Incidence of sex chromosome aneuploidy in a prenatal population: 27-year longitudinal study in Northern Italy.

OBJECTIVES: The availability of cell-free (cf) DNA as a prenatal screening tool affords an opportunity for non-invasive identification of sex chromosome aneuploidy (SCA). The aims of this longitudinal study were to investigate the evolution and frequency of both invasive prenatal diagnostic testing, using amniocentesis and chorionic villus sampling (CVS), and the detection of SCA in cfDNA samples from a large unselected cohort in Northern Italy. METHODS: The results of genetic testing from CVS and amniotic fluid samples received from public and private centers in Italy from 1995 to 2021 were collected. Chromosomal analysis was performed by routine Q-banding karyotype. Regression analyses and descriptive statistics were used to determine population data trends regarding the frequency of prenatal diagnostic testing and the identification of SCA, and these were compared with the changes in indication for prenatal diagnostic tests and available screening options. RESULTS: Over a period of 27 years, there were 13 939 526 recorded births and 231 227 invasive procedures were performed, resulting in the prenatal diagnosis of 933 SCAs. After the commercial introduction of cfDNA use in 2015, the frequency of invasive procedures decreased significantly (P = 0.03), while the frequency of prenatal SCA detection increased significantly (P = 0.007). Between 2016 and 2021, a high-risk cfDNA result was the indication for 31.4% of detected sex chromosome trisomies, second only to advanced maternal age. CONCLUSIONS: Our findings suggest that the inclusion of SCA in prenatal cfDNA screening tests can increase the prenatal diagnosis of affected individuals. As the benefits of early ascertainment are increasingly recognized, it is essential that healthcare providers are equipped with comprehensive and evidence-based information regarding the associated phenotypic differences and the availability of targeted effective interventions to improve neurodevelopmental and health outcomes for affected individuals. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

Performance of a targeted cell-free DNA prenatal test for 22q11.2 deletion in a large clinical cohort.

OBJECTIVE: 22q11.2 deletion is more common than trisomies 18 and 13 combined, yet no routine approach to prenatal screening for this microdeletion has been established. This study evaluated the clinical sensitivity and specificity of a targeted cell-free DNA (cfDNA) test to screen for fetal 22q11.2 deletion in a large cohort, using blinded analysis of prospectively enrolled pregnancies and stored clinical samples. METHODS: In order to ensure that the analysis included a meaningful number of cases with fetal 22q11.2 deletion, maternal plasma samples were obtained by prospective, multicenter enrolment of pregnancies with a fetal cardiac abnormality and from stored clinical samples from a research sample bank. Fetal genetic status, as evaluated by microarray analysis, karyotyping with fluorescence in-situ hybridization or a comparable test, was available for all cases. Samples were processed as described previously for the Harmony prenatal test, with the addition of DANSR (Digital Analysis of Selected Regions) assays targeting the 3.0-Mb region of 22q11.2 associated with 22q11.2 deletion syndrome. Operators were blinded to fetal genetic status. Sensitivity and specificity of the cfDNA test for 22q11.2 deletion were calculated based on concordance between the cfDNA result and fetal genotype. RESULTS: The final study group consisted of 735 clinical samples, including 358 from prospectively enrolled pregnancies and 377 stored clinical samples. Of 46 maternal plasma samples from pregnancies with a 22q11.2 deletion, ranging in size from 1.25 to 3.25 Mb, 32 had a cfDNA result indicating a high probability of 22q11.2 deletion (sensitivity, 69.6% (95% CI, 55.2-80.9%)). All 689 maternal plasma samples without a 22q11.2 deletion were classified correctly by the cfDNA test as having no evidence of a 22q11.2 deletion (specificity, 100% (95% CI, 99.5-100%)). CONCLUSIONS: The results of this large-scale prospective clinical evaluation of the sensitivity and specificity of a targeted cfDNA test for fetal 22q11.2 deletion demonstrate that this test can detect the common and smaller, nested 22q11.2 deletions with a low (0-0.5%) false-positive rate. Although the positive predictive value (PPV) observed in this study population was 100%, the expected PPV in the general pregnant population is estimated to be 12.2% at 99.5% specificity and 41.1% at 99.9% specificity. The use of this cfDNA test to screen for 22q11.2 deletion could enhance identification of pregnancies at risk for 22q11.2 deletion syndrome without significantly increasing the likelihood of maternal anxiety and unnecessary invasive procedures related to a false-positive result. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Rare autosomal trisomies: comparison of detection through cell-free DNA analysis and direct chromosome preparation of chorionic villus samples.

OBJECTIVE: Direct chromosome preparations of chorionic villus samples (CVS) and cell-free DNA (cfDNA) testing both involve analysis of the trophoblastic cell lineage. The aim of this study was to compare the spectrum of rare autosomal trisomies (RATs) detected by these two approaches and assess the available information on their clinical significance. METHODS: Data from 10 reports on genome-wide cfDNA testing were pooled to determine which chromosomes were most frequently involved in RAT-positive cases, and pregnancy outcome information was reviewed. CVS information was obtained from an updated database of 76 102 consecutive CVS analyses performed over a period of 18 years at TOMA laboratory, in which trophoblastic and mesenchymal layers were analyzed and amniotic fluid cell analysis was recommended for RAT-positive cases. Chromosomes involved and presence of confined placental mosaicism, true fetal mosaicism and uniparental disomy (UPD) for imprinted chromosomes were assessed. Also evaluated were the frequency and types of RATs in products of conception. RESULTS: RATs were present in 634 of 196 662 (0.32%) cfDNA samples and 237 of 57 539 (0.41%) CVS trophoblast samples (P < 0.01). The frequency of RATs varied over 8-fold between the cfDNA reports. Confirmation of abnormality through amniocentesis was more likely when RATs were ascertained through cfDNA (14 of 151; 9.3%) than through CVS trophoblasts (seven of 237; 3.0%) (P < 0.01). In cfDNA-ascertained cases, trisomies 15, 16 and 22, which are associated with fetal loss, were identified proportionately more often. Of 151 cases with RAT identified by cfDNA and outcome information available, 41.1% resulted in normal live birth; 27.2% in fetal loss; 7.3% had phenotypic abnormality detected through ultrasound or other follow-up evaluation; 2.0% had a clinically significant UPD; and 14.6% had fetal growth restriction or low birth weight. All autosomes were involved in trisomies in products of conception; the most common RATs detected were trisomies 16, 22 and 15 with a frequency of > 9% each. CONCLUSIONS: Although there are strong parallels between RATs ascertained through cfDNA analysis and direct chromosome preparation of CVS, caution is needed in applying conclusions from CVS analysis to cfDNA testing, and vice versa. RATs identified through genome-wide cfDNA tests have uncertain risks for fetal loss, growth restriction or fetal abnormality. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Prenatal BACs-on-Beads™ : a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

OBJECTIVE: Molecular cytogenetic techniques on uncultured prenatal samples are the sole tests applied in some countries in cases with advanced maternal age (AMA) or increased risk after prenatal screening. Moreover, there is a trend to perform invasive prenatal diagnosis (PD) during the first trimester before ultrasound manifestations, so new rapid and reliable assays are necessary to investigate microdeletions not detectable with the conventional karyotype. We report the validation study of the prenatal bacterial artificial chromosomes-on-Beads™ (BoBs™ ; CE-IVD), a bead-based multiplex assay detecting chromosomes 13, 18, 21, X/Y aneuploidies and nine microdeletion regions having an overall detection rate of 1/1700. METHOD: We retrospectively studied 408 selected samples and prospectively tested 212 consecutive samples ascertained for conventional karyotyping. RESULTS: We did not find false-positive results. Triploidies were not detected. Maternal cell contamination of male samples up to 90% was unmasked inspecting gonosome profiles. Mosaic conditions at 20 to 30% were revealed. Failures were due to low amount of DNA. CONCLUSION: Prenatal BoBs™ is a robust technology for the investigation of fetuses with normal karyotype with or without sonographic abnormalities. Running in parallel with the karyotype analysis, it can be proposed instead of rapid FISH or QF-PCR providing rapid results on common aneuploidies and additional information regarding the microdeletion syndromes.

ESX1 gene expression as a robust marker of residual spermatogenesis in azoospermic men.

BACKGROUND: It would be of value to identify ongoing spermatogenesis molecular markers which can predict successful sperm recovery in patients with non-obstructive azoospermia undergoing conventional or microsurgical testicular sperm extraction (TESE/microTESE). ESX1 is an X-linked homeobox gene expressed in testis, placenta, brain and lung in humans and specifically in pre- and post-meiotic germ cells of the testis in mice. METHODS: We investigated the sequence, expression (by RT-PCR) and epigenetic status (by promoter pyrosequencing) of ESX1 in testicular tissue samples, obtained from 81 azoospermic subjects in the context of surgical sperm extraction, to check a possible association between ESX1 alterations and impaired spermatogenesis, as determined by histological analysis. RESULTS: The ESX1 transcript was detected in 100% of cases diagnosed as obstructive azoospermia (33), hypospermatogenesis (18) and incomplete maturation arrest (MA) (2), and sperm recovery was also successful in 100% of these cases. ESX1 mRNA was also detected in 5 of 6 patients with incomplete Sertoli cell-only syndrome, in 4 of 6 subjects with complete MA but in only 3 of 16 cases of complete Sertoli cell-only syndrome (cSCOS), whereas sperm recovery was successful in 4 of 6, 2 of 6 and 5 of 16 of these patients, respectively. In cases of focal spermatogenesis, ESX1 expression and sperm retrieval were concordant in 14 of 19 (74%) cases subjected to TESE, but in only 3 of 11 (27%) men who underwent microTESE. With TESE, but not with microTESE, both samples originated from adjacent testicular areas. The pyrosequencing of the ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors when compared with non-expressors. CONCLUSIONS: ESX1 emerges as a potentially reliable spermatogenesis molecular marker, whose clinical value as a predictor of successful sperm retrieval warrants further studies.

Detecting the occurrence of indigenous and non-indigenous megafauna through fishermen knowledge: A complementary tool to coastal and port surveys.

Marine bioinvasions and other rapid biodiversity changes require today integrating existing monitoring tools with other complementary detection strategies to provide a more efficient management. Here we explored the efficacy of fishermen observations and traditional port surveys to effectively track the occurrence of both indigenous and non-indigenous megafauna in the Adriatic Sea. This consisted mainly of mobile taxa such as fishes, crustaceans and molluscs. Port surveys using traps and nets within 10 major Adriatic harbours, were compared with the information obtained from 153 interviews with local fishermen. Information gathered by traps and nets varied significantly and generally resulted of limited efficacy in exotic species detection. Interviews allowed tracking the occurrence of new species through time and space, providing complementary knowledge at the low cost. This combined approach improves our capability of being informed on the arrival of species of different origin, providing a more rational, improved basis for environmental management and decision making.

Macrozoobenthos in the Adriatic Sea ports: Soft-bottom communities with an overview of non-indigenous species.

The present paper is a contribution to the first initiative of the Port Baseline Survey (PBS) for Non-indigenous species (NIS) in the Mediterranean Sea. It presents a report on the soft-bottom macrobenthos from the five Adriatic ports: Bari, Ancona (Italy), Koper (Slovenia), Pula, Rijeka (Croatia), with a focus on the presence and contribution of NIS to native assemblages. Out of 451 species identified, only four were common to all ports. A total of eight NIS were recorded, five in surveyed ports and three in the lagoon connected to the Port of Koper. The highest number of NIS was recorded in Bari, and the highest abundance in Ancona and Bari. Generally, the number, abundance and contribution of NIS seems too low to cause a substantial impact on native communities in surveyed ports. The suitability of methods adopted for PBS for soft-bottom NIS was discussed and suggestion for methodological improvement is provided.

Non-indigenous macrozoobenthic species on hard substrata of selected harbours in the Adriatic Sea.

The intense shipping traffic characterising the Adriatic Sea favours the spread of marine organisms. Yet, a study of 12 Adriatic ports (4 on the western side and 8 on the eastern side of the basin) found that non-indigenous species (NIS) accounted for only 4% of the benthic communities settled on hard substrates. The cirripeds Amphibalanus amphitrite and Balanus trigonus, found in 8 harbours, were the most common invaders followed by Amphibalanus eburneus, the ascidian Styela plicata, and the bivalve Magallana gigas. The highest percentage of NIS was recorded in Venice and Ploce, the harbours with the least rich native communities; the lowest percentage was retrieved in Trieste, Koper, Pula, and Rijeka, the harbours hosting the highest species diversity. In contrast, the ports of Bari and Ancona showed both high NIS percentages and highly diversified communities.

Chromosome 11 segmental paternal isodisomy in amniocytes from two fetuses with omphalocoele: new highlights on phenotype-genotype correlations in Beckwith-Wiedemann syndrome.

BACKGROUND: The phenotypic variability in Beckwith-Wiedemann syndrome (BWS) reflects the genetic heterogeneity of the mechanism which by default leads to the deregulation of genes located at 11p15.5. Genotype-phenotype correlation studies have demonstrated an association between omphalocoele and CDKN1C/p57 mutations or hypermethylation. Paternal uniparental disomy 11 (pUPD11) has been described only in the mosaic condition with both uniparental and biparental cell lines, and no association with omphalocoele has been pointed out. METHODS: Two cases are presented here, in which a paternal segmental UPD11 was detected by molecular investigation of amniotic fluid cell cultures after the presence of apparently isolated omphalocoele was revealed in the fetuses by ultrasound scan. Further studies were performed on additional autoptic feto-placental tissues to characterise the distribution of the uniparental cell line and to unmask any biparental lineage in order to document in more detail the as yet unreported association between omphalocoele and pUPD11. RESULTS: Results on the UPD distribution profile showed that the abdominal organs have a predominant uniparental constitution. This condition could mimic the effect of CDKN1C/p57 inactivation, causing the omphalocoele. CONCLUSION: New genotype-phenotype correlations emerge from the investigated cases, suggesting that molecular analysis be extended to all cases with fetal omphalocoele in order to establish the incidence of pUPD11 in complete BWS and in monosymptomatic/mild forms.

Characterization of microplastic litter in the gastrointestinal tract of Solea solea from the Adriatic Sea.

Micro-plastic particles in the world's oceans represent a serious threat to both human health and marine ecosystems. Once released into the aquatic environment plastic litter is broken down to smaller pieces through photo-degradation and the physical actions of waves, wind, etc. The resulting particles may become so small that they are readily taken up by fish, crustaceans and mollusks. There is mounting evidence for the uptake of plastic particles by marine organisms that form part of the human food chain and this is driving urgent calls for further and deeper investigations into this pollution issue. The present study aimed at investigating for the first time the occurrence, amount, typology of microplastic litter in the gastrointestinal tract of Solea solea and its spatial distribution in the northern and central Adriatic Sea. This benthic flatfish was selected as it is a species of high commercial interest within the FAO GFCM (General Fisheries Commission for the Mediterranean) area 37 (Mediterranean and Black Sea) where around 15% of the overall global Solea solea production originates. The digestive tract contents of 533 individuals collected in fall during 2014 and 2015 from 60 sampling sites were examined for microplastics. These were recorded in 95% of sampled fish, with more than one microplastic item found in around 80% of the examined specimens. The most commonly found polymers were polyvinyl chloride, polypropylene, polyethylene, polyester, and polyamide, 72% as fragments and 28% as fibers. The mean number of ingested microplastics was 1.73 ± 0.05 items per fish in 2014 and 1.64 ± 0.1 in 2015. PVC and PA showed the highest densities in the northern Adriatic Sea, both inshore and off-shore while PE, PP and PET were more concentrated in coastal areas with the highest values offshore from the port of Rimini.

A multidisciplinary approach to study the reproductive biology of wild prawns.

This work aims to provide deeper knowledge on reproductive biology of P. kerathurus in a multidisciplinary way. Upon 789 examined females, 285 were found inseminated. The logistic equation enabled to estimate the size at first maturity at 30.7 mm CL for female. The Gono-Somatic Index (GSI) showed a pronounced seasonality, ranged from 0.80 ± 0.34 to 11.24 ± 5.72. Histological analysis highlighted five stages of ovarian development. Gonadal fatty acids analysis performed with gas chromatograph evidenced a pronounced seasonal variation; total lipids varied from 1.7% dry weight (dw) in Winter, to 7.2% dw in Summer. For the first time, a chemometric approach (Principal Component Analysis) was applied to relate GSI with total lipid content and fatty acid composition of gonads. The first two components (PC1 and PC2) showed that seasonality explained about 84% of the variability of all data set. In particular, in the period February-May, lipids were characterized by high PUFAs content, that were probably utilized during embryogenesis as energy source and as constituent of the cell membranes. During the summer season, gonads accumulated saturated FAs, that will be used during embryogenesis and early larval stages, while in the cold season total lipids decreased drastically and the gonad reached a quiescent state.