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Präimplantationsdiagnostik für monogen vererbte Erkrankungen
Abstract
Zusammenfassung
Die Präimplantationsdiagnostik (PID) für monogen vererbte Erkrankungen ist heute neben der Pränataldiagnostik als eine Möglichkeit der Realisierung des Kinderwunsches international fest etabliert. Die Schwangerschaftsraten entsprechen denen einer Behandlung mit intrazytoplasmatischer Spermieninjektion (ICSI) ohne genetische Testung im Rahmen der normalen Kinderwunschbehandlung. Sie erfordert vorab eine umfassende ergebnisoffene genetische und reproduktionsmedizinische Beratung interessierter Paare mit Darstellung der Möglichkeiten der PID, aber auch ihrer Risiken und ihrer begrenzten Erfolgschancen. Von Seiten des PID-Zentrums ist neben einer guten interdisziplinären medizinischen Betreuung ein Qualitätsmanagement für das genetische und In-vitro-Fertilisations(IVF)-Labor inklusive Schnittstellen zu etablieren, welches den Besonderheiten der Einzelzelldiagnostik Rechnung trägt.
Identifying rare genetic forms of infantile cholestasis is challenging due to their similar clinical presentation and their diverse etiology. After exclusion of common non-genetic causes a huge list of rare differential diagnosis remains to be solved. More than 90 genes are associated with monogenic forms of infantile cholestasis, thus preventing routine genetic workup by Sanger sequencing. Here we demonstrate a next generation sequencing approach to discover the underlying cause in clinically well characterized patients in whom common causes of infantile cholestasis have been excluded. After validation of the analytical sensitivity massive parallel sequencing was performed for 93 genes in six prospectively studied patients. Six novel mutations (PKHD1: p.Thr777Met, p.Tyr2260Cys; ABCB11: p.Val1112Phe, c.611+1G>A, p.Gly628Trpfs*3 and NPC1: p.Glu391Lys) and two known pathogenic mutations were detected proving our multi gene panel for infantile cholestasis to be a sensitive and specific method overcoming the complexity of the phenotype-based, candidate gene approach. Three exemplary clinical cases of infants with cholestasis are presented and discussed in the context of their genetic and histopathological findings (autosomal recessive polycystic kidney disease, atypical PFIC and Niemann-Pick syndrome type C1). These case reports highlight the critical impact of integrating clinical, histopathological and genetic data during the process of multi gene panel testing to ultimately pinpoint rare genetic diagnoses.
Copyright © 2015. Published by Elsevier Ltd.
Genetic testing is an integral part of the routine diagnostic workup of subfertile couples. They are
performed according to the German act on genetic diagnostic testing (“Gendiagnostikgesetz”) in a
stepwise and evidence-based manner for the diagnostic evaluation in order to identify genetic alterations
that may potentially have an impact on the results of assisted reproduction techniques (ART)
and hence, the decision of the couple to utilize them. Basic workup includes karyotyping of both
partners, and based on individual clinical and laboratory findings may be complemented by further
specific gene analysis. Identified genetic alterations may also confer increased specific genetic
risks for the prospective offspring, which might be addressed by prenatal or preimplantation genetic
diagnosis. Currently applied procedures for genetic testing might be replaced in the near future
by new, whole genome-based techniques. Their application requires new evidence-based definitions
of suitable medical indications as well as new strategies for genetic counseling and communication
of their results.
Although chromosomal mosaicism in human preimplantation embryos has been described for almost two decades, its exact prevalence is still unknown. The prevalence of mosaicism is important in the context of preimplantation genetic screening in which the chromosomal status of an embryo is determined by the analysis of a single cell from that embryo.
Here we report a systematic review and meta-analysis of studies on the chromosomal constitution of human preimplantation embryos. In 36 studies, out of 2117 citations that met our search criteria, data were provided extensively enough to allow classification of each analysed embryo with prespecified criteria for its chromosomal makeup. The main outcome of this classification was the prevalence of chromosomal mosaicism in human preimplantation embryos.
A total of 815 embryos could be classified. Of these, 177 (22%) were diploid, 599 (73%) were mosaic, of which 480 (59% of the total number of embryos) were diploid-aneuploid mosaic and 119 (14% of the total number of embryos) were aneuploid mosaic, and 39 (5%) contained other numerical chromosomal abnormalities. The distribution of the embryos over these categories was associated with the developmental stage of the embryos, the method used for analysis and the number of chromosomes analysed.
Diploid-aneuploid mosaicism is by far the most common chromosomal constitution in spare human preimplantation embryos after IVF. This undermines the reliable determination of the ploidy status of a cleavage-stage embryo based on the analysis of a single cell. Future research should determine the origin and developmental potential of mosaic embryos.
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.
The 10th report of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium is presented, documenting
cycles collected for the calendar year 2007 and follow-up of the pregnancies and babies born until October 2008 which resulted
from these cycles. Since the beginning of the data collections there has been a steady increase in the number of cycles, pregnancies
and babies reported annually. For data collection X, 57 centres participated, reporting on 5887 cycles to oocyte retrieval
(OR), along with details of the follow-up on 1516 pregnancies and 1206 babies born. A total of 729 OR were reported for chromosomal
abnormalities, 110 OR for sexing for X-linked diseases, 1203 OR for monogenic diseases, 3753 OR for preimplantation genetic
screening and 92 OR for social sexing. Data X is compared with the cumulative data for data collections I–IX.
Human embryo biopsy is performed for preimplantation genetic diagnosis (PGD). The impact of 1- or 2-cell removal at cleavage-stage on future embryo development and implantation capacity is highly debated.
In order to explore this issue further, a cohort of Day 5 single embryo transfers was analysed prospectively for embryological and clinical outcome. All transferred embryos resulted from 8-cell embryos on Day 3, from which subsequently either one cell (group I, n = 182) or two cells (group II, n = 259) were removed, or on which no invasion by means of embryo biopsy was performed (group III, control group, n = 702). RESULTS Blastocyst formation was significantly better in group III compared with group II, and similar to group I. Group I and group II did not differ in Day 3 nor in Day 5 embryo development. The overall live birth rate was significantly higher in group I (37.4%, CI 29.0-47.4%) than in group II (22.4%, CI 17.0-28.9%), and comparable to the reference ICSI population (35.0%, CI 30.8-39.7%).
The clinical outcome of 1-cell biopsy was significantly better than that of 2-cell biopsy, even when adjusted for availability of genetically transferable embryos.
For prospective parents at risk of transmitting a monogenic disease, polar body analysis is an option for pre-conception genetic diagnosis. In Germany, polar body analysis is currently performed in only two centers (Lübeck and Regensburg).
The authors present a clinical series of 9 couples at risk for the transmission of a monogenic disease who underwent in vitro fertilization with polar body analysis.
Nine couples have undergone in vitro fertilization with polar body analysis at the center in Lübeck since 2004. Three healthy children were born after polar body analysis for mucopolysaccharidosis type I, incontinentia pigmenti, and cystic fibrosis. The decision to undergo in vitro fertilization with polar body analysis is not easy for prospective parents to take, even though it often follows years of emotional suffering. Treatment with the methods of reproductive medicine in general, and with polar body analysis in particular, can cause considerable physical and emotional stress.
For prospective parents in Germany at risk of transmitting a monogenic disease, polar body-based preimplantation diagnosis is an alternative to prenatal diagnosis and possible termination of pregnancy. The live birth rate per treatment cycle in this clinical series was 30%, which can be considered satisfactory. Nonetheless, most of the couples who did not achieve pregnancy after a first treatment cycle dropped out of treatment prematurely and did not go on to a second cycle.
Although morally acceptable in theory, preimplantation genetic diagnosis (PGD) for mitochondrial DNA (mtDNA) disorders raises several ethical questions in clinical practice. This paper discusses the major conditions for good clinical practice. Our starting point is that PGD for mtDNA mutations should as far as possible be embedded in a scientific research protocol. For every clinical application of PGD for mtDNA disorders, it is not only important to avoid a 'high risk of serious harm' to the future child, but also to consider to what extent it would be possible, desirable and proportional to try to reduce the health risks and minimize harm. The first issue we discuss is oocyte sampling, which may point out whether PGD is feasible for a specific couple. The second issue is whether one blastomere represents the genetic composition of the embryo as a whole -- and how this could (or should) be investigated. The third issue regards the cutoff points below which embryos are considered to be eligible for transfer. We scrutinize how to determine these cutoff points and how to use these cutoff points in clinical practice -- for example, when parents ask to take more or less risks. The fourth issue regards the number of cycles that can (or should) justifiably be carried out to find the best possible embryo. Fifth, we discuss whether follow-up studies should be conducted, particularly the genetic testing of children born after IVF/PGD. Finally, we offer the main information that is required to obtain a truly informed consent.
Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts.
This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis.
In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born.
This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.
Preimplantation genetic diagnosis (PGD) has been the prevalent method of cleavage-stage embryo biopsy for the past 2 decades. Fluorescent in situ hybridization and polymerase chain reaction have been used for diagnosis after biopsy. The primary indications for PGD have been single gene disorders, inherited chromosome abnormalities, and sexing for X-linked disease. Unlike PGD where most couples are fertile and are at risk for a specific inherited disorder, preimplantation genetic screening (PGS) is performed in infertile or subfertile couples receiving assisted reproductive techniques to achieve pregnancy. PGS uses PGD technology to help select embryos for transfer free of chromosomal abnormalities. The technique has been used in patients with advanced maternal age, repeated implantation failure, repeated miscarriages, and severe male factor infertility. Ten randomized controlled trials have shown that PGS performed in cleavage-stage embryos does not improve delivery rates for a variety of indications. Cells biopsied from cleavage-stage embryos may show high levels of chromosomal mosaicism and, therefore, may not be representative of the rest of the embryo. Although the majority of clinics still use cleavage-stage biopsy for PGD, the popularity of polar body biopsy and blastocyst biopsy has been increasing for specific indications; polar biopsy is being used in countries with laws forbidding biopsy of embryos. An added advantage with blastocyst biopsy has been the introduction of vitrification as a successful method of cryopreserving embryos. Blastocyst culture and transfer are being use routinely in many in vitro fertilization units, with reported higher survival rates, even after biopsy. As with cleavage-stage biopsy, mosaicism is problematic with blastocyst biopsy. In recent years, polymerase chain reaction has become one of the most important methods in genetic testing, allowing for diagnosis of small number of cells for PGD by amplifying and enriching a DNA sample. Further advances in PGD and PGS technology include the introduction of array comparative genomic hybridization and single nucleotide polymorphism arrays (SNP arrays). Both techniques can analyze 24 chromosomes in the embryo. The use of SNP arrays for PGD analysis will be controversial because of ethical concerns over the immense amount of genetic information obtained from each embryo. Randomized controlled trials need to be conducted using both array comparative genomic hybridization and SNP arrays for PGS to determine if either will improve delivery rates.
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.
Introduced more than 20 years ago, the use of polar bodies (PB), involving sequential removal and genetic analysis of the first (PB1) and second (PB2) PB, provides the option for pre-embryonic diagnosis, when the objection to the embryo biopsy procedures makes preimplantation genetic diagnosis (PGD) non-applicable. PB based approach has presently been utilized in PGD for genetic and chromosomal disorders, applied either separately, or together with embryo biopsy approaches, especially if there are two or more PGD indications. We present here the world's largest experience of 938 PGD cycles for single gene disorders performed by PB testing for 146 different monogenic conditions, which resulted in birth of 345 healthy children (8 pregnancies are still ongoing), providing strong evidence that PB based PGD is a reliable and safe procedure, with extremely high accuracy rate of over 99%. With application of microarray technology, PB based approach will be utilized for increasing number of indications, involving simultaneous testing for 24 chromosomes and single gene disorders.
To investigate the incidence of embryos' self-correction during preimplantation development in terms of mosaicism and in correlation with its developmental stage.
Prospective study to compare the chromosome status of embryos on day 3 with that of day 5, in correlation with their developmental stage.
In vitro fertilization unit of a university-affiliated hospital.
Eighty-three aneuploid embryos.
Fluorescence in situ hybridization (FISH) reanalysis.
Day 3 embryos classified as mosaic or chromosomally abnormal by preimplantation genetic screening (PGS) were reanalyzed on day 5. The results were evaluated in correlation with the embryos' developmental stage.
Out of 83 day 3 aneuploid embryos, 15 (18.1%) were diagnosed with mosaicism. The FISH reanalysis on day 5 demonstrated that 27 embryos (32.6%) were partly or entirely normal disomic. Of these 83 aneuploid embryos, 8 (9.7%) underwent complete self-correction. The PGS results demonstrated that 26.5% of the embryos were trisomic, of which 41.0% underwent trisomic rescue by day 5. Self-correction was in correlation with the embryo's developmental stage, i.e., 38.1% of aneuploid embryos that developed to the blastocyst stage underwent self-correction compared with only 12.5% of embryos that only cleaved after biopsy.
Our results demonstrate that self-correction of aneuploid and mosaic embryos occurs probably more significantly during development toward the blastocyst stage than in delayed embryos. In addition, trisomic embryos correct themselves more than other aneuploidies. These findings suggest that PGS results must be interpreted with caution.
In women who are heterozygous for a genetic disease, genetic analysis of the first polar body allows the identification of oocytes that contain the maternal unaffected gene. These oocytes can be fertilized and transferred to the mother without risk of establishing a pregnancy with a genetically abnormal embryo. We have demonstrated that removal of the first polar body has no effect on subsequent fertilization rates or embryonic growth to the blastocyst stage. We have developed a PCR technique to successfully analyze the PI type Z and PI type M genotypes of alpha-1-antitrypsin deficiency and applied this technique for a couple at risk for PI type ZZ alpha-1-antitrypsin deficiency. After standard IVF treatment to stimulate multiple follicle development, eight oocytes were aspirated transvaginally. Polar bodies were removed by micromanipulation from seven oocytes and fertilization occurred in six cases. PCR analysis was successful in five oocytes. One was PI type M, two were PI type Z and two were heterozygous MZ due to crossing over. Embryos from the two oocytes containing the unaffected gene (polar body PI type Z) were transferred in the same cycle 48 h after insemination. No pregnancy was established. The accuracy of the polar body diagnosis was confirmed by polymerase chain reaction (PCR) analysis of an oocyte that failed to fertilize.
Over 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins.
To compare the clinical outcomes after the transfer of blastocysts versus early cleavage embryos in assisted reproduction technologies (ART).
A retrospective analysis of all the ovarian stimulation-in vitro fertilization-embryo transfer cycles performed at the Centre for Human Reproduction, Athens, Greece, between June 1997 and December 2001.
The number of blastocysts transferred per ET was significantly lower compared to that of all early cleavage embryos. The implantation rate of blastocysts was significantly higher compared to that of all other modes of transfer. Clinical pregnancy rate after the transfer of blastocysts was significantly increased compared to that after transfer of any early cleavage embryo. The viable pregnancy rate after the transfer of blastocysts was significantly increased only compared to that after the transfer of day-2 embryos. There were no significant differences regarding the multiple gestation rates among the various modes of transfer.
The use of blastocysts in ART is beneficial when compared to that of day-2 embryos and at least comparable to that of day-3 embryos. Blastocyst culture and transfer remains a favourable and promising option in ART.
Valuable information on the cytogenetic constitution of female gametes has been deduced from the direct, so-called conventional
analysis of oocytes remaining unfertilized in programmes of assisted reproduction. Additional, indirect conclusions have become
possible by PGD of the polar bodies. Both techniques provided evidence for the co-existence of two aneuploidy-causing mechanisms
during first maternal meiosis; non-disjunction (ND) of bivalents results in the loss or gain of whole chromosomes in metaphase
II complements, whereas a precocious division (pre-division, PD) of univalents leads to the loss or gain of single chromatids.
As to the distribution of ND and PD, however, direct oocyte chromosome studies and PGD tell surprisingly different stories.
Moreover, first and second polar body analyses contradict the data derived from DNA polymorphism studies concerning the distribution
of first and second meiotic division errors. An increased awareness of these problems appears necessary because important
decisions are made on the basis of PGD results.
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Board of Trustees (2010) DIR Annual 2009
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Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential
- S Barbash-Hazan
- T Frumkin
- M Malcov
- Barbash-Hazan
Polar body biopsy in the diagnosis of monogenic diseases: the birth of three healthy children
- G Griesinger
- N Bündgen
- D Salmen
- Griesinger
Blastocyst versus early cleavage embryo transfer: a retrospective analysis of 4,165 transfers
- K Pantos
- E Makrakis
- P Karantzis
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