PID in der Reproduktionsmedizin

Preimplantation genetic diagnosis in assisted reproduction Preimplantation genetic diagnosis (PGD) describes the biopsy of one or more cells of an in vitro fertilized embryo and its genetical analysis before the transfer of the embryo into the uterus. Since the modification of the Embryonenschutzgesetz a PGD can be lawfully performed in Germany. Several diagnostic methods can be performed: FISH for the detection of maternal translocations on polar bodies, at the moment the most applied the array‐CGH for the screening of aneuploidies and the detection of translocations and also in the case of monogenic diseases PCR followed by sequencing. In the near future next generation sequencing will possibly replace all mentioned methods.     

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.     

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.     

Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization

Meiotic errors during oocyte maturation are considered the major contributors to embryonic aneuploidy and failures in human IVF treatment. Various technologies have been developed to screen polar bodies, blastomeres and trophectoderm cells for chromosomal aberrations. Array-CGH analysis using bacterial artificial chromosome (BAC) arrays is widely applied for preimplantation genetic diagnosis (PGD) using single cells. Recently, an increase in the pregnancy rate has been demonstrated using array-CGH to evaluate trophectoderm cells. However, in some countries, the analysis of embryonic cells is restricted by law. Therefore, we used BAC array-CGH to assess the impact of polar body analysis on the live birth rate. A disadvantage of polar body aneuploidy screening is the necessity of the analysis of both the first and second polar bodies, resulting in increases in costs for the patient and complex data interpretation. Aneuploidy screening results may sometimes be ambiguous if the first and second polar bodies show reciprocal chromosomal aberrations. To overcome this disadvantage, we tested a strategy involving the pooling of DNA from both polar bodies before DNA amplification. We retrospectively studied 351 patients, of whom 111 underwent polar body array-CGH before embryo transfer. In the group receiving pooled polar body array-CGH (aCGH) analysis, 110 embryos were transferred, and 29 babies were born, corresponding to live birth rates of 26.4% per embryo and 35.7% per patient. In contrast, in the control group, the IVF treatment was performed without preimplantation genetic screening (PGS). For this group, 403 embryos were transferred, and 60 babies were born, resulting in live birth rates of 14.9% per embryo and 22.7% per patient. In conclusion, our data show that in the aCGH group, the use of aneuploidy screening resulted in a significantly higher live birth rate compared with the control group, supporting the benefit of PGS for IVF couples in addition to the suitability and effectiveness of our polar body pooling strategy.     

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

胰岛素样生长因子1(IGF-1)mRNA同源模板竞争性PCR定量方法的建立

胰岛素样生长因子 1(IGF 1)是一种多功能的细胞增殖调控因子 ,其表达水平受多种因素的影响 ,为了研究IGF 1基因在转录水平上的调控机制 ,建立了定量测定IGF 1mRNA的竞争性PCR方法 .同时 ,也建立了一种简便的制备同源性竞争模板的方法 .以构建好的重组pUC IGF 1质粒为基础 ,利用IGF 1mRNA序列上唯一存在 ,但是在pUC18质粒上多拷贝的MspⅠ酶切位点 ,以该限制性内切酶处理重组pUC IGF 1质粒 .在T4DNA连接酶作用下对酶切产物进行随机连接 ,以连接产物作为模板 ,用可扩增IGF 1cDNA的引物进行PCR ,由此得到因含有随机插入序列而与原IGF 1cDNA产生明显长度差别的重组IGF 1.以不同浓度的该DNA片段作为同源竞争模板与大鼠肝组织cDNA在同一反应体系中进行PCR ,对PCR产物进行分析 ,计算出样本中IGF 1cDNA的初始浓度 .成功地建立了IGF 1mRNA的竞争性PCR定量检测方法 ,为研究IGF 1基因的表达调控奠定了基础 ,同时也为对已克隆的基因进行mRNA定量测定提供了一种简便和灵敏的手段     

Preimplantation genetic diagnosis: State of the ART 2011

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.     

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.     

Validating a rapid,real-time,PCR-based direct mutation detection assay for preimplantation genetic diagnosis

Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic l-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.     

Cytogenetics in reproductive medicine: the contribution of comparative genomic hybridization (CGH)

Cytogenetic research has had a major impact on the field of reproductive medicine, providing an insight into the frequency of chromosomal abnormalities that occur during gametogenesis, embryonic development and pregnancy. In humans, aneuploidy has been found to be relatively common during fetal life, necessitating prenatal screening of high-risk pregnancies. Aneuploidy rates are higher still during the preimplantation stage of development. An increasing number of IVF laboratories have attempted to improve pregnancy rates by using preimplantation genetic diagnosis (PGD) to ensure that the embryos transferred to the mother are chromosomally normal. This paper reviews some of the techniques that are key to the detection of aneuploidy in reproductive samples including comparative genomic hybridization (CGH). CGH has provided an unparalleled insight into the nature of chromosome imbalance in human embryos and polar bodies. The clinical application of CGH for the purposes of PGD and the future extensions of the methodology, including DNA microarrays, are discussed.     

Cutting edge: prostaglandin D2 enhances leukotriene C4 synthesis by eosinophils during allergic inflammation: synergistic in vivo role of endogenous eotaxin

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies-potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.     

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly.     

Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes

Individuals carrying translocations suffer from reduced fertility or spontaneous abortions and seek help in form of assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD). While most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are thus unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Thus several methods have been devised to detect translocation imbalance through FISH in single cells for purpose of PGD, among them polar body chromosome painting, interphase FISH with combination of subtelomeric and centromeric probes, breakpoint spanning probes, and cell conversion. Results with PGD indicate a significant decrease in spontaneous abortions, from 81% before PGD to 13% after PGD. They also indicate very high rates of chromosome abnormalities in embryos from translocation carriers, 72% for Robertsonian translocations and 82% for reciprocal translocations. Sperm analysis was found to be a good predictor of IVF and PGD outcome, with samples with more than 60% abnormal forms indicating poor prognosis. Similarly, the predictability from first PGD cycle results for future cycles was 90%. In summary, PGD can help translocation carriers to achieve viable pregnancies, but the success of the process is conversely related to the baseline of unbalanced gametes.     

Current status of preimplantation genetic diagnosis in Japan

This is a retrospective study aimingto clarify the current status of preimplantation genetic diagnosis (PGD) in Japan. Our data were collected from 12 facilities between September 2004 and September 2012, and entered into a database. A majority of PGD in Japan was performed for balanced structural chromosomal abnormalities in couples with recurrent miscarriage. PGD for monogenic diseases was performed only in two facilities. The average maternal age was 38 years for monogenic diseases and 40 years for chromosomal abnormalities. Overall there have been671 cycles to oocyte retrieval reported. Of these cycles, 85% (572 cycles)were for chromosomal abnormalities, and 15% (99 cycles) for monogenic diseases. Diagnosis rates in the current study were 70.8% for monogenic diseases and 94.0% for chromosomal abnormalities. Rates of embryo transfer of PGD were 62.7% for monogenic diseases and 25.5% for chromosomal abnormalities. Clinical pregnancy rates per embryo transfer were 12.0% for monogenic diseases and 35.6% for chromosomal abnormalities. Our study is the first PGD report from all facilities which had the approval of the ethics committee of the Japanese Society of Obstetrics and Gynecology. We have built a basis for gathering continuous PGD data in Japan.     

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy

Conventional PCR methods combined with linkage analysis based on short tandem repeats(STRs) or Karyomapping with single nucleotide polymorphism(SNP) arrays, have been applied to preimplantation genetic diagnosis(PGD) for spinal muscular atrophy(SMA), an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wildtype embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses(MARSALA) is a new method allowing embryo selection by a one-step next-generation sequencing(NGS) procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion(carriers) from the wild-type(normal) ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos(homozygous deletion), which can be regarded as probands for linkage analysis, in case that the affected family member is absent. In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.     

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.     

ERIC-PCR-Generated Genomic Fingerprints and Their Relationship with Pathogenic Variability of Xanthomonas campestris pv. punicae, the Incitant of Bacterial Blight of Pomegranate

Bacterial blight caused by Xanthomonas campestris pv. punicae (Xcp) has emerged as a potential threat in pomegranate (Punica granatum) cultivation in India. Here, we report the genomic fingerprints and their correlation with virulence pattern of Xcp isolates from Maharashtra and Delhi. The genomic fingerprints of Xcp isolates were generated using enterobacterial repetitive intergenic consensus (ERIC) sequence-based primers, and virulence level was based on their reaction upon infiltration to susceptible pomegranate cultivar. Maharashtra isolate PGM1 showed only 50% similarity with Delhi isolate PGD8 forming a distinct genotype, whereas the Delhi isolates PGD5 and PGD6 form a cluster with Maharashtra isolates PGM2 and PGM4. The isolates PGM2, PGM4, PGD5, and PGD6 showing mean disease score of 7.47 were marked as group A or highly virulent. The moderately virulent or group B isolates PGM3 and PGD7 produced mean disease score of 4.19, whereas less virulent or group C isolates PGD8 and PGM1 gave mean disease intensity of 1.91. A correlation between genotypic groups based on ERIC fingerprints and pathogenicity of the isolates was established. The highly virulent isolates PGM2, PGM4, PGD5, and PGD6 formed a single cluster. A unique 900 bp amplicon present in all highly virulent isolates has been identified that can be used as genetic marker to screen isolates for virulence. The less virulent isolates PGD8 and PGM1 formed single cluster at 50% similarity coefficient. This seems to be the first report to establish a correlation between ERIC-PCR fingerprints and their corresponding virulence pattern of the pomegranate bacterial blight pathogen.     

Risk,parental autonomy and the epistemic divide: preimplantation genetic diagnosis in the Australian print news media, 1990–2007

Preimplantation genetic diagnosis (PGD) is a reproductive/genetic technology which has become the subject of public and scholarly debate because it involves the evaluation and consequent selection (and implantation) or destruction of human embryos. This research investigates the way PGD is constituted in the Australian print news media. Foucauldian discourse analysis reveals that proponents draw on their direct knowledge and experience of PGD to support their claims. There is an epistemic divide between consumers and others claiming direct knowledge, and critics and others drawing on indirect or abstract understandings of PGD. This divide characterizes the discourses present in the data and is directly linked to changes in these over the period under analysis.