Molecular Characterization of a Rare Case of Monozygotic Dichorionic Diamniotic Twin Pregnancy after Single Blastocyst Transfer in Preimplantation Genetic Testing (PGT).

Preimplantation genetic testing (PGT) is widely used to select unaffected embryos, increasing the odds of having a healthy baby. During the last few decades, it was accepted that monozygotic dichorionic diamniotic twin pregnancies occurred from the embryo splitting before Day 3 postfertilization according to Corner's dogma. Hence, the occurrence of a dichorionic diamniotic twin pregnancy after a single blastocyst transfer was considered a dizygotic pregnancy resulting from blastocyst transfer and concurrent natural fertilization. In our study, we have provided for the first time molecular proof that a single blastocyst transfer can result in a monozygotic dichorionic diamniotic twin pregnancy, invalidating Corner's dogma. In this case, we recommend systematically assessing the genetic status of dichorionic twins after single blastocyst transfer using prenatal diagnosis to exclude the risk from a potential concurrent spontaneous pregnancy and to ensure that both fetuses are unaffected. To achieve this goal, we have developed here an innovative noninvasive prenatal diagnosis by exclusion of paternal variants with droplet digital PCR, maximizing the reliability of genetic diagnosis. Further multicentric prospective studies using genetic testing are now required to establish the rate of blastocyst splitting leading to dichorionic pregnancy in PGT and to identify the risk factors.

Are ovarian response and pregnancy rates similar in selected FMR1 premutated and mutated patients undergoing preimplantation genetic testing?

PURPOSE: To assess if the ovarian response of FMR1 premutated women undergoing preimplantation genetic testing (PGT) for Fragile X syndrome is lower compared with fully mutated patients, due to their frequent premature ovarian failure. METHODS: In a retrospective cohort study from January 2009 to March 2019, we compared PGT outcomes in 18 FMR1 premutated women and 12 fully mutated women and aimed to identify predictive factors of stimulation outcomes. RESULTS: Eighty-six IVF/PGT-M cycles for FMR1 PGT were analyzed. Premutation and full mutation patients were comparable in terms of age, body mass index (BMI), basal FSH, antral follicular count, and cycle length. However, premutation carriers had significantly lower AMH (1.9 versus 4.0 ng/mL, p = 0.0167). Premutated patients required higher doses of FSH (2740 versus 1944 IU, p = 0.0069) but had similar numbers of metaphase II oocytes (7.1 versus 6.6, p = 0.871) and embryos (5.6 versus 4.9, p = 0. 554). Pregnancy rates (37.1% versus 13.3%, p = 0.1076) were not statistically different in both groups. CONCLUSION: In spite of lower ovarian reserve and thanks to an increased total dose of FSH, FMR1 premutated selected patients seem to have similar ovarian response as fully mutated patients. Neither the number of CGG repeats in FMR1 gene nor FMR1 mutation status was good predictors of the number of retrieved oocytes.

Evolution of post-pulmonary vein isolation atrial fibrillation inducibility at redo ablation: Electrophysiological evidence of extra-pulmonary vein substrate progression.

BACKGROUND: The electrophysiological substrate underlying atrial fibrillation (AF) progression remains difficult to identify. OBJECTIVE: The goals of this study were to study the evolution of post-pulmonary vein isolation (PVI) AF inducibility (AFI) after AF ablation and to compare patients with organized atrial tachycardia recurrence (OATr) versus those with paroxysmal or persistent AF recurrence. METHODS: We studied 99 patients who underwent de novo AF ablation (p1) and redo ablation (p2) for AF recurrence (AFr) or OATr. Stepwise AF ablation was performed at p1 and p2: (1) PVI, (2) coronary sinus defragmentation, and (3) left atrial (LA) defragmentation. Burst pacing followed each step, with AFI defined as sustained AF >5 minutes, triggering the next step. Patients with OATr underwent OAT ablation and inducibility testing post-redo PVI. Inducibility progression (IP) was defined as AFI at further steps of p2 compared to p1. RESULTS: Among patients with AFr, 34 of 72 patients (47%) exhibited post-PVI IP vs 2 of 27 (7.4%) patients with OATr (P = .0002). Stratification for persistent AF/paroxysmal AF/OATr showed a consistent association between recurrence phenotype and IP. Pulmonary vein (PV) reconnection incidence was 90%, without association with recurrence type or IP. LA volume was larger in patients with IP than in those without IP (86.7 ± 25.3 mL vs 72.0 ± 28.9 mL; P = .001). Right atrial dimensions increased between p1 and p2 in patients with IP vs no IP and in patients with AFr vs OATr. CONCLUSION: Patients with AFr after first ablation exhibit IP more frequently at redo ablation than do patients with OATr. IP correlates with more advanced AFr type, larger LA volumes, and progressive right atrial enlargement. PV reconnection is not associated with AFr. Changes in post-PVI AFI may accurately indicate progression of extra-PV AF-maintaining substrate.

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis.

BACKGROUND: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). METHODS: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. RESULTS: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. CONCLUSIONS: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

Thirteen years' experience of 893 PGD cycles for monogenic disorders in a publicly funded, nationally regulated regional hospital service.

This study provides an overview of preimplantation genetic diagnosis (PGD) for single gene diseases and the management of expanding indications in the context of a fully financially covered service at Montpellier's regional hospital centre. Within the framework of a restrictive law ruling PGD in France, only the parental genetic risk can be studied in embryos (concurrent aneuploidy screening is not allowed). PCR-based techniques were developed combining mutation detection and closely linked short tandem repeat markers within or flanking the affected genes, and set up more than 100 different robust fluorescent multiplex assays for 61 monogenic disorders. This strategy was used to analyse blastomeres from cleavage-stage embryos. Overall, 893 cycles were initiated in 384 couples; 727 cycles proceeded to oocyte retrieval and 608 cycles to embryo transfer, resulting in 184 deliveries. Clinical pregnancy rate per transfer, implantation and miscarriage rates were 33.6%, 25.1% and 8.8%, respectively. Our PGD programme resulted in the birth of 214 healthy babies for 162 out of 358 couples (45.3%), constituting a relevant achievement within an organizational framework that does not allow aneuploidy screening but provides equal access to PGD, both geographically and socioeconomically. This is a rare example of a fully free-of-charge PGD service.

Cervical cancer screening in low-resource settings: a smartphone image application as an alternative to colposcopy.

BACKGROUND: Visual inspection after application of acetic acid (VIA) and Lugol's iodine (VILI) is a cervical cancer (CC) screening approach that has recently been adopted in low- and middle-income countries (LMIC). Innovative technologies allow the acquisition of consecutive cervical images of VIA and VILI using a smartphone application. The aim of this study was to evaluate the quality of smartphone images in order to assess the feasibility and usability of a mobile application for CC screening in LMIC. METHODS: Between May and November 2015, women aged 30-65 years were recruited in a CC screening campaign in Madagascar. Human papillomavirus-positive women were invited to undergo VIA/VILI assessment. Pictures of their cervix were taken using a Samsung Galaxy S5 with an application called "Exam", which was designed to obtain high-quality images and to classify them in the following sequence: native, VIA, VILI and posttreatment. Experts in colposcopy were asked to evaluate if the quality of the pictures was sufficient to establish the diagnosis and to assess sharpness, focus and zoom. RESULTS: The application use was simple and intuitive, and 208 pictures were automatically classified and recorded in the patient's file. The quality was judged as adequate for diagnosis in 93.3% of cases. The interobserver agreement was κ =0.45 (0.23-0.58), corresponding to a moderate agreement on the common scale of kappa values. CONCLUSION: This smartphone application allows the acquisition of good quality images for VIA/VILI diagnosis. The classification of images in a patient database makes them accessible to on- and off-site experts, and allows continuous clinical education. Smartphone applications may offer an alternative to colposcopy for CC screening in LMIC.

Germline mosaicism is a pitfall in PGD for X-linked disorders. Single sperm typing detects very low frequency paternal gonadal mosaicism in a case of recurrent chondrodysplasia punctata misattributed to a maternal origin.

This manuscript presents a molecularly demonstrated gonadal mosaicism from paternal origin for X-linked dominant chondrodysplasia punctata by single sperm typing. A couple who had experienced two medical terminations of pregnancy of female fetuses was referred to our pre-implantation genetic diagnosis (PGD) centre with the diagnosis of maternally derived gonadal mosaicism. Indeed, genetic analyses of different DNA samples - including semen - from the healthy parents failed to detect the variant found in the fetuses. Six embryos, all male, were obtained during the PGD cycle. The causative variant was not detected in any embryo, whereas five embryos had inherited the 'at-risk' maternal haplotype. The assumption of a maternal gonadal mosaicism was still possible, but this finding allowed us to consider the possibility of a paternal rather than maternal gonadal mosaicism. It prompted us to perform extensive single sperm analyses, demonstrating a low-frequency paternal germline mosaicism, which led to completely different haplotype phasing and PGD counselling. In conclusion, this case further exemplifies that germline mosaicism is a pitfall in PGD where diagnosis largely relies on linkage analysis and suggests that tracing the parental inheritance through polar body analysis and/or single sperm typing experiments is of major importance for adequate genetic counselling and accurate PGD. © 2016 John Wiley & Sons, Ltd.

The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.

New multiplex PCR-based protocol allowing indirect diagnosis of FSHD on single cells: can PGD be offered despite high risk of recombination?

Molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) involves the heterozygous contraction of the number of tandemly repeated D4Z4 units at chromosome 4q35.2. FSHD is associated with a range of 1-10 D4Z4 units instead of 11-150 in normal controls. Several factors complicate FSHD molecular diagnosis, especially the cis-segregation of D4Z4 contraction with a 4qA allele, whereas D4Z4 shortening is silent both on alleles 4qB and 10q. Discrimination of pathogenic 4q-D4Z4 alleles from highly homologous 10q-D4Z4 arrays requires the use of the conventional Southern blot, which is not suitable at the single-cell level. Preimplantation genetic diagnosis (PGD) is a frequent request from FSHD families with several affected relatives. We aimed to develop a rapid and sensitive PCR-based multiplex approach on single cells to perform an indirect familial segregation study of pathogenic alleles. Among several available polymorphic markers at 4q35.2, the four most proximal (D4S2390, D4S1652, D4S2930 and D4S1523, <1.23 Mb) showing the highest heterozygote frequencies (67-91%) were selected. Five recombination events in the D4S2390-D4S1523 interval were observed among 144 meioses. In the D4S2390-D4Z4 interval, no recombination event occurred among 28 FSHD meioses. Instead, a particular haplotype segregated with both clinical and molecular status, allowing the characterization of an at-risk allele in each tested FSHD family (maximal LOD score 2.98 for theta=0.0). This indirect protocol can easily complement conventional techniques in prenatal diagnosis. Although our multiplex PCR-based approach technically fulfils guidelines for single-cell analysis, the relatively high recombination risk hampers its application to PGD.

Efficient strategies for preimplantation genetic diagnosis of spinal muscular atrophy.

OBJECTIVE: To develop and apply efficient multiplex preimplantation genetic diagnosis (PGD) protocols for spinal muscular atrophy (SMA). DESIGN: Two multiplex PGD protocols were developed allowing the detection of the common homozygous deletion of the telomeric spinal muscular atrophy gene (SMN1), together with two microsatellites located on each side of SMN1. SETTING: The molecular genetics laboratory of the university hospital in Montpellier. PATIENT(S): A couple who had already given birth to a child affected with SMA. INTERVENTION(S): In vitro fertilization using intracytoplasmic sperm injection (ICSI) and blastomere biopsy. MAIN OUTCOME MEASURE(S): Improvement of PGD for SMA. RESULT(S): Two different multiplex protocols were set up on 81 (multiplex A) and 64 single cells (multiplex B) from normal controls, affected patients, and individuals with homozygous SMN2 deletion. In one PGD cycle that used one of these protocols, two embryos were transferred, which resulted in the birth of a healthy baby. CONCLUSION(S): Analysis of microsatellite markers in addition to the SMN1 deletion allows the detection of contamination, the study of ploidy of the biopsied blastomeres, and the performance of an indirect genetic diagnosis, thereby increasing the reliability of the results. This PGD assay may be applied to all families with the common deletion of SMN1 and also to couples in whom one of the partners carries a small intragenic mutation in SMN1, identified in about 6% of affected individuals who do not lack both copies of SMN1.

Large genomic rearrangements in the CFTR gene contribute to CBAVD.

BACKGROUND: By performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole CFTR locus in the 32 CBAVD patients with only one or no mutation. METHODS: We developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing. RESULTS: We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or CFTRdele2], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or CFTRdele 22_24], in two males carrying a typical CBAVD mutation on the other parental CFTR allele. We present the first bioinformatic tool for exon phasing of the CFTR gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame. CONCLUSION: Identification of large rearrangements further expands the CFTR mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.

Rapid detection of the deltaF508 mutation in single cells using DHPLC: implications for preimplantation genetic diagnosis.

PURPOSE: Practice of preimplantation genetic diagnosis (PGD) requires efficient amplification and analysis techniques. We have tested Denaturing High Performance Liquid Chromatography (DHPLC) to screen the deltaF508 mutation in heterozygous single cells in order to assess its usefulness for PGD of cystic fibrosis. METHODS: One hundred and two single lymphocytes--including N/N and N/deltaF508--were studied. F508 locus was amplified by nested PCR followed by the analysis of PCR products by DHPLC in non-denaturing conditions. RESULTS: On the basis of PCR-amplified product analysis, total efficiency of amplification was 98.78% (101/102), and allele dropout (ADO) rate was 3.7% (3/81). For each sample, results were obtained in less than 4 min with high resolution. CONCLUSIONS: DHPLC is a rapid and efficient technique to detect the deltaF508 mutation in single cells and is therefore appropriate for clinical application of preimplantation genetic diagnosis of cystic fibrosis.