increases the consequent implantation rate (6).
Importantly the error rate for embryos in the study by Munn et al. (5) after resampling cells from the biopsied embryos was 6% for those considered normal but then found to be abnormal or vice versa (type 1 false positive or false negative diagnoses). This is probably due to chromosomal mosaicism in cells of the preimplantation human embryo. Re-analysis that identified another aneuploidy to the one originally identified (type 2 error) was also identified in 9% of re-examined embryos. As a consequence, the diagnosis of aneuploidy does involve a degree of error which is at least 6% which may result in pregnancies that include aneuploidy and this has occurred (5). Patients should be advised to confirm preimplantation diagnosis by more conventional prenatal diagnosis, to ensure the complete avoidance of aneuploidy in pregnancy. Skill and experience is needed for accuracy and consistency in preparing and reading FISH for single embryonic cells (7).
Preimplantation genetic diagnosis (PGD) for aneuploidy is normally done on single cells of 6- to 8-cell embryos prior to compaction on day 3 of culture. A hole is usually drilled in the zona pellucida with acid Tyrodes medium, or by laser dissection. Embryos are usually sorted into euploid and aneuploid and those with a normal chromosome number, transferred. If there is an ambiguous reading, or a failure to detect fluorescence, another cell may be taken. Embryos with more normal morphological appearance and the expected cell number have significantly lower rates of aneuploidy (3, 4). In a study involving 663 embryos examined for aneuploidy for nine chromosomes (XY, 13, 16, 18, 21, 14, 15, 22) by FISH, a second cell was required (4% of the time) to obtain a reliable diagnosis (8). The rate of aneuploidy was 61 and 62% comparing six and nine FISH probes and implantation rates were the same (24%). For nine probes another day of embryo culture is required for rehybridization of the additional three FISH probes (chromosomes 14, 15, 21). There appeared little advantage in rehybridization to enable nine chromosomes to be evaluated in this study.
It is considered that culture of embryos to the blastocyst stage may reduce the proportion of aneuploid embryos because these are likely to become blocked during cleavage and development. New data would indicate this is the case but a substantial number of aneuploid embryos are still able to develop to the blastocyst stage in culture. This new data will be discussed.
(1) PLACHOT M, MANDELBAUM J. Oocyte maturation, fertilization and embryonic growth in vitro. Br. Med. Bull. 46: 675-694, 1990.
(2) MUNNÃ S, ALIKANI M, TOMKIN G, ET AL. Embryo morphology, developmental rates and maternal age are correlated with chromosomal abnormalities. Fertil. Steril. 64: 382-391, 1995.
(3) GIANAROLI L, MAGLI MC, MUNNÃ S, FIORENTINO A, MONTANARO N, FERRARETTI AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod. 12: 1762-1767, 1997.
(4) GIANAROLI L, MAGLI MC, FERRARETTI AP. Chromosome abnormalities and embryo viability. Syllabus for ESHRE GÃ¶teborg Pre-Congress Course, 1998.
(5) MUNNÃ S, MAGLI C, BAHCE M, FUNG J, LEGATOR M, MORRISON L, COHERT J, GIANAROLI L. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat. Diagn. 18: 1459-1466, 1998.
(6) GIANAROLI L, MAGLI MC, FERRARETTI AP, FIORENTINO A, GARRISI J, MUNNÃ S. Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril. 68: 1128-1131, 1997.
(7) MUNNÃ S, MÃRQUEZ C, MAGLI C, MORTON P, MORRISON L. Scoring criteria for preimplantation genetic diagnosis of numerical abnormalities for chromosomes X, Y, 13, 16, 18 and 21. Mol. Hum. Reprod. 4: 863-870, 1998.
(8) GIANAROLI L, MAGLI MC, MUNNÃ S, FORTINI D, FERRARETTI AP. Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist. Reprod. Genet. 16: 148-152, 1999.