INTRODUCTION:The study investigated whether the disclosure of the chromosomal anomaly causing early pregnancy loss (EPL) favors the grief process and reduces psychological distress. METHODS:Women experiencing EPL were invited to participate in the study at the time they were offered chorionic villi sampling (CVS) and karyotyping before uterine evacuation. They completed two online surveys: one a week after EPL and another a month later, after receiving cytogenetic results. The surveys measured anxiety (Hospital Anxiety and Depression Scale [HADS]), Beck Depression Inventory (BDI-II) cognitive depression factor items, post-traumatic stress disorder (Impact of the Event Scale-Revised [IES-R]), and rumination (Ruminative Responses Scale [RRS-10]). Participants were divided into groups based on the chromosomal anomaly found at CVS: A1 (autosomal trisomy), A2 (other anomalies), and B (no anomalies). RESULTS:A significant score decline was observed in the four studied psychological scales assessing anxiety, depression, post-traumatic stress, and rumination, between the first and second survey. The proportion of women with a clinical score also demonstrated a significant decline, except for anxiety. When these changes were assessed after karyotypic group stratification, the group with other chromosomal anomalies (A2) showed the highest drop. This group also demonstrated a significantly higher depression score decline at multivariate regression analysis of the median. CONCLUSION:EPL significantly impacts women's mental health, with 19%-51% experiencing manifestations. Disclosing chromosomal anomalies may aid in psychological recovery, particularly in reducing clinical scores for depression.

At 16 + 6-weeks a fetal scan performed in the second pregnancy of a 42 y.o. woman identified a right multicystic dysplastic kidney, left renal agenesis, absent urinary bladder, myocardial hypertrophy, increased nuchal fold, a single umbilical artery, and oligohydramnios. Trio exome sequencing analysis detected a novel pathogenic NONO variant. Postmortem examination after the termination of pregnancy confirmed the ultrasound findings and also revealed pulmonary hypoplasia, retrognathia and low-set ears. The variant was a novel de novo hemizygous pathogenic loss-of-function variant in NONO [NM_007363.5], associated with a rare X-linked recessive neurodevelopmental disorder, named intellectual developmental disorder, X-linked syndromic 34 (OMIM#300967). The postnatal characteristic features of this disorder include intellectual disability, developmental delay, macrocephaly, structural abnormalities involving the corpus callosum and/or cerebellum, left ventricular noncompaction and other congenital heart defects. In the prenatal setting, the phenotype has been poorly described, with all described cases presenting with heart defects. This case highlights the need of further clinical delineation to include renal abnormalities in the prenatal phenotype spectrum.

Objective: The aim of the study was to assess the diagnostic yield of 2 different next-generation sequencing (NGS) approaches: gene panel and “solo” clinical exome sequencing (solo-CES), in fetuses with structural anomalies and normal chromosomal microarray analysis (CMA), in the absence of a known familial mutation. Methodology: Gene panels encompassing from 2 to 140 genes, were applied mainly in persistent nuchal fold/fetal hydrops and in large hyperechogenic kidneys. Solo-CES, which entails sequencing the fetus alone and only interpreting the Online Mendelian Inheritance in Man genes, was performed in multisystem or recurrent structural anomalies. Results: During the study period (2015–2020), 153 NGS studies were performed in 148 structurally abnormal fetuses with a normal CMA. The overall diagnostic yield accounted for 35% (53/153) of samples and 36% (53/148) of the fetuses. Diagnostic yield with the gene panels was 31% (15/49), similar to 37% (38/104) in solo-CES. Conclusions: A monogenic disease was established as the underlying cause in 35% of selected fetal structural anomalies by gene panels and solo-CES.

Steel syndrome (STLS) encompasses characteristic facies, dwarfness, irreducible bilateral hip and radial head dislocation, and carpal bone coalition due to COL27A1 mutations. Two consecutive pregnancies in a non-consanguineous couple were terminated because of severe fetal anomalies. Complete autopsies with microscopic exam were performed on both fetuses. Next-generation-based clinical exome sequencing was applied to the first fetus. Exome sequencing results, parental segregation, and affection of the second fetus were confirmed by Sanger sequencing. Both fetuses had signs consistent with STLS. Bilateral capitulum humeri absence explained radial head dislocation in STLS. Metaphyseal cartilage showed severe disorganization. Resting cartilage was hypercellular, organized in irregular nests limited by acellular matrix. Two variants in COL27A1 (c.2548G>A -p.Gly850Arg- and c.3249+1G> T) were found in both fetuses in compound heterozygosity with parental Mendelian segregation. This is the first report to include histology of STLS. The COL27A1 variants here described increase the number of mutations associated with STLS.

Objective: To explore the use of a new molecular work-up based on the stepwise use of Quantitative Fluorescence PCR (QF-PCR) extended to eight chromosomes and single nucleotide polymorphism array (SNP-array) in chorionic villi obtained by chorionic villi sampling (CVS) offered to women experiencing an early pregnancy loss. Methods: During a 3-year period (January 2016–December 2018), CVS was offered to women experiencing an early pregnancy loss before the evacuation of the products of conception (POC) to retrieve chorionic villi, irrespective of the number of previous losses. A new molecular work-up was prospectively assayed encompassing a first QF-PCR round (with the 21, 18, 13, 7, X, and Y chromosomes), a second QF-PCR round (with the 15, 16, and 22 chromosomes), and a high resolution SNP-array in those cases with normal QF-PCR results. A control group in which POC were collected after surgical uterine evacuation was used to be compared with the intervention group. Results: Around 459 women were enrolled in the intervention group (CVS) and 185 in the control group (POC after uterine evacuation). The QF-PCR testing success rates were significantly higher in the intervention group (98.5%: 452/459) as compared to the control group (74%: 109/147; p < 0.001), while the chromosomal anomaly rate at the two QF-PCR rounds was similar between the two groups: 52% (234/452) in the intervention and 42% (46/109) in the control group ( p = 0.073). The SNP-array was performed in 202 QF-PCR normal samples of the intervention group and revealed 67 (33%) atypical chromosomal anomalies (>10 Mb), 5 (2.5%) submicroscopic pathogenic copy number variants, and 2 (1%) variant of uncertain significance (VOUS). Conclusion: Eighty-two percent of women experiencing an early pregnancy loss opted for a CVS. The testing success rates were higher in the intervention group (CVS; 98%) as compared to the control group (POC; 74%). The overall yields were 52% by QF-PCR (including three complete hydatiform moles), and 16% by SNP-array, including 15% atypical chromosomal anomalies and 1.1% submicroscopic pathogenic copy number variants.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder (1/400-1,000 livebirths). Its genetic diagnosis is well stablished by the presence of heterozygous mutation of the PKD1 (80%) or PKD2 genes and recently new mutations as GANAB, HNF1B and DZIP1L have been described. Early-onset manifestation of the disease is observed only in 2% of patients and most of them (90%) present familial history, being de novo in 10% of the cases. Fetuses affected do not present a typical ultrasound (US) pattern, so the prenatal diagnosis, especially in de novo cases, is a challenge. We describe a 28-year-old primigravida without familial history of kidney diseases, referred to our unit at 32 weeks of GA due to fetal enlarged and echogenic kidneys. The US findings showed both enlarged kidneys (>95 centile) with increased echogenicity and poor corticomedullary differentiation. Neither cysts, urinary tract dilatations or extrarenal anomalies were found. Amniotic fluid index (AFI) was 7cm. Normal karyotype and microarray ruled out aneuploidies and microdeletions syndromes. Next-generation sequencing (NGS) with targeted panel for kidney disease revealed the presence of a frameshift c.2085dupC (p.Ala696fs) heterozygous pathogenic variant in PKD1 de novo associated with ADPKD type I. The patient refused the evaluation of renal function by cordocentesis. The fetus showed stable kidney image and normal values of AFI (7-9cm) in the following controls. The patient had a spontaneous delivery at 40 weeks. A 3,670g male with Apgar score 6-8-10 was born. Newborn follow-up revealed kidney function within normal range and US evaluation confirmed prenatal findings. This case reflects that NGS is a value tool in the diagnosis of prenatal debut of ADPKD, particularly in de novo cases, which makes possible the correct counselling of the families and monitoring the renal function from birth to detect early complications.

Abstract STUDY QUESTION Can maternal plasma cell-free DNA (cfDNA) detect chromosomal anomalies in early pregnancy loss (EPL) and recurrent pregnancy loss (RPL)? SUMMARY ANSWER Genome-wide cfDNA testing can serve as an alternative to cytogenetic analysis in products of conception (POCs) in RPLs and can guide further management. WHAT IS KNOWN ALREADY Random chromosomal anomalies are the single most common cause for EPL and RPL. Cytogenetic analysis in POCs may be used to direct management in RPL because the detection of random chromosomal anomalies can eliminate further unwarranted testing. STUDY DESIGN, SIZE, DURATION This was a prospective diagnostic test study from March 2018 to January 2019 of 109 patients experiencing pregnancy loss before 14 weeks gestation at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS Blood samples were drawn for genome-wide cfDNA testing prior to chorionic villous sampling for cytogenetic analysis of POCs with both short-term cultures (STCs) and long-term cultures (LTCs). Final analysis included 86 patients with non-mosaic cytogenetic results in POCs and available cfDNA results. Aneuploidy detection rates by cfDNA testing and POC cytogenetic analysis were compared. The first 50 samples served as the Training Set to establish pregnancy loss-specific log-likelihood ratio (LLR) thresholds using receiver-operator characteristic (ROC)-like analyses. These were then used for the entire cohort. MAIN RESULTS AND THE ROLE OF CHANCE Seventy-eight samples (71.5%) had results available from both STC and LTC; 12 samples (11%) had a result from STC only, and 7 samples (6.4%) had a result from LTC only. A chromosomal anomaly was detected in 55/86 (64%). The rates of chromosomal anomalies were 61, 72, 73 and 44% in patients undergoing their first, second, third and ≥4th pregnancy losses, respectively. The median cfDNA fetal fraction was 5%. With standard LLR thresholds used for noninvasive prenatal screening, the sensitivity of cfDNA in detecting aneuploidy was 55% (30/55) and with a specificity of 100% (31/31). Using pregnancy loss-specific LLR thresholds, the sensitivity of cfDNA in detecting aneuploidy was 82% (45/55), with a specificity of 90% (28/31). The positive and negative likelihood ratios were 8.46 and 0.20, respectively. Fetal sex was correctly assigned in all cases. LIMITATIONS, REASONS FOR CAUTION Cases with a false-positive result by cfDNA analysis would not receive the indicated RPL workup. Specificity could be improved by using a fetal fraction (FF) cutoff of 4%, but this would result in exclusion of more than a quarter of cases. WIDER IMPLICATIONS OF THE FINDINGS cfDNA-based testing can serve as an alternative to POC cytogenetic analysis and can guide further RPL management: if cfDNA demonstrates aneuploidy, no further action is taken and if no abnormality is detected, the recommended RPL workup is performed. STUDY FUNDING/COMPETING INTEREST(S) Cell-free DNA testing was funded by Illumina, Inc., San Diego, CA. Y.Y. is a member of Illumina’s Clinical Expert Panel and has received travel grants. A.B. has received travel grants from Illumina. All authors have no competing interest to declare.

Objective To assess the frequency of atypical chromosomal and submicroscopic anomalies, as well as fetal structural abnormalities, observed on first-trimester ultrasound scan in fetuses with nuchal translucency (NT) thickness > 99(th) centile, in order to evaluate the suitability of using standard cell-free DNA (cfDNA) testing as the sole screening test in these pregnancies. Methods This was a retrospective cohort study of 226 fetuses with NT > 99(th) centile at 11-14 weeks' gestation, between January 2013 and December 2017, in a clinical setting in which greater than 95% of pregnant women receive first-trimester combined screening. All patients underwent genetic testing by means of quantitative fluorescence polymerase chain reaction and chromosomal microarray analysis, mainly in chorionic villus samples. We assessed the theoretical yield of two cfDNA testing models, targeted cfDNA (chromosomes 21, 18 and 13) and extended cfDNA (chromosomes 21, 18, 13 and sex chromosomes), and compared it with that of cytogenetic testing and ultrasound assessment in the first and second or third trimesters. Results In the 226 fetuses analyzed, cytogenetic testing revealed 84 (37%) anomalies, including 68 typical aneuploidies (involving chromosomes 13, 18 or 21), six sex chromosome aneuploidies (four cases of monosomy X and two of trisomy X), three clinically relevant atypical chromosomal anomalies (one trisomy 22, one trisomy 21 mosaicism and one unbalanced translocation), five submicroscopic pathogenic variants and two cases with Noonan syndrome. Targeted and extended cfDNA testing would miss at least 12% (10/84) and 19% (16/84), respectively, of genetic anomalies, accounting for 4.4% and 7.1% of the fetuses with an increased NT, respectively. Finally, of the 142 fetuses with no identified genetic anomaly, a major fetal malformation was observed in 15 (10.6%) fetuses at the early anomaly scan, and in 19 (13.4%) in the second or third trimester. Conclusions cfDNA does not appear to be the appropriate genetic test in fetuses with NT > 99(th) centile, given that it would miss 12-19% of genetic anomalies in this group. Additionally, first-trimester ultrasound will identify a major structural abnormality in 11% of the fetuses with NT > 99(th) centile and no genetic anomaly. Copyright (c) 2019 ISUOG. Published by John Wiley & Sons Ltd.

To assess the clinical implication of chromosomal microarray analysis (CMA) and nex-generation sequencing (NGS) in prenatal diagnosis of Congenital Anomalies of the Kidney and the Urinary Tract (CAKUT). We review 428 cases of CAKUT followed by prenatal ultrasound in our Fetal Nephrourological Unit from January 2015 until December 2018. CAKUT cases were classified into 3 groups: 308 (72%) collecting system anomalies (including upper urinary tract dilatations, duplex collector system, megacystis and LUTO); 57 (13%) number, fusion or location anomalies and 43 (10%) renal dysplasias (including echogenic, hypoplastic, polycystic and multicystic kidneys). Invasive test (amniocentesis or chorionic villous sampling) for CMA were always offered except when it was isolated mild pelvic dilatation. In some selected cases, NSG of gens panel was added to complete the study. From a total of 164 (38%) genetic studies performed, 3 VOUS and 17 (10%) anomalies were found: 7 (42%) of them in dysplastic and echogenic kidneys, 5 (29%) in collecting system anomalies and 5 (29%) in number, fusion or location anomalies. Of this 17 chromosomal anomalies, only 4 cases could be diagnosed by conventional karyotype (two cases of T21, one T13 and one case of 47,XYY) and the remaining 13 anomalies were microdeletions/microduplications syndromes highlighting 4 deletion 17q12 in echogenic kidneys and 3 monogenic disorders (Bardet-Biedl, Steel and CHARGE syndromes). CMA and NSG increased the detection rate of genetic disorders by 8% in our CAKUT series. CMA and NGS are a valuable tool in the diagnosis and prenatal counseling of CAKUT, particularly in those cases that represent a diagnostic dilemma such as echogenic/dysplastic kidneys with normal amniotic fluid volume. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Steel syndrome (STLS) is an autosomal recessive disease caused by mutations in the COL27A1 gene, located on chromosome 9q32. It is characterized by dislocated hips and radial heads, carpal coalition, characteristic facies, short stature, scoliosis, and cervical spine anomalies. It was first described by Steel et al on 1993 in Puerto Rican children. We describe two STLS cases in consecutive pregnancies diagnosed in 2018 and 2019. First case was diagnosed at post-mortem studies. Distal arthrogryposis and horseshoe kidney were suspected at the second trimester scan. Amniocentesis was performed for array-CGH analysis obtaining a normal result. Parents opted for termination of the pregnancy at 22 weeks. At post-mortem studies, bilateral dislocated hips and carpal coalition were observed and STLS was suspected. To confirm diagnosis, NGS was performed, founding two heterozygous mutations in COL27A1 gene: c.2548G > A and c.3249 + 1G > T. Parental segregation studies showed that the first mutation was inherited from the father and the second one from the mother. The second case was suspected on first trimester ultrasound due to the finding of malposition of the lower limbs and a single umbilical artery. A transcervical chorionic villus sampling was performed in order to look for the previously found gene mutations in the COL27A1 gene. The diagnosis of STLS was confirmed by molecular analysis. These are the first prenatally diagnosed STLS cases reported. Previously described mutations in children in the COL27A1 gene are a homozygous missense mutation (G697R; 608461.0001), heterozygous nonsense (R707X; 608461.0002) and a frameshift (608461.0003) mutation and homozygous splice site mutation (608461.0004). Neither one of the found mutations was previously described on other STLS cases. c.3249 + 1G > T was only found in one individual on the general population. Therefore, these two cases provide new information on the molecular genetics of STLS. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Recurrent pregnancy loss (RPL) is defined by ≥2 failed clinical pregnancies. The recommended workup (parental karyotyping, uterine cavity assessment, endocrine imbalances and autoimmune factors) will detect a probable cause in only ∼50% of cases. However, the most common cause of miscarriage, even in RPLs, is random chromosomal aberrations (trisomy, monosomy, triploidy). Chromosomal analysis of products of conception (POCs) has been shown to be cost-saving when used to guide further workup. This study was undertaken to assess whether maternal serum cell free (cf)DNA can be used to detect chromosomal abnormalities in RPLs. The study included 102 patients experiencing pregnancy loss at < 14 weeks. Blood samples were drawn for genome-wide cfDNA testing. Patients then underwent chorionic villous sampling (CVS) for karyotyping of both short-term and long-term cultures. Quantitative fluorescent PCR was also performed for some chromosomes. The first 50 cases served as a training set to determine appropriate thresholds for making an aneuploidy call. These were used for the entire cohort. The mean gestational age at embryo demise was 9.8 ± 2.1 weeks and the fetal fraction was 5.2 ± 3.7%. A chromosomal anomaly was detected in 68 cases. The rates of chromosomal anomalies were 67%, 74%, 73% and 42% in patients undergoing their 1st, 2nd, 3rd, and ≥4th losses, respectively. Of the 54 cases with a single autosomal trisomy or monosomy X, the detection rate was 83%. Of the 14 cases with complex abnormalities, monosomy, triploidy or mosaic, only 2 were detected. There were 4 false positive cases. The overall sensitivity of cfDNA-based testing was 69%. We suggest that cfDNA-based testing serve to guide management in RPLs: if cfDNA in the 2nd and subsequent RPL demonstrates aneuploidy, no further action is taken; if cfDNA demonstrates an unbalanced rearrangement, parental karyotypes is recommended; and if no abnormality is detected, then the recommended RPL workup is performed.

To explore a new molecular work-up based on the stepwise use of Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR) extended to 9 chromosomes and SNP-array in chorionic villi retrieved by chorionic villi sampling (CVS) after routine offering to women with an early pregnancy loss. During a 3-year period (October 2015-October 2018), a new molecular work-up was prospectively assayed in chorionic villi, based on two QF-PCR rounds including the 21,18,13,7,X,Y,15,16 and 22 chromosomes, as the first step, and a high-resolution SNP-array as a second step. CVS was offered to all women with an early pregnancy loss before evacuation of the products of conception (POC), irrespectively of the number of previous losses, and routine genetic testing includes also karyotyping after short and long-term cultures. Among 459 women with an early pregnancy loss that consent to undergo CVS and the new work-up, chorionic villi were successfully retrieved in 454 (99%). In the first step, aneuploidies and triploidies were found in 227 (50%) cases including 3 complete uniparental disomies (complete moles). In the half of the losses with a normal QF-PCR result, routine karyotyping revealed 75 (17% of the total) atypical chromosomal anomalies, 15 (3.3%of the total) had an insufficient amount of remaining DNA, and 134 underwent SNP-arrays, revealing 5 (3.7%) pathogenic submicroscopic anomalies and 2 (1.4%) variants of unknown of significance. The use of a two-step molecular work-up in early pregnancy loss allowed to detect typical chromosomal anomalies in 50% of the cases, and 1.5% submicroscopic anomalies, in addition to 17% atypical chromosomal anomalies. Detection of maternal cell contamination, complete moles, and 3.7% failure rate are additional benefits over conventional karyotyping.

Autosomal trisomies and triploidies are the most common chromosome abnormalities in humans, and the leading genetic cause of miscarriage. The aim of our study was to determine the parental origin of trisomies and triploidies diagnosed in our centre from 2016 to 2019 by quantitative fluorescent polimarase chain reaction (QF-PCR). The parental origin of 97 autosomal trisomies and 32 triploidies diagnosed in spontaneous miscarriages or high risk ongoing pregnancies was determined by QF-PCR applied to a fetal sample obtained by chorionic villi sampling or amniocentesis and a maternal saliva sample. There were 63 (49%) nonviable intrauterine pregnancies (34 trisomies and 29 triploidies) and 66 (51%) ongoing pregnancies (63 trisomies and 3 triploidies) sampled because of a high risk of chromosomal anomalies. In the vast majority (96%: 93/97) of trisomies, the extra chromosome was maternally inherited, and only few trisomies 21 (7%) and 22 (12%) were of paternal origin (table). Regarding the allelic patterns of trisomies, 109 cases (96%) were triallelic and 5 (4.4%) were diallelic. Among the 32 triploidies, 18 (56%) had a paternal origin and they corresponded to partial moles. Maternal origin was the most likely source of the additional chromosome in autosomal trisomies. Nevertheless, in triploidies both maternal and paternal origin had a similar frequency, with a slightly higher implication of the paternal provenience. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The aim of this study is to assess the clinical implication of chromosomal microarray analysis (CMA) in the diagnosis of chromosomal disorders in fetal isolated echogenic kidneys. We review 10 cases of isolated echogenic kidneys with normal amniotic fluid volume followed in our Fetal Nephrourological Unit from January 2015 to December 2018. A detailed fetal ultrasound scan was done to rule out possible associated malformations and invasive test was offered in all cases to investigate chromosomal anomalies. Two women refused the amniocentesis, and eight (seven CMA and one karyotype) studies were performed. From eight (80%) genetic studies performed, five (63%) chromosomal anomalies were observed: four (50%) 17q12 microdeletions and one 47,XYY (in karyotype). After birth, all children with 17q12 deletion have been followed in Nephro-Pediatric, Genetics and Neuropediatrics Units presenting preserved renal function until now, but persistent anomalous appearance of kidneys in ultrasound. One of them present a mild neurodevelopment delay and a pre-diabetic status. A high proportion (63%) chromosomal anomalies are present in fetuses with isolated echogenic kidneys. CMA strongly improves genetic disorders detection and, it should be offered in order to rule out 17q12 microdeletion syndrome, which has a high correlation with echogenic kidneys during fetal life. This syndrome is a cause of renal abnormalities with uncertain further renal function, maturity-onset diabetes of the young (MODY-5) and a wide spectrum of neurodevelopement disorders. Early diagnosis of 17q12 microdeletion allows a closer follow-up of these children from birth. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.