The importance of determining the limit of detection of non-invasive prenatal testing methods.

OBJECTIVE: Several non-invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell-free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥ 4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing-based NIPT method and evaluated its performance in testing samples with low FF. METHOD: An experimental model, involving the creation of artificial plasma mixtures with a final aneuploid FF ranging from 1% to 4%, simulated samples at different proportions of fetal cfDNA. We then analyzed 7103 blood samples, from pregnant women undergoing NIPT, to assess the impact of low FF on the performance of cfDNA testing. RESULTS: Detection of common aneuploidies in samples with an FF as low as 2% is well within the ability of this technology. Of 105 pregnancies confirmed chromosomally abnormal, 25 (23.8%) involving a 2% < FF < 4% were consistently detected. These high-risk pregnancies would have not been identified using the suggested 4% FF cut-off. CONCLUSION: This study underscores the importance of determining the actual LOD for each specific NIPT methodology. It may reduce the incidence of test cancelations and shorten the time required for the diagnosis of aneuploidy. © 2016 John Wiley & Sons, Ltd.

Introducing array comparative genomic hybridization into routine prenatal diagnosis practice: a prospective study on over 1000 consecutive clinical cases.

OBJECTIVE: To assess the feasibility of offering array-based comparative genomic hybridization testing for prenatal diagnosis as a first-line test, a prospective study was performed, comparing the results achieved from array comparative genomic hybridization (aCGH) with those obtained from conventional karyotype. METHOD: Women undergoing amniocentesis or chorionic villus sampling were offered aCGH analysis. A total of 1037 prenatal samples were processed in parallel using both aCGH and G-banding for standard karyotyping. Specimen types included amniotic fluid (89.0%), chorionic villus sampling (9.5%) and cultured amniocytes (1.5%). RESULTS: Chromosomal abnormalities were identified in 34 (3.3%) samples; in 9 out of 34 cases (26.5%) aCGH detected pathogenic copy number variations that would not have been found if only a standard karyotype had been performed. aCGH was also able to detect chromosomal mosaicism at as low as a 10% level. There was complete concordance between the conventional karyotyping and aCGH results, except for 2 cases that were only correctly diagnosed by aCGH. CONCLUSIONS: This study demonstrates that aCGH represents an improved diagnostic tool for prenatal detection of chromosomal abnormalities. Although larger studies are needed, our results provide further evidence on the feasibility of introducing aCGH as a first-line diagnostic test in routine prenatal diagnosis practice.

Cross-Validation of Next-Generation Sequencing Technologies for Diagnosis of Chromosomal Mosaicism and Segmental Aneuploidies in Preimplantation Embryos Model.

Detection of mosaic embryos is crucial to offer more possibilities of success to women undergoing in vitro fertilization (IVF) treatment. Next Generation Sequencing (NGS)-based preimplantation genetic testing are increasingly used for this purpose since their higher capability to detect chromosomal mosaicism in human embryos. In the recent years, new NGS systems were released, however their performance for chromosomal mosaicism are variable. We performed a cross-validation analysis of two different NGS platforms in order to assess the feasibility of these techniques and provide standard parameters for the detection of such aneuploidies. The study evaluated the performance of MiseqTM Veriseq (Illumina, San Diego, CA, USA) and Ion Torrent Personal Genome Machine PGMTM ReproSeq (Thermo Fisher, Waltham, MA, USA) for the detection of whole and segmental mosaic aneuploidies. Reconstructed samples with known percentage of mosaicism were analyzed with both platforms and sensitivity and specificity were determined. Both platforms had high level of specificity and sensitivity with a Limit Of Detection (LOD) at ≥30% of mosaicism and a showed a ≥5.0 Mb resolution for segmental abnormalities. Our findings demonstrated that NGS methodologies are capable of accurately detecting chromosomal mosaicism and segmental aneuploidies. The knowledge of LOD for each NGS platform has the potential to reduce false-negative and false-positive diagnoses when applied to detect chromosomal mosaicism in a clinical setting.

The level of adrenomedullin immunoreactivity in seminal fluid is higher in oligozoospermic subjects and correlates with semen biochemical parameters.

OBJECTIVE: The newly discovered vasoactive peptide, adrenomedullin, and its receptors are widely distributed in various non-vascular tissues. Recent studies have suggested the possible regulatory role of adrenomedullin (AM) at several levels of the pituitary-gonadal axis. We determined the level of adrenomedullin-like immunoreactivity in the seminal fluid and examined its possible correlation with routine semen parameters, semen biochemical levels or plasma levels of FSH, LH, testosterone or prolactin. MATERIALS AND METHODS: A total of 51 males were divided into three groups according to semen analysis: (i) normospermic (n=19); (ii) oligozoospermic (n=17); (iii) azoospermic (n=15). All the subjects were submitted to hormone analysis (LH, FSH, testosterone, prolactin), routine semen parameters and semen biochemical levels (fructosio, citric acid, L-carnitine, nitric oxide) evaluation. AM was determined in plasma and seminal fluid using a specific radioimmunoassay. RESULTS: Mean AM concentration in seminal plasma was higher in oligozoospermic subjects than in normospermic males. In patients with non-obstructive azoospermia AM in semen was significantly lower than in patients with obstructive azoospermia. Semen AM levels correlated negatively with citric acid concentrations in oligozoospermic subjects. In patients with obstructive azoospermia AM in seminal fluid was correlated with citric acid levels. There was a relationship between plasma AM and prolactin. CONCLUSIONS: We conclude that in human seminal fluid AM concentration is increased in infertile oligozoospermic patients and derives very likely from the prostate. Its role in the regulation of male fertility, however has to be understood.

Adrenomedullin in human male reproductive system.

OBJECTIVE: To investigate adrenomedullin (AM) localization and distribution in human male reproductive system and to determine whether seminal fluid AM concentration correlates with sperm parameters. STUDY DESIGN: Plasma and semen samples (n = 19) obtained from healthy volunteers with normal seminal fluid parameters were assayed for AM using a specific RIA. AM immunostaining was sought on sections of penile cavernous bodies and testicular tissues obtained postmortem from four young males after accidental death using a polyclonal antibody to AM 1-52. RESULTS: Mean AM concentration in seminal plasma was 209.4+/-46.6 pg/ml, 8-9-fold higher than in circulating plasma and correlated with sperm motility (r = 0.715, p < 0.01). Endothelial cells of cavernous vessels stained for AM. Intense AM immunostaining was found in germinal cells and in peritubular myocytes and Leydig cells in the testis. CONCLUSIONS: These findings demonstrated for the first time that AM is localized in human male reproductive system. The local secretion of AM suggests that AM may contribute either in the penile erection and in the regulation of testicular function and sperm motility.

Chromosomal microarray analysis as a first-line test in pregnancies with a priori low risk for the detection of submicroscopic chromosomal abnormalities.

In this study, we aimed to explore the utility of chromosomal microarray analysis (CMA) in groups of pregnancies with a priori low risk for detection of submicroscopic chromosome abnormalities, usually not considered an indication for testing, in order to assess whether CMA improves the detection rate of prenatal chromosomal aberrations. A total of 3000 prenatal samples were processed in parallel using both whole-genome CMA and conventional karyotyping. The indications for prenatal testing included: advanced maternal age, maternal serum screening test abnormality, abnormal ultrasound findings, known abnormal fetal karyotype, parental anxiety, family history of a genetic condition and cell culture failure. The use of CMA resulted in an increased detection rate regardless of the indication for analysis. This was evident in high risk groups (abnormal ultrasound findings and abnormal fetal karyotype), in which the percentage of detection was 5.8% (7/120), and also in low risk groups, such as advanced maternal age (6/1118, 0.5%), and parental anxiety (11/1674, 0.7%). A total of 24 (0.8%) fetal conditions would have remained undiagnosed if only a standard karyotype had been performed. Importantly, 17 (0.6%) of such findings would have otherwise been overlooked if CMA was offered only to high risk pregnancies.The results of this study suggest that more widespread CMA testing of fetuses would result in a higher detection of clinically relevant chromosome abnormalities, even in low risk pregnancies. Our findings provide substantial evidence for the introduction of CMA as a first-line diagnostic test for all pregnant women undergoing invasive prenatal testing, regardless of risk factors.

Polymerase chain reaction-based detection of chromosomal imbalances on embryos: the evolution of preimplantation genetic diagnosis for chromosomal translocations.

OBJECTIVE: To develop and assess a polymerase chain reaction (PCR)-based preimplantation genetic diagnosis (PGD) approach for detection of chromosomal imbalances in embryos. DESIGN: A prospective study of embryos derived from chromosome translocation carriers that have undergone PGD using a novel molecular-based approach. SETTING: A reference molecular genetics laboratory specialized in the provision of transport PGD services and a private IVF clinic. PATIENT(S): Twenty-seven couples carrying 12 different reciprocal translocations and 2 Robertsonian translocations. INTERVENTION(S): Preimplantation genetic diagnosis from chromosome translocation carriers on blastomeres biopsied from cleavage stage embryos. MAIN OUTCOME MEASURE(S): Embryo diagnosis rate, pregnancy rate (PR), implantation rate, take-home-baby rate. RESULT(S): Overall, 241/251 (96.0%) embryos were successfully diagnosed for chromosome rearrangements. Preimplantation genetic screening was included in the protocol of 12 couples, involving analysis of 90 embryos, 84 (93.3%) of which were successfully diagnosed and 53 (63.1%) showed aneuploidies. Embryos suitable for transfer were identified in 24 cycles. Eighteen couples achieved a clinical pregnancy (75.0% PR/embryo transfer), with a total of 31 embryos implanted (59.6% implantation rate). Ten patients (1 triplet, 1 twin, and 8 singleton pregnancies) have delivered 13 healthy babies, and the other patients (3 twins and 5 singletons) have currently ongoing pregnancies. CONCLUSION(S): The PCR-based PGD protocol for translocations has the potential to overcome several inherent limitations of fluorescence in situ hybridization-based tests, providing potential improvements in terms of test performance, automation, turnaround time, sensitivity, and reliability.