Search Results (96)

Objectives: Noninvasive prenatal testing relies on the analysis of total cell-free DNA (cfDNA) in maternal plasma, where fetal-derived DNA constitutes only a minor fraction. This study aimed to preliminarily compare a modified TPY cfDNA extraction protocol with two commercial extraction kits for the downstream detection of Y-chromosome-specific markers in pregnancies carrying male fetuses. Methods: Plasma samples were obtained from 52 singleton pregnancies between 10 and 30 weeks of gestation with male fetal sex confirmed by ultrasonography. Total cfDNA was extracted from aliquots of the same maternal plasma samples using the modified TPY protocol, the QIAamp DSP Virus Kit, and the MagMAX™ Cell-Free DNA Isolation Kit. Quantitative real-time PCR was performed for the Y-chromosome-specific markers SRY and DYS14. At the same time, GLO was used as a reference marker to reflect the total cfDNA background. Extraction performance was assessed primarily using total cfDNA concentration and Ct values obtained from amplification of fetal-specific Y-chromosome markers. Results: Total cfDNA concentrations varied among the extraction methods, with the commercial kits yielding higher total cfDNA concentrations than the modified TPY protocol. In contrast, the TPY protocol yielded slightly lower mean Ct values for SRY and DYS14 than the commercial kits. SRY and DYS14 amplification was detected in 90.4% and 94.2% of samples, respectively. However, these Ct differences should be interpreted cautiously because fetal fraction, maternal DNA contamination, extraction recovery, and fragment size distribution were not directly measured. Conclusions: The modified TPY protocol showed preliminary technical feasibility for extracting total cfDNA from maternal plasma and enabling downstream amplification of Y-chromosome-specific markers in male pregnancies. Nevertheless, the observed lower Ct values do not establish selective fetal DNA enrichment, reduced maternal DNA contamination, or clinical superiority over commercial methods. Further analytical validation using standardized fetal fraction measurement, recovery efficiency testing, fragment size analysis, fetal-to-maternal DNA ratio assessment, and larger cohorts including both male and female pregnancies is required before broader clinical applicability can be determined. Full article

Background: Non-invasive prenatal testing (NIPT) based on analysis of Cell-Free Fetal DNA has transformed screening for common aneuploidies and is increasingly extended to genome-wide detection of copy number variants (CNVs). However, CNV detection remains constrained by analytical limitations and biological signal complexity. Methods: This review evaluates the analytical validity, biological constraints, and clinical interpretation challenges of CNV detection by NIPT, framing it as a probabilistic genomic inference rather than a direct measure of fetal copy number. Results: Performance depends on sequencing depth, bin resolution, fetal fraction, guanine–cytosine correction, and reference modeling, leading to variable detection thresholds. The predominantly placental origin of cfDNA introduces discordance through Confined Placental Mosaicism, post-zygotic events, and clonal variation. Maternal CNVs, mosaicism, vanishing twin, and occult malignancy further complicate interpretation and may cause false positives. Clinical validity is heterogeneous, with positive predictive value dependent on CNV size, genomic context, and prevalence. Reporting practices remain inconsistent. Conclusions: CNV detection by NIPT is fundamentally limited by interpretation of a composite maternal–placental signal. Progress requires improved tissue-of-origin discrimination, multi-omic integration, and standardized reporting to ensure responsible clinical implementation. Full article

First-trimester sonographic examination remains a fundamental part of antenatal care, providing crucial information for the well-being of both the mother and fetus and leading to the best possible perinatal outcomes. This study aimed to review and compare the most recently published guidelines on first-trimester ultrasound. Therefore, a descriptive review of guidelines from the American Institute of Ultrasound in Medicine (AIUM), the Australasian Society of Ultrasound in Medicine (ASUM), the Association of the Scientific Medical Societies in Germany (AWMF), the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG), the Society of Obstetrician and Gynecologists of Canada (SOGC) and the World Association of Perinatal Medicine (WAPM) regarding first-trimester scans was conducted. There is a consensus regarding the main parameters that should be evaluated, the qualifications of the examiner and specifications of the ultrasound machine, as well as the importance of informed consent. Additionally, the importance of careful visualization of fetal anatomy is discussed, with minor discrepancies regarding the appropriate protocol used. The use of combined first-trimester examination is encouraged by all the reviewed medical societies, although cell-free DNA testing is addressed only by a few, with similar indications. Controversy exists regarding the indications and appropriate gestational age at which the first-trimester scan should be performed, as well as the proper establishment of pregnancy dating. Notably, recommendations regarding fetal growth restriction (FGR) and diagnostic invasive procedures are made only by few medical societies, with the AWMF and SOGC addressing screening for FGR. Furthermore, invasive diagnostic testing is discussed by the AIUM, AWMF and SOGC, with differentiations among them regarding the indications for pursuing such procedures. First-trimester sonographic examination is essential for assessing fetal viability, establishing accurate pregnancy dating, evaluating fetal and maternal anatomy and calculating the risk of various fetal and maternal conditions. The implementation of evidence-based, unified protocols would advance both maternal and fetal outcomes. Full article

Background/Objectives: Non-invasive prenatal testing (NIPT) for fetal aneuploidy requires accurate trisomy detection together with reliable fetal fraction assessment. This study evaluated the clinical feasibility of a 106-plex digital PCR (dPCR) NIPT assay for trisomies 13, 18, and 21 with internal fetal fraction quantification. Methods: We consecutively recruited 470 women with high-risk singleton pregnancies. Fetal trisomies were detected using dPCR-NIPT and confirmed by invasive prenatal diagnosis. Pregnancies with negative prenatal diagnostic results were followed to birth. Analytical performance and quality control were assessed using trisomic DNA. The euploid cut-off and diagnostic performance were evaluated in two independent maternal plasma sample sets, using invasive diagnosis and clinical outcome as the reference standard. Results: dPCR-NIPT measured fetal fraction irrespective of fetal sex and detected trisomies at fetal fractions ≥3% using 5 ng DNA. A total of 12 of 470 plasma samples failed cell-free DNA quality control and were excluded before dPCR testing. Of the remaining 458 samples, 5 had fetal fractions below 3% and were classified as failed tests, yielding a nonreportable rate of 1.1%. Using a cut-off of 6.9 established in 103 training samples, no false-positive or false-negative trisomy calls were observed in the 350-sample testing set, corresponding to 100% sensitivity (95% confidence interval [CI], 85.18–100%) and 100% specificity (95% CI, 98.88–100%) for 23 confirmed trisomies. Conclusions: This proof-of-principle study supports the feasibility of fetal fraction-informed dPCR-NIPT for trisomy detection in high-risk singleton pregnancies. Larger prospective studies in average-risk and earlier-gestation populations are required. Full article

Preterm birth is a major cause of neonatal morbidity and mortality, and many spontaneous cases remain idiopathic. Increasing evidence suggests that intrauterine inflammation may occur in the absence of detectable infection, leading to the recognition of sterile intrauterine inflammation as an important mechanism contributing to threatened preterm labor and spontaneous preterm birth. This review summarizes current knowledge regarding the role of damage-associated molecular patterns (DAMPs), alarmins, pattern recognition receptors, inflammasome activation, cellular senescence, and pyroptosis in the initiation of sterile inflammatory pathways associated with labor. Key mediators including HMGB1, IL-1α, fetal cell-free DNA, platelet-activating factor, and S100 proteins appear to promote inflammatory activation within fetal membranes and the amniotic cavity. The review also discusses the emerging contribution of fetal immune activation, maternal–fetal immune dysregulation, maternal microchimerism, and epigenetic mechanisms to idiopathic preterm birth. Current diagnostic and therapeutic options remain limited, and no targeted treatment for sterile intrauterine inflammation has yet been established. Future approaches may include precision biomarkers, multiomics-based risk stratification, targeted immunomodulatory therapies, and modulation of maternal–fetal immune interactions. Improved understanding of sterile inflammatory mechanisms may ultimately support development of personalized strategies to prevent preterm birth and improve perinatal outcomes. Full article

Background: Non-invasive prenatal testing (NIPT) examines cell-free DNA (cfDNA) in maternal serum, which includes both maternal DNA and apoptotic placental DNA. The presence of multiple aneuploidies or widespread abnormal patterns of gains and losses across chromosomes in a structurally normal fetus has been linked to maternal cancer. Case Presentation: The patient was a 22-year-old G1P0 with a history of classical Hodgkin’s lymphoma in remission. Her NIPT collected at 14 weeks and 3 days was reported as a “no call”. A second NIPT at a different laboratory showed multiple chromosomal aneuploidies (trisomy 18, 21, and monosomy X) with normal fetal anatomy on ultrasound. The patient was asymptomatic and was referred to hematology–oncology specifically to address the concern that these NIPT results could be related to cancer recurrence. Imaging was deferred as she was already on an established surveillance protocol for her Hodgkin’s lymphoma. At 26 weeks of gestation, the patient presented with a cough and dyspnea. Chest x-ray raised concern for disease recurrence, and biopsy confirmed recurrent Hodgkin’s lymphoma. She received two cycles of ICE chemotherapy. Cesarean delivery at 34 weeks and 2 days was performed for non-reassuring fetal heart tones. She continued chemotherapy, followed by BEAM conditioning and autologous stem cell transplantation. Genetic testing of the neonate revealed a normal karyotype; the placenta karyotype yielded no interpretable results. Discussion and Conclusions: Certain patterns of abnormal NIPT results may be associated with maternal malignancy and warrant further investigation. The absence of standardized protocols for reporting such NIPT results can complicate timely interdisciplinary evaluation and treatment. However, diagnostic testing should be offered with a positive NIPT result, a no-call or test failure, and abnormal ultrasound results, even with a “low-risk” NIPT result. Full article

Feto-maternal microchimerism (Mc) refers to the exchange of cells between the fetus and mother, and fetal–fetal Mc to the exchange between fetuses during pregnancy. This phenomenon occurs across mammalian species, including humans, mice, and cattle. Key data on Mc cells and theoretical considerations regarding the presence of fetal-derived material, such as trophoblast cells, cell-free fetal DNA (cffDNA), and exosomes in maternal blood are summarized. This review aims to first, synthesize current knowledge on feto-maternal and fetal–fetal Mc across mammals, second, address three core questions: how and where Mc has been demonstrated in animals, what techniques have been used over time to detect fetal-derived material and Mc, and how placental structures influence the frequency of Mc. Finally, it aims to identify gaps in the literature for species such as horses, goats, and pigs. This article concludes that Mc is a widespread phenomenon among mammals, but detection methods and reported frequencies vary significantly by species and placental type. A biological model is presented in this article in which multinucleated trophoblast cells undergo apoptosis, releasing cffDNA that enters the maternal blood circulation after multinucleated trophoblast invasion. Advances in molecular biology technology have improved the ability to detect fetal-derived material, cells, DNA, and exosomes in maternal blood. However, notable research gaps remain for Mc in horses, goats, and pigs, highlighting the need for targeted studies to better understand species-specific patterns or a general biological model. Full article

Preeclampsia (PE), pregnancy-associated high blood pressure linked to organ damage, affects 3–8% of all pregnancies and results worldwide in 70,000 maternal and 500,000 perinatal deaths each year. Untreated PE may progress to eclampsia with long-term health implications for both mother and child. Non-invasive prenatal diagnosis or screening applies cell-free DNA approaches and offers a less invasive and more economical method for early diagnosis and prediction of various pregnancy complications. Recently, cell-free assays, particularly blood-based cell-free DNA and RNA analysis, have shown great potential in early PE prediction and diagnosis. Here, we provide an updated review of the current understanding and discoveries of PE, focusing on recent publications (1 January 2019–30 December 2025) of liquid biopsy-derived circulating fetal cells (circulating trophoblasts and fetal nucleated red blood cells), cell-free DNA, cell-free RNA and small extracellular vesicles (i.e., exosomes). We aim to discuss the conceptual framework and technical evolution of liquid biopsy applications in preeclampsia pathogenesis, prediction and diagnosis. Progressing novel screening and diagnostic molecular biomarkers have high potential to facilitate early detection for patients at risk of PE. Liquid biopsy-based screening strategies may aid in providing timely intervention and treatment. Full article

Therapeutic termination of pregnancy (TToP) represents an intervention that is performed for medical reasons, such as risks to maternal health or severe fetal anomalies. Advances in prenatal screening and diagnostic tools—including serum markers, ultrasound, cell-free fetal DNA, chorionic villus sampling and amniocentesis—have significantly improved early detection and clinical decision-making. This narrative review synthesizes current knowledge on the genetic, environmental and psychosocial determinants that influence the decision of the patients to pursue TToP. The literature search was performed primarily using PubMed database, while Scopus and Google Scholar were used to identify additional relevant studies. Some of the selected studies, as well as certain sections of this review, address both therapeutic and voluntary termination of pregnancy, whereas others focus exclusively on TToP. Moreover, this review describes the types of abortion (medical or surgical/aspiration) along with their management strategies to prevent or address potential complications. It is well known that demographic, cultural and socio-economic factors continue to influence the access to TToP, as well as the perceptions of it. Psychiatric comorbidities (such as anxiety, affective and psychotic disorders) are observed with a higher prevalence among women undergoing TToP and may influence both the decision and psychological outcomes post-procedure. While most women report emotional relief after TToP, some of them experience depression, post-traumatic stress disorder or substance misuse. Legal and ethical considerations further complicate access to safe abortion, leading to situations where patients may resort to unsafe procedures, which result in higher rates of morbidity and mortality. Data from the EUROCAT network show rising trends in congenital anomalies like trisomy 13, trisomy 18 and caudal regression syndrome (conditions commonly associated with TToP). Therefore, it is mandatory to form a multidisciplinary team in these cases, integrating medical, psychological and ethical dimensions. Ensuring safe, evidence-based and compassionate access to TToP remains a critical component of reproductive healthcare. Full article

Background: Maternal serum screening remains widely implemented for prenatal aneuploidy assessment despite increased uptake of cell-free DNA testing. Evidence suggests that fetal sex may influence placental endocrine function and maternal serum biomarker levels; however, available studies are inconsistent and often limited by sample size or incomplete adjustment for maternal factors. Objective: The aim of this study was to determine whether fetal sex independently modifies first- and second-trimester maternal serum marker Multiple of the Median (MoM) values and whether sex-related biochemical variation affects trisomy-21 screen-positive classification. Methods: A retrospective cohort was identified from institutional screening records (10,384 screened pregnancies), of which 5040 first-trimester and 1476 second-trimester cases had complete biochemical data. First-trimester PAPP-A and free β-hCG, as well as second-trimester AFP, uE3, and free β-hCG, were measured. Implausible MoM values (<0.10 or >5.00) were excluded. Multivariable linear and logistic regression models adjusted for maternal age, maternal weight, gestational age at sampling, and parity assessed independent associations. Results: Pregnancies with female fetuses showed significantly higher MoM values for first-trimester PAPP-A and free β-hCG as well as second-trimester AFP and uE3. The magnitude of these differences was small, and no significant differences were observed in trisomy-21 screen-positive rates between fetal sex groups. Conclusions: Fetal sex independently influences several maternal serum markers across both trimesters but does not result in clinically meaningful differences in trisomy-21 screening outcomes under current algorithms. Any potential relevance of fetal sex for risk interpretation should be regarded as hypothesis-generating and requires outcome-validated investigation before clinical application. Full article

Pregnancies complicated by diabetes, including pregestational and gestational diabetes mellitus, are associated with increased maternal and fetal morbidity. Early identification of at-risk pregnancies is crucial for timely intervention and improved outcomes. Emerging evidence highlights the interplay of genetic predisposition, epigenetic modifications, and non-invasive biomarkers in the early detection of diabetic pregnancies. Genetic factors influencing insulin signaling, glucose metabolism, and pancreatic β-cell function may contribute to susceptibility to gestational hyperglycemia. Concurrently, epigenetic alterations, such as DNA methylation and histone modifications in maternal and placental tissues, have been linked to dysregulated metabolic pathways and adverse pregnancy outcomes. Non-invasive biomarkers, including circulating cell-free DNA and microRNAs in maternal blood, show promise for early diagnosis by offering a safer and more practical alternative to invasive testing. Integrating genetic, epigenetic, and molecular marker data could enhance risk stratification and enable personalized monitoring and management strategies. This review synthesizes current knowledge on the molecular underpinnings of diabetic pregnancies, evaluates the potential of emerging biomarkers for early diagnosis, and discusses the challenges and future perspectives for translating these findings into clinical practice. Understanding these mechanisms may pave the way for precision medicine approaches, ultimately improving maternal and neonatal outcomes in pregnancies affected by diabetes. Full article

Objective: To evaluate the association between low and high fetal fraction (FF) of cell-free fetal DNA on non-invasive prenatal screening (NIPS) and placental pathology. Methods: We undertook a prospective cohort study of patients undergoing NIPS between July 2022 and July 2023 through Natera Inc. Based on the FF percentile, the study cohort was divided into three groups: high FF (≥95th%), low FF (≤5th%), and a control group (FF 6th–94th%). Our primary study outcome was a composite of high-risk placental lesions. We compared the occurrence of the primary study outcome across the study groups using the chi² test. Multivariable regression analyses were performed to predict the likelihood of the primary outcome based on the FF percentile. Selected obstetric and neonatal outcomes were assessed as secondary outcomes. Results: The primary outcome was present in 11 (50.0%), 19 (48.7%), and 11 (35.5%) of participants in the low FF, high FF, and control cohorts, respectively (p = 0.46). In an adjusted model, the FF percentile was not associated with the primary outcome (aOR 2.41 (0.72–8.42) for low FF, aOR 1.55 (0.51–4.82) for high FF). Chorangiosis (p = 0.02) and fetal inflammatory response (p = 0.002) were seen more commonly in the low and high FF groups. Spontaneous preterm birth was more common in the low FF group (p = 0.04). Conclusions: Our study did not identify a correlation between high-risk histopathological patterns and extremely low or high FF when compared to a control cohort. Chorangiosis and fetal inflammatory response were found more commonly in the low and high FF groups. Full article

Background: Non-invasive prenatal testing (NIPT) based on cell-free fetal DNA (cfDNA) in maternal blood has revolutionized prenatal screening for trisomies 21, 18, and 13. This approach, based on next-generation sequencing (NGS), usually allows the detection of other chromosomal abnormalities; however, their clinical value in routine practice requires further evidence. Objectives: This study aimed to assess the experience and clinical utility of genome-wide NIPT in pregnant women at intermediate risk in the autonomous communities of Aragón and Valencia, Spain. Methods: For this purpose, a retrospective cohort study was conducted between 2020 and 2024 across two public hospitals. Pregnant women at intermediate risk for trisomies 21, 18, or 13, were included, as well as those meeting specific clinical criteria. Participants were offered either basic or expanded NIPT, and positive results were confirmed by invasive prenatal testing or placental analysis. Results: Among 9,059 expanded NIPT tests, 132 (1.45%) indicated a high-risk result for less common chromosomal anomalies, comprising 60 rare autosomal aneuploidies (RAAs), 39 copy number variants (CNVs), 23 sex chromosome aneuploidies (SCAs), and 10 multiple abnormalities. The positive predictive value (PPV) was 5.5% for RAAs in the fetus, 12.8% for CNVs (31% for deletions), and 58% for SCAs. Conclusions: Several confirmed anomalies were clinically significant and would not have been detected through conventional screening. Opportunistic use of expanded NIPT enables the detection of additional clinically relevant abnormalities, potentially improving obstetric management without substantially increasing invasive testing. Full article

Advancements in genomic technologies have transformed prenatal genetic testing, offering more accurate, comprehensive, and noninvasive approaches to reproductive care. This review provides an in-depth overview of current methodologies and emerging innovations, including expanded carrier screening (ECS), cell-free DNA (cfDNA) testing, chromosomal microarray analysis (CMA), and sequencing-based diagnostics. We highlight how next-generation sequencing (NGS) technologies have revolutionized carrier screening and fetal genome analysis, enabling detection of a broad spectrum of genetic conditions. The clinical implementation of cfDNA has expanded from common aneuploidies to include copy number variants (CNVs), and single-gene disorders. Diagnostic testing has similarly evolved, with genome sequencing outperforming traditional CMA and exome sequencing through its ability to detect both sequence and structural variants in a single assay. Emerging tools such as optical genome mapping, RNA sequencing, and long-read sequencing further enhance diagnostic yield and variant interpretation. This review summarizes major technological advancements, assesses their clinical utility and limitations, and outlines future directions in prenatal genomics. Full article

Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results. Full article