Quality control standards in PGD and PGS.

Preimplantation genetic diagnosis (PGD) aims to test the embryo for specific conditions before implantation in couples at risk of transmitting genetic abnormality to their offspring. The couple must undergo IVF procedures to generate embryos in vitro. The embryos can be biopsied at either the zygote, cleavage or blastocyst stage. Preimplantation genetic screening uses the same technology to screen for chromosome abnormalities in embryos from patients undergoing IVF procedures as a method of embryo selection. Fluorescence in-situ hybridization was originally used for chromosome analysis, but has now been replaced by array comparative genomic hybridization or next generation sequencing. For the diagnosis of single gene defects, polymerase chain reaction is used and has become highly developed; however, single nucleotide polymorphism arrays for karyomapping have recently been introduced. A partnership between IVF laboratories and diagnostic centres is required to carry out PGD and preimplantation genetic screening. Accreditation of PGD diagnostic laboratories is important. Accreditation gives IVF centres an assurance that the diagnostic tests conform to specified standards. ISO 15189 is an international laboratory standard specific for medical laboratories. A requirement for accreditation is to participate in external quality assessment schemes.

Investigation of microRNA expression and DNA repair gene transcripts in human oocytes and blastocysts.

PURPOSE: The aim of the study is to investigate the regulation of DNA repair genes by microRNAs (miRNAs). miRNAs are short non-coding RNAs that regulate transcriptional and post-transcriptional gene silencing. Several miRNAs that are expressed during preimplantation embryo development have been shown or are predicted to target genes that regulate cell cycle checkpoints and DNA repair in response to DNA damage. METHODS: This study compares the expression level of 20 miRNAs and 9 target transcripts involved in DNA repair. The statistical significance of differential miRNA expression between oocytes and blastocysts was determined by t test analysis using the GraphPad Prism v6 software. The possible regulatory roles of miRNAs on their target messenger RNAs (mRNAs) were analysed using a Pearson correlation test. RESULTS: This study shows for the first time that several miRNAs are expressed in human oocytes and blastocysts that target key genes involved in DNA repair and cell cycle checkpoints. Blastocysts exhibited statistically significant lower expression levels for the majority of miRNAs compared to oocytes (p < 0.05). Correlation analyses showed that there was both inverse and direct association between miRNAs and their target mRNAs. CONCLUSIONS: miRNAs target many mRNAs including ones involved in DNA repair mechanisms. This study suggests that miRNAs and their target mRNAs involved in DNA repair are expressed in preimplantation embryos. Similar to the miRNAs expressed in adult tissues, these miRNAs seem to have regulatory roles on their target DNA repair mRNAs during preimplantation embryo development.

ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011.

STUDY QUESTION: How do data in the 13th annual data collection (Data XIII) of the European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium compare with the cumulative data for collections I-XII? SUMMARY ANSWER: The 13th retrospective collection represents valuable data on PGD/PGS cycles, pregnancies and children: the main trend observed is the decrease in the routine implementation of PGS. WHAT IS KNOWN ALREADY: Since 1999, the PGD Consortium has collected, analysed and published 12 data sets and an overview of the first 10 years of data collections. STUDY DESIGN, SIZE, DURATION: Data were collected from each participating centre using a FileMaker Pro database (versions 5-11). Separate predesigned FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar year 2010 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2011). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were submitted by 62 centres (full PGD Consortium members). The submitted data were thoroughly analysed to identify incomplete data entries and corrections were requested from the participating centres. Records remaining with incomplete or inconsistent data were excluded from the calculations. Corrections, calculations and tables were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XIII, 62 centres reported data for 5780 cycles with oocyte retrieval (OR), along with details of the follow-up on 1503 pregnancies and 1152 babies born. A total of 1071 OR were reported for chromosomal abnormalities, 108 OR for sexing for X-linked diseases, 1574 OR for monogenic diseases, 2979 OR for preimplantation genetic screening and 48 OR for social sexing. LIMITATIONS, REASONS FOR CAUTION: The findings apply to the 62 participating centres and may not represent worldwide trends in PGD. WIDER IMPLICATIONS OF THE FINDINGS: The annual data collections provide an important resource for data mining and for following trends in PGD practice. STUDY FUNDING/COMPETING INTERESTS: None.

ESHRE PGD Consortium data collection XII: cycles from January to December 2009 with pregnancy follow-up to October 2010.

STUDY QUESTION: How do data in the 12th annual data collection (Data XII) of the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis (PGD) Consortium compare with the cumulative data for collections I-XI? SUMMARY ANSWER: Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. WHAT IS KNOWN ALREADY: The PGD Consortium has collected, analysed and published 11 previous data sets since 1997. STUDY DESIGN, SIZE, DURATION: Data were collected from each participating centre using a pre-designed FileMaker Pro database (versions 5-10). Separate FileMaker Pro files were used for the cycles, pregnancies and baby records. The study documented cycles performed during the calendar year 2009 and follow-up of the pregnancies and babies born which resulted from these cycles (until October 2010). PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were submitted by 60 centres (full PGD Consortium members), and the blank files were distributed to each PGD Consortium member centre at the end of 2008. The submitted data were thoroughly analysed to identify incomplete data entries and corrections were requested from the participating centres. Records remaining with incomplete data were excluded from the calculations. Corrections, tables and calculations were made by expert co-authors. MAIN RESULTS AND THE ROLE OF CHANCE: For data collection XII, 60 centres reported data for 6160 cycles with oocyte retrieval (OR), along with details of the follow-up on 1607 pregnancies and 1238 babies born. A total of 870 OR were reported for chromosomal abnormalities, 113 OR for sexing for X-linked diseases, 1597 OR for monogenic diseases, 3551 OR for preimplantation genetic screening and 29 OR for social sexing. LIMITATIONS, REASONS FOR CAUTION: These data cannot include every PGD cycle performed annually, and only indicate the trends in PGD worldwide. WIDER IMPLICATION OF THE FINDINGS: The annual data collections provide an extremely valuable resource for data mining and for following trends in PGD practice. STUDY FUNDING/COMPETING INTEREST(S): None.

The ESHRE PGD Consortium: 10 years of data collection.

BACKGROUND: Since it was established in 1997, the ESHRE PGD Consortium has been collecting data from international preimplantation genetic diagnosis (PGD) centres. Ten papers have been published, including data from January 1997 to December 2007. METHODS: The data collection originally used a hard-copy format, then an excel database and finally a FileMaker Pro database. The indications are divided into five categories: PGD for chromosome abnormalities, sexing for X-linked disease, PGD for single gene defects, preimplantation genetic screening (PGS) and PGD for social sexing. The main end-points are pregnancy outcome and follow-up of deliveries. RESULTS: In data collection I, 16 centres contributed data, which increased to 57 centres by data X (average of 39 centres per data collection). These centres contributed data on over 27 000 cycles that reached oocyte retrieval. Of these cycles, 61% were for aneuploidy screening, 17% for single gene disorders, 16% for chromosomal abnormalities, 4% for sexing of X-linked disease and 2% for social sexing. Cumulatively, 5187 clinical pregnancies gave rise to 4140 deliveries and 5135 newborns (singletons: 3182, twins: 921, triplets: 37). CONCLUSIONS: In this paper, we present an overview of the first 10 years of PGD data, highlighting trends. These include the introduction of laser-assisted biopsy, an increase in polar body and trophectoderm biopsy, new strategies, methodologies and technologies for diagnosis, including recently arrays, and the more frequent use of freezing biopsied embryos. The Consortium data reports represent a valuable resource for information about the practice of PGD.

The nature and origin of binucleate cells in human preimplantation embryos: relevance to placental mesenchymal dysplasia.

Cleavage-stage embryos often have nuclear abnormalities, one of the most common being binucleate blastomeres, which may contain two diploid or two haploid nuclei. Biopsied cells from preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) cycles were studied to determine the relative frequency of binucleate cells with two haploid versus two diploid nuclei. The frequency of mononucleate haploid biopsied blastomeres was also recorded. In the chromosomal PGD cycles 45.2% of the biopsied binucleate cells were overall diploid and 38.7% were overall tetraploid, compared with 50.0% and 29.2% for the PGS group, respectively. Placental mesenchymal dysplasia is a rare condition associated with intrauterine growth restriction, prematurity and intrauterine death. Recent work suggests that androgenetic diploid/haploid mosaicism may be a causal mechanism. There are two possible origins of haploid nuclei, either the cell contained only one parental genome initially or they may be derived from the cytokinesis of binucleate cells with two haploid nuclei. Binucleate formation therefore may be a way of doubling up the haploid genome, to produce diploid cells of androgenetic origin as seen in placental mesenchymal dysplasia.

Pathogenesis and clinical management of hereditary non-polyposis colorectal cancer.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) is an inherited genetic condition associated with microsatellite instability; it accounts for around 5 per cent of all cases of colorectal cancer. This review examines recent data on management strategies for this condition. METHODS: A Medline-based literature search was performed using the keywords 'HNPCC' and 'microsatellite instability'. Additional original papers were obtained from citations in articles identified by the initial search. RESULTS AND CONCLUSION: The Amsterdam criteria identify patients in whom the presence of an inherited mutation should be investigated. Those with a mutation should be offered counselling and screening. The role of prophylactic surgery has been superseded by regular colonoscopy, which dramatically reduces the risk of colorectal cancer. Screening for extracolonic malignancy is also advocated, but the benefits are uncertain. Chemoprevention may be of value in lowering the incidence of bowel cancer in affected patients, but further studies are required.

Genetic instability in patients with metachronous colorectal cancers.

BACKGROUND: Nearly 7 per cent of patients who undergo resection for colorectal cancer develop metachronous cancers several years later. A molecular marker that could identify patients susceptible to metachronous cancers would be of clinical importance. METHODS: Twenty-four colorectal cancers from 15 individuals with metachronous colorectal cancer were investigated for microsatellite instability at five loci by single stranded conformational polymorphism analysis. A control group of 14 colorectal cancers from individuals who had only developed one sporadic colorectal cancer each was analysed similarly. RESULTS: Microsatellite instability was demonstrated in 17 of 24 cancers from individuals with metachronous cancer compared with one of 14 cancers from individuals with a single colorectal cancer. CONCLUSION: These results suggest that testing for microsatellite instability may be useful in recognizing patients at high risk of developing metachronous colorectal cancers.

Regionally clustered APC mutations are associated with a severe phenotype and occur at a high frequency in new mutation cases of adenomatous polyposis coli.

Germline mutation in APC at 5q21-22 results in the dominantly inherited syndrome adenomatous polyposis coli (APC). Somatic mutation in this gene is an early event in colorectal tumourigenesis. Both types of mutation are concentrated in the 5' half of exon 15. We have used single strand conformational polymorphism (SSCP) and heteroduplex analysis to screen for variants in this region of the gene in a total of 45 affected but unrelated individuals. Eighteen patients had no family history of the disease; of these 11 were classified as having a severe phenotype, based on an early age at presentation or cancer development. This compared with 6 of 27 familial cases. A 5 bp deletion at codon 1309 reported to occur in 10-15% of unselected APC patients worldwide, was found in 5 of the 18 new mutation cases and 4 of the 27 familial cases: all nine were classed as severe. A further 3 new mutations and 1 familial mutation were located downstream from codon 1309, these individuals similarly being classed as phenotypically severe. In contrast all of the APC mutations detected in affected individuals with an average phenotype were located prior to codon 1309. The frequent association of a severe phenotype with fresh mutation may explain the apparent conflict of a high mutation rate (20-30%) in a condition, which on average, is lethal at a post-reproductive age.