Chromosomal Problems Affect Nearly All Human Embryos

Discovery may explain low fertility rates in humans Wednesday, 01 July 2009 Scientists have shown that chromosomal abnormalities are present in more than 90% of IVF embryos, even those produced by young, fertile couples. Ms Evelyne Vanneste, a PhD student in the Centre for Human Genetics and the University Fertility Center, Leuven University, Belgium, told the 25th annual conference of the European Society for Human Reproduction and Embryology today, that the surprising finding meant that current techniques used in preimplantation genetic screening (PGS), where embryos are screened genetically in order to select the best embryo for transfer, do nothing to improve pregnancy and live birth rates. Indeed, it can lead to potentially viable embryos being discarded, she said. Ms Vanneste and her team studied each cell from 23 three or four day-old IVF embryos from young (less than 35 years old), fertile couples who had asked for preimplantation genetic diagnosis (PGD). PGD is carried out where one or both parents have a known genetic abnormality, in this case an X-linked disorder or the microdeletions (loss of a tiny piece of a chromosome) that can cause such disorders as the cancer predisposition syndrome neurofibromatosis type 1. The embryos are screened to avoid the implantation of one carrying that abnormality. Such embryos are the most representative of normal human embryogenesis, the process that begins once an egg has been fertilised. Using new technologies that can detect chromosomal aberrations in the whole genome (all human chromosomes) of a single cell, the team was able to screen embryonic cells at a much higher resolution than previously. Therefore, they could identify more chromosomal abnormalities than has been possible using the current technique, fluorescent in situ hybridisation (FISH), which can only analyse ten of the approximately 32,000 genetic regions at the same time. "Until now, the majority of studies analysing the genetic composition of human embryos used low resolution techniques on embryos derived from couples with fertility problems who are at risk for embryonic aneuploidy, an aberrant number of chromosomes, such as three copies of chromosome 21 that results in Down's syndrome. Therefore, little was known about the frequency and type of chromosomal imbalances in embryos from normal, fertile women," said Ms Vanneste. "Our new technique has enabled us to show that chromosomal abnormalities are far more common and complex than previously anticipated, even in embryos from young, normal fertile couples. This leads us to believe that such abnormalities must be present in all human IVF-ICSI embryos.” "Although in vitro culture conditions are known to have a limited influence on the rate of chromosomal imbalances in IVF/ICSI embryos, it is probable that the chromosome instability observed in vitro also occurs in spontaneous pregnancies since, at most, 30% of human conceptions result in a live birth and more than 50% of spontaneous abortions carry chromosomal aberrations. The high rate of chromosomal abnormalities is almost certainly responsible for the low fecundity of humans compared with other mammals," she added. The scientists say that their work has important implications for preimplantation genetic screening (PGS) in fertility treatment. PGS is routinely used in many fertility centres for couples who encounter problems with conception, particularly for advanced maternal age, repeated failure of implantation, repeated miscarriages, or severe male fertility problems. In PGS, a single cell is removed from the early embryo for genetic testing, since it is hypothesised that the selection of chromosomally normal embryos for uterine transfer would increase the live birth rate and decrease the spontaneous abortion rate per embryo transferred. "Although PGS is promoted as a way of increasing the chances of a successful pregnancy," said Ms Vanneste, "there has never been any significant evidence that it does, in fact, increase live birth rates after IVF. Our findings have shown that almost every cell of a human embryo carries a different genetic composition; consequently, the one cell that is analysed genetically is not representative of the rest of the embryo. If the tested cell is genetically abnormal, the embryo will not be transferred. But the rest of the embryo might be normal and develop into a healthy person. Therefore, the use of PGS means that potentially viable embryos will be discarded. The prevalent chromosomal instability in all early human IVF embryos explains the failure of PGS to improve the live birth rate per embryo transferred.” "I think that we have made a crucial breakthrough that will change the way we do preimplantation genetic diagnosis and PGS and help to advance our ability to improve human fertility," said Ms Vanneste. ......... ZenMaster

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Human Embryonic Stem Cells Developed from 4-cell Embryo

World first may lessen ethical concerns Wednesday, 09 July 2008 For the first time in the world scientists have succeeded in developing human embryonic stem cells (hESCs) from a single cell, or blastomere, of a 4-cell stage embryo, the 24th annual conference of the European Society of Human Reproduction and Embryology heard today. Dr. Hilde Van de Velde, from the Vrije Universiteit Brussel (VUB), Brussels, Belgium, said that their research meant that it might be possible in the future to produce hESC lines at an earlier stage without destroying the embryo. Blastomeres are formed in the very early stages of embryonic development. About 24 hours after fertilisation the egg divides into two cells. Division into four cells occurs after 48 hours. After 96 hours, at the morulae stage, the fertilized egg has divided four to five times. During this time the size of the embryo does not increase, so the cells become smaller and smaller and they are strongly attached to each other which makes them more difficult to manipulate. At this early stage important decisions are taken: inner cells will become the foetus (including germ cells) and outer cells will become trophoblast (the outermost layer of the embryo that attaches to the wall of the uterus and serves as a nutritive pathway). There was, until now, uncertainty about which stage of early development the blastomeres ceased to be totipotent, i.e. able to develop into all cell types of the body. Worldwide, the majority of established hESC lines have been derived from the inner cells at the blastocyst stage; these are said to be pluripotent. "Previously, scientists have been able to derive hESC lines at the 8-cell stage," said Dr. Van de Velde, "but success rates were variable and it was necessary to culture them by mixing with established hESC lines. We have been able to derive hESCs at an earlier stage of embryonic development, and without the need for co-culture with established hESC lines. Now we have derived a second hESC from one cell of a 4-cell stage embryo. Given the complex nature of earlier attempts, we were pleased that we could develop a technique that seemed simple and was also reproducible." The scientists used mature eggs donated by couples being treated at the University's IVF centre. Embryos were obtained after ICSI using sperm from a consenting donor. Three, good quality 4-cell stage embryos were split into 12 single blastomeres and allowed to grow in vitro to produce twelve morulaes that were cultured in the conventional way for hESC derivation. From these twelve, one resulted in a stable hESC line. The scientists concluded that at least one cell was pluripotent. These new data confirm their recent report that at the 4-cell stage the cells are equal and totipotent. "Now we will try to derive four hESC lines from the same embryo in order to compare the potency capacity of all four cells," said Dr. Van de Velde. The work could have major ramifications for preimplantation genetic diagnosis (PGD), by enabling the biopsy of one cell from a 4-cell stage embryo, allowing the remaining three cells to develop into a blastocyst (five day embryo) which could be transferred into the uterus and develop into a healthy baby. "Currently, PGD is performed at the 8-cell stage, when one or two cells are removed; others have derived stable hESC lines at this stage but with low efficiency. If hESC derivation at the 4-cell stage turns out to be more efficient then at the 8-cell stage, we might consider to change our PGD policy in cases where we perform human leukocyte antigen (HLA) typing," said Dr. Van de Velde. HLA molecules play an important role in the immune system by ensuring that our bodies recognise their own cells as their own. By being able to select an HLA-identical embryo, the cord blood of the 'saviour siblings' could help cure an older brother or sister suffering from a genetic disease affecting the production and/or function of hematopoietic (blood producing) stem cells. "We could also combine the pregnancy of an HLA-matched healthy baby with the derivation of a personal hESC line that could be used to generate in vitro hematopoietic stem cells as an additional source of hematopoietic stem cells," she said. "We need to determine whether the removal of one cell at the 4-cell stage impairs the capacity of the embryo to develop into a healthy child in comparison with the removal of one/two cells at the 8-cell stage. We understand that some people may have ethical concerns about the production of hESCs," said Dr. Van de Velde, "but we already know from cryo-preserved embryos that the loss of one cell from a 4-cell stage embryo does not affect its capacity to implant in the womb. We believe that by making it possible to intervene at an earlier stage, and without destruction of the embryo, these ethical concerns will be diminished." ......... ZenMaster

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Genetics: Lost In Translation

Language barrier adds confusion to prenatal testing Monday, 09 June 2008 Many people struggle to understand the complexities of genetic problems in pregnancy and find medical language difficult to understand, particularly when faced with major decisions such as whether to terminate a pregnancy. A recent study, funded by the Economic and Social Research Council (ESRC), investigated how Britain's Bangladeshi community understand the disorders, and make decisions about testing and screening in the light of health care and religious opinion. The researchers found language difficulties added a great deal of misunderstanding about the nature and cause of disorders. There were difficulties, for example, over the distinction between being 'affected' and being a 'carrier'. The nature of risk, and the kind of inferences that can be made from genetic testing, can also be a source of confusion. While earlier studies have found that similar confusions are common among the general public as well, the difficulties of translation can make minority groups, such as the Bangladeshis, especially vulnerable to such misunderstandings. Those who have English as a second language are not alone in struggling to understand the complexities of medical terminology. Researchers find that these misunderstandings are not solely connected to language as a barrier but commonly to misinterpreting medical information. Such as a 75% chance of a having a child that is not being affected by a particular condition can be interpreted as having a child that is 75% normal. Problems linked to use of interpreters are compounded by the fact that there may be medical terms for which there is no appropriate translation. Women with limited English may be entirely reliant on their husband, or another family member, for an explanation of what consultants or genetics counsellors have said. As a result, information they receive may be inaccurate, misunderstood, or incomplete. Senior Research Fellow, Dr Santi Rozario, said: "Genetic disorder is likely to be understood by Bangladeshi Muslims in Britain, at least initially, as a biomedical problem for which conventional medical treatment is appropriate, and indeed fard (obligatory) as an Islamic duty. Bangladeshi families will therefore almost always look to the British medical system for assistance." The research shows us that the issue is not simply one of numbers or availability of interpreters and it is a complex and difficult time for patients. Greater understanding of the language barrier and possible misunderstanding need to be considered when dealing with patients whose first language is not English. Read more at: Genetics, Religion and Identity: a study of Bangladeshi Muslims in Britain Sophie Gilliat-Ray and Santi Rozario The School of Religious and Theological Studies, Cardiff University ......... ZenMaster

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Prenatal Screening Misses Half of Chromosomal Abnormalities

Women should be better informed before deciding to undertake it Monday, 02 June 2008 Prenatal biochemical screening tests are widely used to look for chromosomal abnormalities in the foetus which can lead to serious handicap or even death during gestation or in the first few days after birth. But these tests are only able to detect fewer than half of the total chromosomal abnormalities in the foetus, a scientist will tell the annual conference of the European Society of Human Genetics today (Monday 2 June) Dr. Francesca R. Grati, of the TOMA Laboratory, Busto Arsizio, Italy, says that these findings mean that women should be better informed on the limitations of such diagnostic tests. The researchers studied 115,576 prenatal diagnoses carried out during the last fourteen years. 84,847 were amniocenteses, usually carried out around the 16th week of pregnancy, and 30,729 chorionic villus samplings, which can be undertaken from 12 weeks into the pregnancy. Both these tests carry an increased risk of miscarriage, so the decision on whether or not to undertake them can be difficult to weigh up. "Since our sample included a large number of women aged less than 35 who underwent invasive prenatal diagnosis without any pathological indication to do so, we felt that the results could be useful in helping to inform pre-test counselling of such women", says Dr. Grati. "Up until now, the information we had came from smaller studies which only looked at the performance of these tests in detecting a limited number of chromosomal abnormalities." After analysing the results of the chromosomal abnormalities from their own dataset, the researchers combined them with the official detection rates for these abnormalities published by SURUSS and FASTER consortia. These are multi-centre research groups involved in the investigation of screening and diagnostic tests performed in pregnancy, whose results are being used to optimise prenatal care for pregnant patients. They found that current screening procedures were only able to detect half the total chromosomal abnormalities in women both younger and older than 35. The TOMA laboratory is particularly suited to carry out this kind of research, says Dr. Grati, because it was among the first in the world to deal with prenatal diagnosis, and has a vast number of prenatal diagnostic samples at its disposal. Current tests do not detect all foetal chromosomal abnormalities, but only trisomies 21 (Down syndrome), 18 (Edward's syndrome), and 13 (Patau syndrome), monosomy X (Turner syndrome), and triploids (conceptuses with 69 chromosomes instead of 46). "These are common vital chromosomal abnormalities, but there are many others which are not picked up by these tests", says Dr. Grati. "And the tests do not even detect 100% of the common abnormalities." At conception, 23 chromosomes from each parent combine to create a foetus with 46 chromosomes in all its cells. Trisomy occurs when the foetus has one additional chromosome (47 instead 46). The extra genetic material from the additional chromosome causes a range of problems of varying severity. In Down syndrome, for example, where the foetus has three copies of chromosome 21, babies are usually born with impaired cognitive ability and physical growth, cardiac defects and a characteristic facial appearance. Unlike many other such abnormalities, however, babies born with Down syndrome are able to lead relatively normal lives and their life expectancy is around 50 years. Other than trisomy, the foetus can also have the loss of genetic material (deletions) or chromosomal abnormalities in a non-homogeneous form, where there is a mixture of two cell lines, one normal and the other abnormal. "Some of these disorders are relatively common in the foetus, which may have as much chance of surviving as children who are born with Down syndrome, and it is worrying that current biochemical tests are not always able to detect them," says Dr. Grati. "Our research confirms that it is fundamental for doctors to counsel patients about the limitations of current screening methods, so that they can make an informed decision on whether or not to undergo invasive diagnostic testing." ......... ZenMaster

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Human ESC's derived from PGD embryos with Fragile X

hESCs derived from PGD embryos with Fragile X Wednesday, 14 November 2007 A human stem cell line derived from embryos that were identified by preimplantation genetic diagnosis (PGD) to carry the mutation for fragile X syndrome has provided an unprecedented view of early events associated with this disease. In addition to giving scientists fresh insight into fragile X, results from this unique model system have emphasized the value of this new source of embryonic stem cells and may have a significant impact on the way that genetic diseases are studied in the future. The research is published in the November issue of the journal Cell Stem Cell, published by Cell Press. Fragile X syndrome, the most common cause of inherited mental impairment and of autism, is caused by the absence of the fragile X mental retardation protein (FMRP). Most individuals with fragile X exhibit a specific mutation in the fragile X mental retardation 1 (FMR1) gene that usually coincides with epigenetic DNA modifications. However, the developmental timing and mechanisms associated with acquisition of these characteristics are not clear due to the absence of appropriate cellular and animal models. To examine developmentally regulated events involved in fragile X pathogenesis, Dr. Nissim Benvenisty and Dr. Rachel Eiges from the Hebrew University Department of Genetics in Jerusalem, Israel, together with Dr. Dalit Ben-Yosef from the IVF unit at the Tel-Aviv Sourasky Medical Center, established a human embryonic stem cell (hESC) line from a preimplantation fragile X-affected embryo identified by PGD. The fragile X cell line, called HEFX, displayed all characteristics typical of an hESC line and possessed the full genetic mutation observed in fragile X patients. The work "highlights the value of [human embryonic stem cells] as a model system for early human embryo development," the study's co-author, Rachel Eiges, told The Scientist. "We show that it can be used as a powerful tool to analyze the effect of a specific mutation on particular developmental events, allowing exploring processes which are otherwise inaccessible for research." The researchers found that undifferentiated HEFX cells transcribed FMR1 and expressed FMRP, suggesting that the fragile X mutation by itself is not sufficient to cause FMR1 inactivation. The research team went on to show that differentiated derivatives of HEFX cells exhibited a decrease in FMRI transcription and FMRP expression along with an increase in epigenetic modifications associated with fragile X syndrome. “The fact that FMR1 inactivation and other modifications take place after differentiation suggests that it might be possible to prevent some of these events as an attempt to rescue the abnormal phenotype in cells with the full fragile X mutation,” suggests Dr. Benvenisty. HEFX cells represent an excellent model for examination of early embryogenesis and will contribute to a clearer understanding of the molecular mechanisms underlying fragile X pathogenesis. This research is also compelling on a more general level in that it validates the usefulness of hESCs derived from embryos that have been screened for specific mutations with PGD. hESC lines derived in this manner represent a potent tool for the study of a variety of human diseases and the development of new therapeutic strategies.

"Certainly, stem cell lines such as this will help science unravel the mechanisms associated with human genetic disorders, and hopefully lead to new therapeutic treatments and interventions in the future," said Robert Lanza of Advanced Cell Technology in Los Angeles, CA, who was not involved in the research. Such an approach has been overshadowed by the focus on developing stem cells as treatments for various disorders, he noted. ......... ZenMaster

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Changes in chromosomal constitution of preimplantation embryos suggest caution in genetic screening

Changes in chromosomal constitution of preimplantation embryos suggest caution in genetic screening June 18 2007 Embryos that are selected out as abnormal can undergo chromosomal modifications, a scientist will tell the annual conference of the European Society of Human Genetics today. Ms Tsvia Frumkin, from the Racine IVF unit, LIS Maternity Hospital, Tel Aviv Sourasky Medical Centre, Tel Aviv, Israel, will tell the conference that her team’s findings meant that the results of preimplantation genetic screening (PGS) for chromosomal abnormalities were not always reliable and should be interpreted with caution. PGS is offered to women with recurrent IVF failures as well as repeated miscarriages. It is based on the concept that the entire chromosomal constitution of an embryo can be represented by a single cell, which is removed from the embryo. If one biopsied cell is found to be abnormal, there is a 90% chance that the rest of the embryo is also abnormal or mosaic, where two or more cells with different chromosomal constitution exist in a single embryo. Ms Frumkin analysed 8 cell embryos at day 3 of development using the FISH (fluorescence in situ hybridization) technique. Two cells from each embryo were analysed, and between 5 and 9 chromosomes were investigated. The abnormal embryos were re-analysed on day 5, using the same method. “By comparing FISH results of day 5 embryos to the abnormal results of the same embryos on day 3, we could elucidate the origin of the chromosomal aberrations and follow different chromosomal modifications as they occurred during preimplantation period. The timing is significant because embryos used in IVF are normally transferred at between 3 and 5 days old”, says Ms Frumkin. “We found that embryos which were abnormal on day 3 demonstrated a high rate of mosaicism However, on day 5 some of them had undergone ‘self-correction’ into normal embryos. Others kept the same abnormalities, while some had acquired additional chromosomal abnormalities”, she says. Following the research, Ms Frumkin’s hospital has decided to offer PGS only to patients after they have undergone more than 6 previous failed IVF cycles, been checked for and found to have normal chromosomal make-up, and produced more than 6 good quality embryos. “Even in cases that fulfil these conditions, we nevertheless prefer transferring more embryos back to the uterus rather than carrying out PGS biopsies for them on day 3, assuming that natural selection will usually favour the normal embryos for implantation,” she says. “Our results can explain why PGS would not be able to increase pregnancy potential but rather can serve as a prognostic tool in a limited number of cases”, says Ms Frumkin. “It can also help us make optimal decisions about the value of switching to a different assisted reproduction technique, for example egg donation.” .........

ZenMaster

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