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1.
The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders 总被引:11,自引:0,他引:11
Fiorentino F Magli MC Podini D Ferraretti AP Nuccitelli A Vitale N Baldi M Gianaroli L 《Molecular human reproduction》2003,9(7):399-410
We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods. 相似文献
2.
Findlay Ian; Ray Pierre; Quirke Phil; Rutherford Anthony; Lilford Richard 《Human reproduction (Oxford, England)》1995,10(6):1609-1618
Previously the diagnosis of sex and cystic fibrosis status hasbeen studied on single cells using the polymerase chain reaction(PCR). It has been suggested that allelic drop-out (PCR failureof one allele) and/or preferential amplification (hypo-amplificationof one allele) may contribute to poor reliability and misdiagnosis,although this remains controversial as some reports suggestthat allelic drop-out does not occur. We investigated an improvedmethod of diagnosing sex and cystic fibrosis in single cellsusing a new technology (fluorescent PCR) to determine the baselevel of PCR artefacts (allelic drop-out and preferential amplification)which, in combination with improved sensitivity, should improvePCR reliability and accuracy. Fluorescent PCR gives high reliability(97%) and accuracy rates (97%) in somatic cells for both sexand cystic fibrosis diagnosis and its lower detection thresholdallows allelic drop-out and preferential amplification to beeasily distinguished. We also achieved high reliability andaccuracy in diagnosing cystic fibrosis in human blastomeres.This study confirms earlier reports of both allelic drop-outand preferential amplification in single cell analysis. We demonstratethat both allelic drop-out and preferential amplification occurin somatic cells and suggest these are separate phenomena. Preferentialamplification appeared common in single cell PCR while allelicdropout apparently occurred at random in each allele. Preferentialamplification was mainly amplification of the larger allele.We suggest that some inaccuracy/misdiagnosis may be due to bothpreferential amplification as well as allelic drop-out. Otherfindings were variability in drop-out between PCR and that amplificationof signals from human blastomeres may be linked to embryo quality.We suggest that allelic drop-out is dependent on the numberof cells within the sample. 相似文献
3.
Handyside AH Robinson MD Simpson RJ Omar MB Shaw MA Grudzinskas JG Rutherford A 《Molecular human reproduction》2004,10(10):767-772
Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions. 相似文献
4.
Chamayou S Alecci C Ragolia C Giambona A Siciliano S Maggio A Fichera M Guglielmino A 《Human reproduction (Oxford, England)》2002,17(5):1158-1165
BACKGROUND: In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. METHODS AND RESULTS: The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. CONCLUSIONS: These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy. 相似文献
5.
Using PCR in preimplantation genetic disease diagnosis. 总被引:10,自引:1,他引:9
Preimplantation diagnosis of genetic disease can be accomplished by embryo biopsy or polar body analysis using in-vitro gene amplification (PCR). PCR analysis of single cells is subject to a number of errors which decrease the reliability of the diagnosis. Using realistic assumptions about error rates based on experimental data, we analyse some of the practical consequences to be faced by whose wishing to use this diagnostic procedure. We considered both autosomal dominant and recessive diseases. We calculate the probability of making mistakes in the diagnosis, assuming a realistic range in the magnitude of PCR efficiency, cell transfer, and contamination errors. We conclude that, in general, analysing blastomeres is subject to less mis-diagnosis than polar body analysis, except in the case of dominant diseases which are caused by genes which lie extremely close to the centromere. We also show that typing multiple blastomeres from a single embryo or combining polar body typing with blastomere analysis results in significantly lower levels of mis-diagnosis with unacceptable consequences. The preimplantation diagnosis of X-linked diseases based upon Y chromosome sequence analysis is also discussed. 相似文献
6.
Vrettou C Traeger-Synodinos J Tzetis M Palmer G Sofocleous C Kanavakis E 《Human mutation》2004,23(5):513-521
Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application. 相似文献
7.
New advances in human embryology: implications of the preimplantation diagnosis of genetic disease 总被引:3,自引:1,他引:2
The diagnosis of genetic disease in preimplantation embryosis discussed. The typing of spermatozoa may be feasible forfactors such as the presence of an X and Y chromosome. Embryosmight be typed by non-invasive methods, by assessing their uptakeof metabolites although the widest opportunities may arise bythe use of invasive methods which involve the removal of oneor a small number of cells. The methods of diagnosis are discussed,including enzyme assays and the use of DNA probes, preliminaryresults with human embryos are presented and the difficultiesrelated to these techniques are debated. The low rate of implantationof replaced embryos will mean that many embryos will have tobe diagnosed, and certain embryological factors such as thehigh incidence of chromosomal imbalance and the problems ofimprinting might obscure certain diagnoses. Theadvantages and disadvantages of the method are discussed. 相似文献
8.
Fiorentino F Biricik A Karadayi H Berkil H Karlikaya G Sertyel S Podini D Baldi M Magli MC Gianaroli L Kahraman S 《Molecular human reproduction》2004,10(6):445-460
Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder. In this paper we describe a strategy optimized for preimplantation genetic diagnosis (PGD) of haemoglobinopathies combined with HLA matching. This procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The above assay can also be used for preimplantation HLA matching as a primary indication. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassaemia, one for Wiscott-Aldrich syndrome and two for leukaemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. Minisequencing-based HLA typing combined with HLA STR haplotyping has been shown to be a reliable strategy for preimplantation HLA matching. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case. 相似文献
9.
Thornhill AR deDie-Smulders CE Geraedts JP Harper JC Harton GL Lavery SA Moutou C Robinson MD Schmutzler AG Scriven PN Sermon KD Wilton L;ESHRE PGD Consortium 《Human reproduction (Oxford, England)》2005,20(1):35-48
Among the many educational materials produced by the European Society of Human Reproduction and Embryology (ESHRE) are guidelines. ESHRE guidelines may be developed for many reasons but their intent is always to promote best quality practices in reproductive medicine. In an era in which preimplantation genetic diagnosis (PGD) has become a reality, we must strive to maintain its efficacy and credibility by offering the safest and most effective treatment available. The dominant motivators for the development of current comprehensive guidelines for best PGD practice were (i) the absence of guidelines and/or regulation for PGD in many countries and (ii) the observation that no consensus exists on many of the clinical and technical aspects of PGD. As a consequence, the ESHRE PGD Consortium undertook to draw up guidelines aimed at giving information, support and guidance to potential, fledgling and established PGD centres. The success of a PGD treatment cycle is the result of great attention to detail. We have strived to provide a similar level of detail in this document and hope that it will assist staff in achieving the best clinical outcome for their patients. 相似文献
10.
Reproductive genetic counselling in non-mosaic 47,XXY patients: implications for preimplantation or prenatal diagnosis: Case report and review 总被引:3,自引:0,他引:3
Tachdjian G Frydman N Morichon-Delvallez N Dû AL Fanchin R Vekemans M Frydman R 《Human reproduction (Oxford, England)》2003,18(2):271-275
With an incidence of approximately 1 in 500 male newborns, the 47,XXY genotype is one the most common sex chromosome anomalies. It is also the most frequent genetic cause of human infertility. Some non-mosaic 47,XXY patients have sperm production which allows infertility treatment to be offered by ICSI. Therefore, the risk of transmitting a chromosome anomaly to the next generation is an important problem in reproductive genetic counselling of these patients. Here, we report on a twin pregnancy where two karyotypically normal neonates 46,XX and 46,XY were born after the use of ICSI in assisted reproduction of a patient with a non-mosaic 47,XXY syndrome. To date, only 38 evolving pregnancies including the present cases, have been reported after ICSI using sperm from non-mosaic 47,XXY patients. Although these data are scarce, they suggest that the risk of chromosome anomaly in the offspring of these patients is low; hence, their reproductive genetic counselling can be reassuring, and management of the pregnancy can proceed with caution. 相似文献
11.
Cytogenetic analysis of biopsied preimplantation mouse embryos: implications for prenatal diagnosis 总被引:1,自引:0,他引:1
Genetic diagnosis of enzyme deficiencies have been developed in mouse models using biopsied preimplantation embryos. A technique for preimplantation cytogenetic analysis was developed using embryos which were biopsied at the 4-cell, 8-cell and morula stage. Three culture methods were compared, using exposure directly to colchicine or preculture or overnight culture with subsequent exposure to colchicine. Some biopsies were lost through cell death (4.7-20.3%) and technical problems (11.1-27.8%), whilst others remained in interphase (13.0-46.5%). Air-dried preparations were successfully G-banded and karyotyped by modifying routine banding procedures. Biopsies placed directly into colchicine yielded the greatest mitotic index. However, chromosome morphology was unsuitable for G-banded analysis. After a short preculture, 71.4% of fixed 8-cell biopsies were karyotyped. The success of analysis of 8-cell biopsies is probably partly related to the higher mitotic index of intact embryos at this stage. Loss of chromosomes through scatter and chromosome overlapping limited analysis, particularly in biopsies with only one metaphase. The present success rate is not acceptable for the application of this technique to human prenatal diagnosis. However, it presents a useful tool for the study of chromosomal error at the research level. 相似文献
12.
Goossens V Sermon K Lissens W De Rycke M Saerens B De Vos A Henderix P Van de Velde H Platteau P Van Steirteghem A Devroey P Liebaers I 《Molecular human reproduction》2003,9(9):559-567
Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage. 相似文献
13.
Goossens V De Rycke M De Vos A Staessen C Michiels A Verpoest W Van Steirteghem A Bertrand C Liebaers I Devroey P Sermon K 《Human reproduction (Oxford, England)》2008,23(3):481-492
BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different. 相似文献
14.
Dreesen J.C.F.M.; Dumoulin J.C.M.; Evers J.L.H.; Geraedts J.P.M.; Pieters M.H.E.C. 《Human reproduction (Oxford, England)》1995,10(3):743-748
Using a multiplex nested polymerase chain reaction (PCR) methodwith single copy genes and a dinucleotide repeat locus for themouse Y and X chromosomes respectively, it was possible to discriminatebetween single cells derived from male and female embryos. Usingsingle cells, amplification of Sry and Zfy sequences was notevident in all cases. It could be calculated that, with thePCR method used, 0.04% [95% (confidence interval 0.002.03)]of the male embryos would erroneously be diagnosed as femaleif analysis is performed on two cells. The calculated chancefor total amplification failure, if two cells are used for analysis,would be 1.4% [95% (confidence interval 0.048.04)]. Themouse embryo model proved to be a helpful tool to develop skillsin the application of PCR for preimplantation genetic diagnosisat the single cell level. 相似文献
15.
多重置换扩增是一种新兴的全基因组扩增技术,能对单个细胞进行全基因扩增,产生大量的优质DNA,具有高扩增效率和高保真性等特点.多重置换扩增联合常规PCR已被成功用于植入前遗传学诊断,进一步扩展了后者的应用范围. 相似文献
16.
Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in new uses of preimplantation genetic diagnosis 总被引:6,自引:0,他引:6
Robertson JA 《Human reproduction (Oxford, England)》2003,18(3):465-471
The use of preimplantation genetic diagnosis (PGD) to screen embryos for aneuploidy and genetic disease is growing. New uses of PGD have been reported in the past year for screening embryos for susceptibility to cancer, for late-onset diseases, for HLA-matching for existing children, and for gender. These extensions have raised questions about their ethical acceptability and the adequacy of regulatory structures to review new uses. This article describes current and likely future uses of PGD, and then analyses the ethical issues posed by new uses of PGD to screen embryos for susceptibility and late-onset conditions, for HLA-matching for tissue donation to an existing child, and for gender selection. It also addresses ethical issues that would arise in more speculative scenarios of selecting embryos for hearing ability or sexual orientation. The article concludes that except for sex selection of the first child, most current extensions of PGD are ethically acceptable, and provides a framework for evaluating future extensions for nonmedical purposes that are still speculative. 相似文献
17.
Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with mutational heterogeneity. The scarcity of DNA from single cells in preimplantation genetic diagnosis (PGD) for DMD limits comprehensive genetic testing. Multiple displacement amplification (MDA) is reported to generate large amounts of template and give the most complete coverage and unbiased amplification to date. Here, we developed mutation and haplotype analysis in conjunction with gender determination on MDA products of single cells providing a generic approach that widens availability of PGD for female carriers with varied mutations. MDA amplified with 98.5% success for single lymphocytes and 94.2% success for single blastomeres, which was evaluated on 60 lymphocytes and 40 blastomeres. A total of six commonly mutant exons, eight short tandem repeat markers within dystrophin gene and amelogenin were incorporated into subsequent singleplex PCR assays. The mean allele dropout rate was 9.0% for single lymphocytes and 25.5% for single blastomeres. None of the blank controls gave a positive signal. Genotyping of each pedigree for three families provided 2-3 fully informative alleles per dystrophin haplotype besides specific mutant exons and amelogenin. We suggest that this approach is reliable to identify non-carrier female embryos other than unaffected male embryos and reduce the risk of misdiagnosis. 相似文献
18.
Donoso P Verpoest W Papanikolaou EG Liebaers I Fatemi HM Sermon K Staessen C Van der Elst J Devroey P 《Human reproduction (Oxford, England)》2007,22(4):1021-1025
BACKGROUND: The Belgian legislation imposes single embryo transfer (SET) on women of <36 years in their first treatment cycle to avoid multiple pregnancies. The aim of this study is to assess the impact of this legislation on the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases in young women undergoing SET. METHODS: A retrospective analysis of PGD cycles for monogenic disorders and translocations in women <36 years on their first treatment cycle. Two groups of patients were defined according to the implementation of the Belgian legislation: (i) double embryo transfer (DET), January 2001-June 2003 (ii) SET, July 2003-June 2005. The primary and secondary outcome measures were delivery per embryo transfer and multiple pregnancy rates, respectively. A subgroup analysis for monogenic disorders and translocations was performed. RESULTS: 62 cycles were included in the DET group and 73 cycles in the SET group. The mean age, number of cumulus-oocyte complexes, number of fertilized oocytes, number of biopsied and cryopreserved embryos were comparable between both groups. There was no significant difference in the delivery rates between the DET and the SET groups (33.9% versus 27.4%, respectively). Multiple pregnancies were avoided when SET was performed. When monogenic disorders and chromosomal translocations were separately evaluated, no significant difference in the delivery rate after SET was observed. CONCLUSIONS: The implementation of a SET policy in young women undergoing PGD for monogenic disorders and translocations enables a significant reduction of multiple pregnancies without significantly affecting the delivery rate. 相似文献
19.
Borkenhagen A Brähler E Wisch S Stöbel-Richter Y Strauss B Kentenich H 《Human reproduction (Oxford, England)》2007,22(7):2051-2057
BACKGROUND: In Germany, preimplantation genetic diagnosis (PGD) is currently not legal, but there is still a controversial debate about legalization. Studies about the attitudes of infertile couples towards PGD are rare. METHODS: A survey was conducted with 265 German infertile couples about knowledge, attitudes and prospective use of PGD. The influence of independent variables associated with approval of PGD is analysed by binary logistic regression. RESULTS: Sixty percent of respondents have heard about PGD. Eighty-seven percent support a general legalization of PGD in Germany for severe, early-onset genetic diseases. Seventy-four percent consider PGD morally acceptable. Sixty percent supported legalizing PGD for HLA-matching. But only a minority approved PGD to test for non-health-related traits. Respondents with a higher education level were the least supportive to all uses of PGD. CONCLUSIONS: Our results suggest that German infertile couples are as liberal towards PGD for health-related uses as in other western countries. They would legalize and use PGD to raise the rates to get pregnant and to avoid severe diseases of the offspring. Taking the opinions of German infertile couples into consideration could help redefine and reframe the public debate towards legalization of PGD and the moral status of the embryo in Germany. 相似文献
20.
Turetsky T Aizenman E Gil Y Weinberg N Shufaro Y Revel A Laufer N Simon A Abeliovich D Reubinoff BE 《Human reproduction (Oxford, England)》2008,23(1):46-53
BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications. 相似文献