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Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds | Montag M.,University of Heidelberg | Magli M.C.,SISMER | And 7 more authors.
European Journal of Human Genetics | Year: 2012

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes. © 2012 Macmillan Publishers Limited All rights reserved.


Magli M.C.,SISMER | Montag M.,University of Bonn | Kster M.,University of Bonn | Muzi L.,SISMER | And 11 more authors.
Human Reproduction | Year: 2011

Background The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridization analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser). Methods Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical-or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification. Results The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency. Conclusions Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical-and laser-assisted biopsy give the same performance in terms of diagnostic efficiency. © 2011 The Author.


Hens K.,Maastricht University | Hens K.,Center for Society and the Life science | Dondorp W.,Maastricht University | Handyside A.H.,Gynaecology and Genetics Center | And 8 more authors.
Human Reproduction Update | Year: 2013

Background: Genetic testing of preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Microarray technology is being introduced in both these contexts, and whole genome sequencing of blastomeres is also expeted to become possible soon. The amount of extra information such tests will yield may prove to be beneficial for embryo selection, will also raise various ethical issues. We present an overview of the developments and an agenda-setting exploration of the ethical issues. Methods: The paper is a joint endeavour by the presenters at an explorative 'campus meeting' organized by the European Society of Human Reproduction and Embryology in cooperation with the department of Health, Ethics & Society of the Maastricht University (The Netherlands). Results: The increasing amount and detail of information that new screening techniques such as microarrays and whole genome sequencing offer does not automatically coincide with an increasing understanding of the prospects of an embryo. From a technical point of view, the future of comprehensive embryo testing may go together with developments in preconception carrier screening. From an ethical point of view, the increasing complexity and amount of information yielded by comprehensive testing techniques will lead to challenges to the principle of reproductive autonomy and the right of the child to an open future, and may imply a possible larger responsibility of the clinician towards the welfare of the future child. Combinations of preconception carrier testing and embryo testing may solve some of these ethical questions but could introduce others. Conclusions: As comprehensive testing techniques are entering the IVF clinic, there is a need for a thorough rethinking of traditional ethical paradigms regarding medically assisted reproduction. The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.


Harton G.L.,Reprogenetics LLC | Harton G.L.,Genetics and IVF Institute | De Rycke M.,Universitair Ziekenhuis | Fiorentino F.,Genoma Laboratories | And 5 more authors.
Human Reproduction | Year: 2011

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. Organization of a PGD centre, fluorescence in situ hybridization-based testing, Amplification-based testing and Polar Body and Embryo Biopsy for PGD/preimplantation genetic screening. Here, we have updated the sections that pertain to amplification-based PGD. Topics covered in this guideline include inclusion/exclusion criteria for amplification-based PGD testing, preclinical validation of tests, amplification-based testing methods, tubing of cells for analysis, set-up of local IVF centre and Transport PGD centres, quality control/quality assurance and diagnostic confirmation of untransferred embryos. © 2010 The Author.


Geraedts J.,Maastricht University | Montag M.,University of Bonn | Magli M.C.,SISMER | Repping S.,University of Amsterdam | And 10 more authors.
Human Reproduction | Year: 2011

Background Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. Methods In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. Results Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 115) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86): 55 were euploid (28) and 140 were aneuploid (72). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17 per cycle and 30 per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24) were euploid and 118 (76) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6), the results were discordant. Conclusions This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs. © 2011 The Author.


Harton G.L.,Reprogenetics LLC | Harper J.C.,University College London | Harper J.C.,Center for Reproductive and Genetic Health | Coonen E.,Maastricht University | And 3 more authors.
Human Reproduction | Year: 2011

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of new technologies as well as evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. organization of a PGD centre, fluorescence in situ hybridization (FISH)-based testing, amplification-based testing and polar body and embryo biopsy for PGD/preimplantation genetic screening (PGS). Here, we have updated the sections that pertain to FISH-based PGD. PGS has become a highly controversial technique. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Whereas some believe that PGS does not have a place in clinical medicine, others disagree; therefore, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. Topics covered in this guideline include inclusion/exclusion criteria for FISH-based PGD testing, referrals and genetic counselling, preclinical validation of tests, FISH-based testing methods, spreading of cells for analysis, set-up of local IVF centre and transport PGD centres, quality control/ quality assurance and diagnostic confirmation of untransferred embryos. © 2010 The Author.


Brown R.,University College London | Harper J.,University College London | Harper J.,Center for Reproductive and Genetic Health
Reproductive BioMedicine Online | Year: 2012

Since the first birth by IVF was achieved in 1978, the techniques involved in assisted reproductive technology have grown at an enormous rate. However, new technology has rarely been robustly validated before clinical use and developing scientific understanding of the available techniques has done little to alter their use. Furthermore, there are inconsistencies in the available clinical studies and endpoints. The benefits of some technologies already established for routine use are currently dubious and there are clear ethical concerns with providing them to patients when their scientific basis is not clear. As the uptake of assisted reproductive technology increases and newer technologies continue to push the boundaries of science, it is important to consider the clinical benefits and safety of all assisted reproductive technologies. This review will discuss aspects of some of the more recent techniques, including sperm DNA-damage tests, intracytoplasmic morphologically selected sperm injection, amino acid and metabolomics profiling, preimplantation genetic screening and time-lapse imaging, and those that may have substantial impacts on the field of reproductive medicine in the future including artificial gametes, ovarian transplantation and gene therapy. In 1978, the first child conceived by IVF was born. In the following 33 years, numerous technologies and techniques have been developed to further aid the ability to achieve pregnancies in couples for whom natural conception has failed. However, these techniques have rarely been robustly tested and approved before they are routinely offered to infertile couples. In other cases, a development in our scientific understanding of a technique has failed to be quickly incorporated into clinical changes. This raises the concern that some of the techniques offered to some patients offer little or no benefit, and in the worse cases is not confirmed to be safe. This is a particular concern as many of the techniques discussed here are often reserved for already vulnerable patients, such as those with recurrent IVF failure, This review begins by discussing some of the techniques already available to patients and questioning to what end they increase the likelihood of a live birth. The review then goes on to discuss the scientific developments that, although not currently in the clinic, could have substantial implications in the future. Since these newly developing techniques could be considered more controversial, discussion about their benefit and safety is urgently needed. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.


Harper J.C.,University College London | Harper J.C.,Center for Reproductive and Genetic Health | Sengupta S.B.,University College London
Human Genetics | Year: 2012

For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1-2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally 'best' embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates. © 2011 Springer-Verlag.


Abdel Raheem A.,University College London | Abdel Raheem A.,Cairo University | Garaffa G.,University College London | Rushwan N.,University College London | And 8 more authors.
BJU International | Year: 2013

What's known on the subject? and What does the study add? The management of patients with non-obstructive azoospermia (NOA) involves testicular sperm extraction (TESE or microdissection TESE) combined with intracytoplasmic sperm injection (ICSI). Sperm retrieval is successful in up to 50% of men with NOA; however, there is no single clinical finding or investigation that can accurately predict a positive outcome. Several studies have concluded that testicular biopsy is the best predictor of a successful TESE. The present study shows that the strongest predictor of the success of TESE is when tubules with mature spermatozoa (Johnsen score ≥8) are found in the histopathology specimen, irrespective of the overall state of spermatogenesis. The findings suggest that a lower limit threshold value of 2% of tubules with spermatogenesis in the histopathology specimen will result in a positive sperm retrieval. However, it is not practical to perform a diagnostic biopsy before TESE because this would mean that patients undergo two surgeries, which adds to the cost and increases the complications. The diagnostic biopsy is best coupled with an initial TESE before starting the ICSI cycle. Based on the findings of the histopathology specimen, patients may be then offered a repeat TESE if more sperm is needed on the day of ovum pick-up and ICSI. Also, if the initial TESE was negative, the biopsy result will help in the decision to offer a repeat TESE. This regimen is more cost-effective because the ICSI cycle will be started only if adequate sperm is retrieved. Objective To assess whether testicular histopathology can predict the outcome of testicular sperm extraction (TESE) in men with non-obstructive azoospermia (NOA) and therefore the role of preoperative diagnostic testis biopsy. Patients and Methods The study comprised a retrospective analysis of 388 patients with azoospermia who were referred from 2005 to 2010. Information collected included a clinical history and an examination including age and testicular size, serum follicle-stimulating hormone, two semen analyses and testicular histology collected at the time of surgical sperm retrieval (TESE or microdissection TESE). Results In total, 388 patients with a mean (range) age of 37 (18-66) years were included in the present study. Based on the history, clinical and laboratory findings, 112 patients had obstructive azoospermia and 276 patients had NOA. All patients in the obstructed group had a positive sperm retrieval. The sperm retrieval rate for the NOA group was 50%. An analysis of the results showed that the best predictor of a positive sperm retrieval was when tubules with mature spermatozoa were seen at biopsy, irrespective of the overall state of spermatogenesis (P < 0.001). Conclusions The presence of tubules with spermatazoa on biospy is the best predictor of a positive surgical sperm retrieval in patients with NOA. The diagnostic biopsy is best coupled with an initial TESE before starting the intracytoplasmic sperm injection (ICSI) cycle. Based on the findings of the histopathology specimen, patients may be offered a repeat TESE if more sperm is needed on the day of ovum pick-up and ICSI, or a redo TESE if the initial TESE was negative. © 2012 BJU International.


Chronopoulou E.,University College London | Harper J.C.,University College London | Harper J.C.,Center for Reproductive and Genetic Health
Human Reproduction Update | Year: 2015

Background: The advances in the world of IVF during the last decades have been rapid and impressive and culture media play a major role in this success. Until the 1980s fertility centers made their media in house. Nowadays, there are numerous commercially available culture media that contain various components including nutrients, vitamins and growth factors. This reviewgoes through the past, present and future of IVF culture media and explores their composition and quality assessment. Methods: A computerized search was performed in PubMed regarding IVF culture media including results from 1929 until March 2014. Information was gathered from the websites of companies who market culture media, advertising material, instructions for use and certificates of analysis. The regulation regarding IVF media mainly in the European Union (EU) but also in non-European countries was explored. Results: The keyword 'IVF culture media' gave 923 results in PubMed and 'embryo culture media' 12 068 results dating from1912 until March 2014, depicting the increased scientific activity in this field. The commercialization of IVF culture media has increased the standards bringing a great variety of options into clinical practice. However, it has led to reduced transparency and comparisons of brand names that do not facilitate the scientific dialogue. Furthermore, there is some evidence suggesting that suboptimal culture conditions could cause long-term reprogramming in the embryo as the periconception period is particularly susceptible to epigenetic alterations. IVF media are now classified as class III medical devices and only CE (Conformité Européene)-marked media should be used in the EU. Conclusion: The CE marking of IVF culture media is a significant development in the field. However, the quality and efficiency of culture media should be monitored closely. Well-designed randomized controlled trials, large epidemiological studies and full transparency should be the next steps. Reliable, standardized models assessing multiple end-points and post-implantation development should replace the mouse embryo assay. Structured long-term follow-up of children conceived by assisted reproduction technologies and traceability are of paramount importance. © The Author 2014.

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