Twelve years of assessing the quality of preimplantation genetic testing for monogenic disorders.

OBJECTIVE: Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. METHOD: The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. RESULTS: The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. CONCLUSION: EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.

Preimplantation genetic testing in patients with genetic susceptibility to cancer.

Data on preimplantation genetic testing (PGT-M) in patients with genetic susceptibility to cancer are scarce in the literature, while there is, in our experience, a growing familiarity with assisted reproduction techniques (ART) among pathogenic variant heterozygotes. We performed a retrospective multicenter study of PGT-M outcomes among French patients with genetic susceptibility to cancer. Our objectives were to collect data on this complex issue, and to help cancer geneticists counsel their patients of reproductive age. We also wanted to increase awareness regarding PGT-M among cancer genetics professionals. Patients from three university hospital cancer genetics clinics who had requested PGT-M between 2000 and 2019 were included retrospectively. Data were extracted from medical records. Patients were then contacted directly to collect missing and up-to-date information. Out of 41 eligible patients, 28 agreed explicitly to participate when contacted and were therefore included. They carried PV in VHL (n = 9), APC (n = 8), CDH1 (n = 5), STK11 (n = 2), AXIN2, BRCA1, MEN1, and FH (n = 1). Seven patients were denied PGT-M based on multidisciplinary team meetings or subsequently by the ART hospital teams, two changed their minds, and two were yet to start the process. PGT-M was successful in seven patients (25%), with a mean age at PGT-M request of 27. Most had von Hippel-Lindau. PGT-M failed in the remaining ten, with a mean age at PGT-M request of 32. The main reason for failure was non-implantation of the embryo. Of these, four patients were pursuing PGT-M at the time of last contact. PGT-M outcomes in patients with cancer susceptibility syndromes were satisfactory. These patients should be informed about PGT-M more systematically, which would imply greater awareness among cancer genetics professionals regarding ART. Our series was not representative of cancer susceptibility syndromes in general; the predominance of cases with syndromes characterized by early-onset, highly penetrant disease is explained by the restrictive French guidelines.

ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders.

The field of preimplantation genetic testing (PGT) is evolving fast and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for monogenic/single-gene defects (PGT-M) and covers recommendations on basic methods for PGT-M and testing strategies. Furthermore, some specific recommendations are formulated for special cases, including de novo pathogenic variants, consanguineous couples, HLA typing, exclusion testing and disorders caused by pathogenic variants in the mitochondrial DNA. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for chromosomal structural rearrangements/aneuploidies. Together, these papers should assist scientists interested in PGT in developing the best laboratory and clinical practice possible.

ESHRE PGT Consortium good practice recommendations for the organisation of PGT.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for preimplantation genetic diagnosis, published in 2005 and 2011, are considered outdated and the development of new papers outlining recommendations for good practice in PGT was necessary. The current updated version of the recommendations for good practice is, similar to the 2011 version, split into four documents, one of which covers the organisation of a PGT centre. The other documents focus on the different technical aspects of embryo biopsy, PGT for monogenic/single-gene defects (PGT-M) and PGT for chromosomal structural rearrangements/aneuploidies (PGT-SR/PGT-A). The current document outlines the steps prior to starting a PGT cycle, with details on patient inclusion and exclusion, and counselling and information provision. Also, recommendations are provided on the follow-up of PGT pregnancies and babies. Finally, some further recommendations are made on the practical organisation of an IVF/PGT centre, including basic requirements, transport PGT and quality management. This document, together with the documents on embryo biopsy, PGT-M and PGT-SR/PGT-A, should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.

Single Circulating Fetal Trophoblastic Cells Eligible for Non Invasive Prenatal Diagnosis: the Exception Rather than the Rule.

Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington's disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation.

Improving preimplantation genetic diagnosis for Fragile X syndrome: two new powerful single-round multiplex indirect and direct tests.

Fragile X syndrome (FraX) is caused by the expansion of an unstable CGG repeat located in the Fragile X mental retardation 1 gene (FMR1) gene. Preimplantation genetic diagnosis (PGD) can be proposed to couples at risk of transmitting the disease, that is, when the female carries a premutation or a full mutation. We describe two new single-cell, single-round multiplex PCR for indirect and direct diagnosis of FraX on biopsied embryos. These tests include five unpublished, highly heterozygous simple sequence repeats, and the co-amplification of non-expanded CGG repeats for the direct test. Heterozygosity of the new markers ranged from 69 to 81%. The mean rate of non-informative marker included in the tests was low (26% and 23% for the new indirect and direct tests, respectively). This strategy allows offering a PGD for FraX to 96% of couples requesting it in our centre. A conclusive genotype was obtained in all cells with a rate of cells presenting an allele dropout ranging from 17% for the indirect test to 26% for the direct test. The new indirect test was applied for eight PGD cycles: 32 embryos were analysed, 9 were transferred and 3 healthy babies were born. By multiplexing these highly informative markers, robustness of the diagnosis is improved and the loss of potentially healthy embryos (because they are non-diagnosed or misdiagnosed) is limited. This may increase the chances of success of couples requesting a PGD for FraX, in particular, when premature ovarian insufficiency in premutated women leads to a reduced number of embryos available for analysis.

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study.

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.

The experience of 3 years of external quality assessment of preimplantation genetic diagnosis for cystic fibrosis.

Preimplantation genetic diagnosis (PGD) was first performed over 20 years ago and has become an accepted part of genetic testing and assisted reproduction worldwide. The techniques and protocols necessary to carry out genetic testing at the single-cell level can be difficult to master and have been developed independently by the laboratories worldwide offering preimplantation testing. These factors indicated the need for an external quality assessment (EQA) scheme for monogenic disease PGD. Toward this end, the European Society for Human Reproduction and Embryology came together with United Kingdom National External Quality Assessment Services for Molecular Genetics, to create a pilot EQA scheme followed by practical EQA schemes for all interested parties. Here, we detail the development of the pilot scheme as well as development and findings from the practical (clinical) schemes that have followed. Results were generally acceptable and there was marked improvement in results and laboratory scores for those labs that participated in multiple schemes. Data from the first three schemes indicate that the EQA scheme is working as planned and has helped laboratories improve their techniques and result reporting. The EQA scheme for monogenic PGD will continue to be developed to offer assessment for other monogenic disorders.

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.

High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis.

Pre-implantation genetic diagnosis allows the characterisation of embryos that carry a gene responsible for a severe monogenic disease and to transfer to the mother's uterus only the unaffected one(s). The genetically affected embryos can be used to establish human embryonic stem cell (hESC) lines. We are currently establishing a cell bank of ESC lines carrying specific disease-causing mutant genes. These cell lines are available to the scientific community. For this purpose, we have designed a technique that requires only minimal manipulation of the embryos. At the blastocyst stage, we just removed the zona pellucida before seeding the embryo as a whole on a layer of feeder cells. This approach gave a good success rate (>20%), whatever the quality of the embryos, and allowed us to derive 11 new hESC lines, representing seven different pathologies. Full phenotypic validation of the cell lines according to ISCI guidelines confirmed their pluripotent nature, as they were positive for hESC markers and able to differentiate in vitro in all three germ layers derivatives. Nine out of 11 stem cell lines had normal karyotypes. Our results indicate that inner cell mass isolation is not mandatory for hESC derivation and that minimal manipulation of embryos can lead to high success rate.

What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee.

Since 2004, there have been 11 randomized controlled trials (RCTs) mainly for advanced maternal age (AMA), which have shown no benefit of performing preimplantation genetic screening (PGS). Ten of the RCTs have been performed at the cleavage stage and one at the blastocyst stage. It is probable that the high levels of chromosomal mosaicism at cleavage stages, which may result in the tested cell not being representative of the embryo, and the inability to examine all of the chromosomes using fluorescence in situ hybridization, have contributed to the lack of positive outcome from the RCTs. We suggest that future RCTs should examine alternative biopsy timing (polar body and/or trophectoderm biopsy), and should apply technologies that allow more comprehensive testing to include all chromosomes (microarray-based testing) to determine if PGS shows an improvement in delivery rate. Currently there is no evidence that routine PGS is beneficial for patients with AMA and conclusive data (RCTs) on repeated miscarriage, implantation failure and severe male factor are missing. To evaluate benefits of PGS, an ESHRE trial has recently been started on patients with AMA using polar body biopsy and array-comparative genomic hybridization, which should bring more information on this patient group in the near future.

[The acrosome: comparative morphology and development, contribution of a human familial globozoospermia case report]. / L'acrosome: Développement et comparaison entre espèces, apport d'un cas familial de globozoospermie.

Sperm acrosome is known to play a role in the fertilization of the majority of animal species studied. As a general rule, the acrosome appeared as soon as the fertilization occurred out of aquaeous phase. The biochemical content of acrosome as well as its release mode could suggest it is a simple lysosome. But this would by pass its important morphogenic role in spermiogenesis. Its development is strongly linked to the development of the microtubules manchette system. Molecular data of animal mutagenesis contribute to the understanding of acrosome biogenesis mechanisms. Globozoospermia is a rare but severe human teratozoospermia, characterized by ejaculates entirely consisting of round-headed spermatozoa that lack an acrosome. It originates from a disturbed acrosome biogenesis. Recently, the genetic study of a familial globozoospermia led to highlight a homozygote mutation of the gene SPATA16, linked to the globozoospermic phenotype. This study contributes to the understanding of the mechanisms implied in human acrosome formation.

Birth after pre-implantation genetic diagnosis (PGD) of spinocerebellar ataxia 2 (Sca2).

BACKGROUND: Spinocerebellar ataxia 2 (SCA2) is an autosomal-dominant neurodegenerative disease caused by an extended polyglutamine sequence in the ATXN2 protein. We describe the development of a new single-cell multiplex PCR protocol for pre-implantation genetic diagnosis (PGD) of SCA2 and its successful clinical application. METHODS: Three duplex tests have been developed, one, which combines the detection of the CAG repeats in addition to the D12S821 microsatellite, another, the amplification of the CAG repeats and the D12S1333 microsatellite and the last, the combination of both microsatellites D12S821 and D12S1333. RESULTS: PCR conditions were established using 226 single lymphoblasts or patient lymphocysts. Amplification was obtained in an average of 99.6%, a complete genotype in 86%, a conclusive result in 96% and an allelic drop-out (ADO) rate of 10.7% was observed. PGD for SCA2 was performed for a couple with a paternal risk of transmitting the pathology. Two cycles were done from which 18 embryos were biopsied, 8 were diagnosed as unaffected, 9 as affected and 1 gave no results. In both cycles 2 embryos were transferred, with no pregnancy at the first attempt, and a twin pregnancy at the second attempt. The patient delivered one girl and one boy at 36 weeks and 3 days.

What next for preimplantation genetic screening?

Preimplantation genetic diagnosis for aneuploidy screening (preimplantation genetic screening-PGS) has been used to detect chromosomally normal embryos from subfertile patients. The main indications are advanced maternal age (AMA), repeated implantation failure, repeated miscarriages and severe male factor infertility. Many non-randomized PGS studies have been published and report an increase in implantation rate, and/or a decrease in miscarriage rate. Recently, two randomized controlled trials have been conducted on patients with AMA as the only indication. Neither study showed a benefit in performing PGS using live birth rate as the measure of success. The debate on the usefulness of PGS is ongoing; the only effective way to resolve the debate is to perform more well-designed and well-executed randomized clinical trials.

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia.

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene.

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90-98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene.

New tools for preimplantation genetic diagnosis of Huntington's disease and their clinical applications.

Huntington's disease (HD) is a late-onset neurodegenerative disorder transmitted as an autosomal dominant trait. The causative mutation was characterised in 1993. For HD carriers willing to create a family, prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) based on the mutation identification can be offered. For at-risk persons who do not want to undergo presymptomatic testing (PT), an exclusion test can be proposed. With such a test, only foetuses or embryos that inherit an allele from the unaffected grandparent are considered as unaffected. In cases of PND, if the foetus has one allele of the affected grandparent, termination of pregnancy is proposed. In cases of PGD, only not at-risk embryos are transferred. Since the beginning of our PGD activity, we have had 43 PGD referrals for HD, of which 24 were from patients who know their genetic status and 19 from patients who do not wish to perform PT. We have developed 12 multiplex fluorescent PCR protocols applied at the single-cell level for PGD, some of which target the CAG repeat while others use two different polymorphic microsatellites. We present here these different protocols and their clinical applications, as well as the characterisation and use of a new highly polymorphic intragenic marker. Between May 2001 and December 2003, 39 PGD cycles have been performed for 17 couples, 11 of whom had a known genetic status and six who did not wish to perform PT, resulting in four pregnancies.

Duplex, triplex and quadruplex PCR for the preimplantation genetic diagnosis (PGD) of cystic fibrosis (CF), an exhaustive approach.

Most of cystic fibrosis (CF) pre-implantation genetic diagnosis (PGD) cases described to date are limited to the detection of DeltaF508. Beside this predominant mutation, over 1000 mutations have been identified, rendering the development of a mutation-based PGD protocol impracticable. This is the reason why we, as well as the others, have developed PGD strategies on the basis of the identification of the pathogenic haplotype instead of the mutation(s). In a previous article, we reported the conditions for the co-amplification of two intragenic polymorphic markers and the F508 locus. Here we describe an improved protocol allowing the additional amplification of two new intragenic markers, intron 1 CA repeat (I1CA) and IVS17bTA. This new protocol should, theoretically, allow us to provide a diagnosis to all couples requiring PGD for CF. Using single lymphoblasts, we have tested four different PCR configurations, including one duplex, two triplexes and one quadruplex PCR. All of them gave results compatible with a clinical application. The number of single lymphoblasts tested in each series varied from 89 to 155. PCR efficiency ranged from 95.4 to 100%. A complete haplotype was achieved for 83.2 to 90.7% of the tested cells, with an allele drop out (ADO) rate comprised between 6.0 and 11.6%. We present here three cases that we performed either with the former test (one case using the triplex PCR combining F508, IVS8CA and IVS17bCA) or with the new one (one case using the triplex combining F508, I1CA and IVS17bTA and one case using a quadruplex test). We obtained two single pregnancies.

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy.

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued.

Preimplantation genetic diagnosis for achondroplasia: genetics and gynaecological limits and difficulties.

BACKGROUND: We report the first attempts at preimplantation genetic diagnosis (PGD) and IVF and their accompanying difficulties for achondroplasia (ACH) patients. METHODS: A PGD test was developed using fluorescent single cell PCR on lymphoblasts from patients and controls and from blastomeres from surplus IVF embryos. A specific digestion control based on the use of two fluorochromes was elaborated. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols. RESULTS: We performed 88 single cell tests for which amplification was obtained in 86 (97.7%) single lymphoblasts. Allele drop out (ADO) was observed in two out of 53 (3.7%) heterozygous lymphoblasts. If we combine the results from the blastomere testing from surplus embryos with those from PGD cycles and re-analysis after PGD, we obtained a PCR signal in 84% of cases of which 91% were correctly diagnosed at the G380 locus. A total of six cycles were performed resulting in three embryo transfers. We observed difficulties in ovarian stimulation and oocyte retrieval with affected female patients. No pregnancy was obtained. CONCLUSION: A PGD test for ACH is now available at our centre but our initial practice raises questions on the feasibility of such a test, specially with affected female patients.