Blurring boundaries. Interviews with PGT couples about comprehensive chromosome screening.

OBJECTIVE: Comprehensive chromosome examination is a promising approach to Preimplantation Genetic Testing (PGT). Next to testing of specific chromosomes, such as in the case of reduced fertility due to chromosomal translocations, it allows testing of all chromosomes. Hence it potentially reduces the time to pregnancy and the risk of miscarriage. But comprehensive testing also introduces some ethical issues. For example, what is the role of the professional in the decision making regarding embryos with chromosomal abnormalities that are potentially viable? Which chromosomal abnormalities should be communicated to people undergoing fertility treatment? With this paper we wanted to explore the ethical issues related to comprehensive chromosome screening in Preimplantation Genetic Testing. DESIGN: In order to explore these issues, we interviewed seven couples undergoing PGT for chromosomal translocations at the VUB University Hospital, Belgium. We presented them with three fictional cases: the transfer of an embryo with trisomy 21, of an embryo with a sex chromosome aneuploidy and of an embryo with a chromosomal microdeletion. RESULTS: We found that opinions regarding the role of fertility professionals in deciding which embryos to transfer were mixed. Moreover, where to draw the line between healthy and unhealthy embryos was unclear. We also found that couples, although they thought that comprehensive chromosome testing had certain benefits, also considered the increased waiting time for transfer a heavy burden. CONCLUSIONS: In the light of comprehensive chromosome screening of embryos, persons undergoing fertility treatment may have views on the burdens and benefits of the techniques that are not analogous to the views of professionals.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy.

STUDY QUESTION: How has the interface between genetics and assisted reproduction technology (ART) evolved since 2005? SUMMARY ANSWER: The interface between ART and genetics has become more entwined as we increase our understanding about the genetics of infertility and we are able to perform more comprehensive genetic testing. WHAT IS KNOWN ALREADY: In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and ART and published an extended background paper, recommendations and two Editorials. STUDY DESIGN, SIZE, DURATION: An interdisciplinary workshop was held, involving representatives of both professional societies and experts from the European Union Eurogentest2 Coordination Action Project. PARTICIPANTS/MATERIALS, SETTING, METHODS: In March 2012, a group of experts from the European Society of Human Genetics, the European Society of Human Reproduction and Embryology and the EuroGentest2 Coordination Action Project met to discuss developments at the interface between clinical genetics and ART. MAIN RESULTS AND THE ROLE OF CHANCE: As more genetic causes of reproductive failure are now recognized and an increasing number of patients undergo testing of their genome prior to conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and PGD may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from RCTs to substantiate that the technique is both effective and efficient. Whole genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. LIMITATIONS, REASONS FOR CAUTION: The legal landscape regarding assisted reproduction is evolving, but still remains very heterogeneous and often contradictory. The lack of legal harmonization and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe, and beyond. WIDER IMPLICATIONS OF THE FINDINGS: This continually evolving field requires communication between the clinical genetics and IVF teams and patients to ensure that they are fully informed and can make well-considered choices. STUDY FUNDING/COMPETING INTERESTS: Funding was received from ESHRE, ESHG and EuroGentest2 European Union Coordination Action project (FP7 - HEALTH-F4-2010-26146) to support attendance at this meeting.

In vitro screening of embryos by whole-genome sequencing: now, in the future or never?

STUDY QUESTION: What are the analytical and clinical validity and the clinical utility of in vitro screening of embryos by whole-genome sequencing? SUMMARY ANSWER: At present there are still many limitations in terms of analytical and clinical validity and utility and many ethical questions remain. WHAT IS KNOWN ALREADY: Whole-genome sequencing of IVF/ICSI embryos is technically possible. Many loss-of-function mutations exist in the general population without serious effects on the phenotype of the individual. Moreover, annotations of genes and the reference genome are still not 100% correct. STUDY DESIGN, SIZE, DURATION: We used publicly available samples from the 1000 Genomes project and Complete Genomics, together with 42 samples from in-house research samples of parents from trios to investigate the presence of loss-of-function mutations in healthy individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the samples, we looked for mutations in genes that are associated with a selection of severe Mendelian disorders with a known molecular basis. We looked for mutations predicted to be damaging by PolyPhen and SIFT and for mutations annotated as disease causing in Human Genome Mutation Database (HGMD). MAIN RESULTS AND THE ROLE OF CHANCE: More than 40% of individuals who can be considered healthy have mutations that are predicted to be damaging in genes associated with severe Mendelian disorders or are annotated as disease causing. LIMITATIONS, REASONS FOR CAUTION: The analysis relies on current knowledge and databases are continuously updated to reflect our increasing knowledge about the genome. In the process of our analysis several updates were already made. WIDER IMPLICATIONS OF THE FINDINGS: At this moment it is not advisable to use whole-genome sequencing as a tool to set up health profiles to select embryos for transfer. We also raise some ethical questions that have to be addressed before this technology can be used for embryo selection. TRIAL REGISTRATION NUMBER: N/A.

Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy. European Society of Human Genetics and European Society of Human Reproduction and Embryology.

In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and assisted reproductive technology (ART), and published an extended background paper, recommendations and two Editorials. Seven years later, in March 2012, a follow-up interdisciplinary workshop was held, involving representatives of both professional societies, including experts from the European Union Eurogentest2 Coordination Action Project. The main goal of this meeting was to discuss developments at the interface between clinical genetics and ARTs. As more genetic causes of reproductive failure are now recognised and an increasing number of patients undergo testing of their genome before conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and preimplantation genetic diagnosis (PGD) may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from randomised clinical trials to substantiate that the technique is both effective and efficient. Whole-genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (International Standards Organisation - ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. The legal landscape regarding assisted reproduction is evolving but still remains very heterogeneous and often contradictory. The lack of legal harmonisation and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe and beyond. The aim of this paper is to complement previous publications and provide an update of selected topics that have evolved since 2005.

Human trophectoderm cells are not yet committed.

STUDY QUESTION: Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? SUMMARY ANSWER: Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker NANOG, thus they are not yet committed. WHAT IS KNOWN ALREADY: Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4 as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite. STUDY DESIGN, SIZE, DURATION: The potency of human blastocyst TE cells was investigated by determining their in vitro capacity to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human preimplantation embryos were used for research after obtaining informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the 'Belgian Federal Committee on medical and scientific research on embryos in vitro'. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted embryos were analyzed by immunocytochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Isolated and reaggregated TE cells from full human blastocysts are able to develop into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express NANOG. LIMITATIONS, REASONS FOR CAUTION: Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are a source of pluripotent cells is to obtain human embryonic stem cell 'like' line(s), which will allow full characterization of the cells. WIDER IMPLICATIONS OF THE FINDINGS: Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. STUDY FUNDING/COMPETING INTEREST(S): Scientific Research Foundation-Flanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.

Preimplantation genetic diagnosis in female and male carriers of reciprocal translocations: clinical outcome until delivery of 312 cycles.

Carriers of reciprocal translocations (rcp) are known to be at risk for reproductive difficulties. Preimplantation genetic diagnosis (PGD) is one of the options these carriers have to try in order to fulfil their desire to have a child. In the present study, we retrospectively looked at the results of 11 years (1997-2007) of PGD for rcp in our center to improve the reproductive counseling of these carriers. During this period 312 cycles were performed for 69 male and 73 female carriers. The mean female age was 32.8 years, the mean male age 35.8 years. Most carriers were diagnosed with a translocation because of fertility problems or recurrent miscarriages, and most of them opted for PGD to avoid these problems. In 150 of the 312 cycles, embryo transfer (ET) was feasible and 40 women had a successful singleton or twin pregnancy. This gives a live birth delivery rate of 12.8% per started cycle and of 26.7% per cycle with ET. Owing to the large number of abnormal embryos, PGD cycles for rcp often lead to cancellation of ET, explaining the low success rate when expressed per cycle with oocyte pick-up. Once ET was feasible, the live birth delivery rate was similar to that of PGD in general at our center. PGD is therefore an established option for specific reciprocal translocation carriers.

Preimplantation genetic diagnosis (PGD) for Huntington's disease: the experience of three European centres.

This study provides an overview of 13 years of experience of preimplantation genetic diagnosis (PGD) for Huntington's disease (HD) at three European PGD centres in Brussels, Maastricht and Strasbourg. Information on all 331 PGD intakes for HD, couples' reproductive history, PGD approach, treatment cycles and outcomes between 1995 and 2008 were collected prospectively. Of 331 couples for intake, 68% requested direct testing and 32% exclusion testing (with a preponderance of French couples). At the time of PGD intake, 39% of women had experienced one or more pregnancies. A history of pregnancy termination after prenatal diagnosis was observed more frequently in the direct testing group (25%) than in the exclusion group (10%; P=0.0027). PGD workup was based on two approaches: (1) direct testing of the CAG-triplet repeat and (2) linkage analysis using intragenic or flanking microsatellite markers of the HTT gene. In total, 257 couples had started workup and 174 couples (70% direct testing, 30% exclusion testing) completed at least one PGD cycle. In total, 389 cycles continued to oocyte retrieval (OR). The delivery rates per OR were 19.8%, and per embryo transfer 24.8%, resulting in 77 deliveries and the birth of 90 children. We conclude that PGD is a valuable and safe reproductive option for HD carriers and couples at risk of transmitting HD.

Pregnancy outcome in carriers of Robertsonian translocations.

Robertsonian translocation carriers are at increased risk for infertility, spontaneous abortions, or chromosomally unbalanced offspring. Reproductive counseling of these carriers is challenging. We performed a retrospective analysis of all prenatal diagnoses from Robertsonian translocation carriers during the time period January 1, 1992 through December 31, 2007. Data on the carriers and the results of their prenatal analyses were retrieved as well as data on their previous pregnancies. We identified 28 female and 20 male carriers of Robertsonian translocations and results on 79 prenatal samples were obtained. Among female carriers, 10.3% of chorionic villus sampling and 5.9% of amniocentesis results were unbalanced, whereas for male carriers, this was 3.6% and 0%, respectively. When considering all pregnancies involving carriers, 52.7% of those to female carriers and 61.8% of those to male carriers led to the birth of a healthy child. Male carriers in whom the translocation was ascertained because of infertility or recurrent miscarriages appear to be at higher risk, whereas carriers in whom ascertainment was because of a family history are at lower risk. We conclude that pregnancies of Robertsonian translocation carriers are at increased risk for chromosomal imbalance, and prenatal chromosomal testing should be discussed. More than half of the pregnancies led to the birth of a healthy child, but prediction of which couples will be successful in obtaining a pregnancy with or without assisted reproductive technologies and/or embryo selection remains difficult. The reason for ascertainment of the translocation should be taken into account when counseling these couples. The possibility of preimplantation genetic diagnosis should also be discussed with the couples.

No relationship between the type of pituitary suppression for IVF and chromosomal abnormality rates of blastomeres: an observational study.

OBJECTIVE: To investigate whether the incidence of chromosomally abnormal blastomeres is related to the type of pituitary suppression used in ovarian stimulation. DESIGN: Retrospective study. SETTING: Tertiary referral center. PATIENT(S): The study involved 694 consecutive cycles; 320 belonged to agonist group and 374 to antagonist group, of patients' ≤ 37 years of age who underwent preimplantation genetic screening between October 1, 1992 until December 31, 2006. All of them (and their partners) had normal karyotyping results. Only the data of patients who had at least one embryo biopsy were analyzed. INTERVENTION(S): Preimplantation genetic screening (PGS). MAIN OUTCOME MEASURE(S): The primary outcome measure was detection of abnormal blastomeres on the total number of embryos analyzed. RESULT(S): The total abnormal ratio was statistically similar between the embryos of the two study groups (49.9 ± 28.1 vs. 50.2 ± 26.6). Likewise, a multivariate (linear regression) analysis indicated that the total abnormality ratio was not influenced by the type of stimulation when simultaneously adjusting for age, rank of trials, indication for preimplantation genetic screening, total gonadotropin amount, number of cumulus-oocyte complexes, and number of two pronuclear oocytes embryos. No difference was observed in ongoing pregnancy rates between agonists and antagonists (26.6% vs. 23.3%, respectively). CONCLUSION(S): Based on our findings there is no difference in the proportion of abnormal blastomeres either when using gonadotropin-releasing hormone (GnRH) agonist, or antagonist protocol.