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The articles in this issue cover a wide range of topics, including the role of the maternal microbiome in fetal growth restriction, the effectiveness of therapy for mixed vaginitis, management strategies for pregnant women with COVID-19, the impact of environmental factors on reproductive health, thrombotic complications of chemotherapy, the connection between the microbiome and obesity, as well as rare clinical cases. Innovations in diagnostics, treatment, and screening are explored, from non-invasive prenatal testing to mathematical models for studying autism. The articles emphasize the importance of a comprehensive approach and the integration of modern technologies.

Background: This prospective study was done to evaluate the benefit of Real-time Polymerase Chain Reaction in the determination of RHD and SRY of the fetuses in the plasma of Rhesus D-negative enrolled ladies. Methods: Thirty-nine pregnant Rh-negative females with (RhD+) partners were registered in this study. Blood samples were taken from those participants whose pregnancy age was from 20 -28 weeks for the purposes of fetal RHD genotyping and DNA extraction/purification. Realtime PCR was used to determine the RH and sex genotype using particular RHD, SRY, and GLO gene primers and probes. Results: Out of the total 39 samples, 28/39(71.8 %) were positive for Rhesus D antigen and 11/39 (28.2 %) were negative. The results of prenatal fetal RHD genotyping based on exons 7, and 10 combined were concordant with postnatal RhD phenotyping in 94.9% of cases (37/39) with a sensitivity & specificity of 93.3 and 100% respectively, and precision was 100%. For the gene SYR, there was a concordance rate of 92.3% between the fetal sex genotype and the newborn sex with one false-positive (2.6%) and two false negatives (5.1%) results leading to a precision of 95.2%. Conclusion: The study demonstrated great accuracy of the Real-Time PCR technique through the usage of cffDNA for the genetic study of the fetal RHD and SYR genes and implicates the effectiveness of this procedure for predicting the necessity of Anti-D immunoprophylaxis in pregnant women whose fetuses have a peril of hemolytic anemia of the newborn due to RhD incompatibility between partners. [ABSTRACT FROM AUTHOR]

BackgroundMutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus.MethodsA small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma.ResultsHere we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these.ConclusionOur NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell‐free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD‐M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity‐by‐descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity‐by‐descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD‐M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach. [ABSTRACT FROM AUTHOR]

Cell-free fetal DNA (cffDNA) analysis is a non-invasive prenatal diagnostic test with a fundamental role for the screening of chromosomic or monogenic pathologies of the fetus. Its administration is performed by fetal DNA detection in the mother's blood from the fourth week of gestation. Given the great interest regarding its validation as a diagnostic tool, the authors have set out to undertake a critical appraisal based on a wide-ranging narrative review of 45 total studies centered around such techniques. Both chromosomopathies and monogenic diseases were taken into account and systematically discussed and elucidated. Not surprisingly, cell-free fetal DNA analysis for screening purposes is already rather well-established. At the same time, considerable interest in its diagnostic value has emerged from this literature review, which recommends the elaboration of appropriate validation studies, as well as a broad discourse, involving all stakeholders, to address the legal and ethical complexities that such techniques entail. [ABSTRACT FROM AUTHOR]

ABSTRACT Hyperglycemia caused by mutations in the glucokinase gene, GCK, is the most common form of monogenic diabetes. Prenatal diagnosis is important, as it impacts on treatment. This study reports a monogenic non‐invasive prenatal diagnostic (NIPD‐M) test on cell‐free DNA in maternal plasma using the relative haplotype dosage. In three pregnancies of two families with known maternal GCK mutations, the fetal genotype was determined unambiguously already at 12 weeks of gestation. In summary, proof is provided of the feasibility for NIPD‐M in GCK diabetes.

Background: In pregnant women at risk of autosomal recessive (AR) disorders, prenatal diagnosis of AR disorders primarily involves invasive procedures, such as chorionic villus sampling and amniocentesis. Methods: We collected blood samples from four pregnant women in their first trimester who presented a risk of having a child with an AR disorder. Cell-free DNA (cfDNA) was extracted, amplified, and double-purified to reduce maternal DNA interference. Additionally, whole-genome amplification was performed for traces of residual purified cfDNA for utilization in subsequent applications. Results: Based on our findings, we detected the fetal status with the family corresponding different genes, i.e., LZTR1, DVL2, HBB, RNASEH2B, and MYO7A, as homozygous affected, wild-type, and heterozygous carriers, respectively. Results were subsequently confirmed by prenatal amniocentesis. The results of AmpFLSTR™ Identifiler™ presented a distinct profile from the corresponding mother profile, thereby corroborating the result reflecting the genetic material of the fetus. Conclusion: Herein, we detected AR disease mutations in the first trimester of pregnancy while surmounting limitations associated with maternal genetic material interference. Importantly, such detection strategies would allow the screening of pregnant women for common AR diseases, especially in highly consanguineous marriage populations. This technique would open avenues for the early detection and prevention of recessive diseases among the population. [ABSTRACT FROM AUTHOR]

Background: In pregnant women at risk of autosomal recessive (AR) disorders, prenatal diagnosis of AR disorders primarily involves invasive procedures, such as chorionic villus sampling and amniocentesis.Methods: We collected blood samples from four pregnant women in their first trimester who presented a risk of having a child with an AR disorder. Cell-free DNA (cfDNA) was extracted, amplified, and double-purified to reduce maternal DNA interference. Additionally, whole-genome amplification was performed for traces of residual purified cfDNA for utilization in subsequent applications.Results: Based on our findings, we detected the fetal status with the family corresponding different genes, i.e., LZTR1, DVL2, HBB, RNASEH2B, and MYO7A, as homozygous affected, wild-type, and heterozygous carriers, respectively. Results were subsequently confirmed by prenatal amniocentesis. The results of AmpFLSTR™ Identifiler™ presented a distinct profile from the corresponding mother profile, thereby corroborating the result reflecting the genetic material of the fetus.Conclusion: Herein, we detected AR disease mutations in the first trimester of pregnancy while surmounting limitations associated with maternal genetic material interference. Importantly, such detection strategies would allow the screening of pregnant women for common AR diseases, especially in highly consanguineous marriage populations. This technique would open avenues for the early detection and prevention of recessive diseases among the population.

Background and Objectives: The study aimed to evaluate the ability defining the risk of developing preeclampsia by a screening test carried out in the first trimester (between 11 + 0 and 13 + 6 weeks of gestational age), in order to identify high-risk women requiring more intensive health surveillance. The secondary objective was to evaluate the ability of this test to predict the risk of adverse obstetric outcomes such as fetal growth restriction, intrauterine fetal death, gestational hypertension, HELLP syndrome, placental abruption, and preterm birth. Materials and Methods: This was a single-center study, conducted at the Operative Unit of Obstetrics of the State Hospital of the Republic of San Marino. Medical history was collected at the time of enrolment in writing. Subsequently, obstetric outcomes were collected for each enrolled woman, through the analysis of medical records. Results: From October 2014 to May 2019, 589 pregnant women were recruited, of whom, 474 (80.5%) were included in the "low-risk" group, and 115 (19.5%) in the "high-risk" group. At the time of analysis of this population, the obstetric outcomes were available for 498 women (84.5%), while 91 cases (15.5%) were current pregnancies. The PI of the uterine arteries was not significantly different between the two study groups. Otherwise, a significant difference was highlighted for MAP, which is higher in the case of pregnancies at high risk based on the risk factors only, and for PAPP-A, higher in the case of low-risk pregnancies. Regarding the percentage of fetal DNA, according to the most recent literature data, in our series, we report a statistically significant difference of the average between the low and high-risk groups. Conclusions: In our study, we demonstrate that the multiparametric screening test for early PE performed well in identifying women at high risk of early PE, which certainly has the most severe maternal–fetal outcomes. The data reported that ASA intake at low doses is significantly higher in the population with high-risk tests for both early PE and late PE suggest once again that anamnestic evaluation plays an essential role in women's screening. [ABSTRACT FROM AUTHOR]

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted. [ABSTRACT FROM AUTHOR]

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

Hyperglycemia caused by mutations in the glucokinase gene, GCK, is the most common form of monogenic diabetes. Prenatal diagnosis is important, as it impacts on treatment. This study reports a monogenic non‐invasive prenatal diagnostic (NIPD‐M) test on cell‐free DNA in maternal plasma using the relative haplotype dosage. In three pregnancies of two families with known maternal GCK mutations, the fetal genotype was determined unambiguously already at 12 weeks of gestation. In summary, proof is provided of the feasibility for NIPD‐M in GCK diabetes. [ABSTRACT FROM AUTHOR]

Background: Non-invasive prenatal testing (NIPT) for aneuploidy in pregnant women screening has been recently established in Saudi Arabia. We aim from this study to report our experience in the implementation of this new technology in clinical practice and to assess factors influencing cell-free fetal (cffDNA) fraction and successful NIPT reporting.Methods: In total, 200 pregnant women were subjected to the NIPT test using standard methods. Next-generation sequencing (NGS) was used to analyze cffDNA in maternal plasma.Results: Out of the 200 NIPT cases, the average age of pregnant women was 35 ± 6 years (range: 21–48 years). The average cffDNA fraction of reported cases was 13.72% (range: 3–31%). Out of these 200 cases, 187 (93.5%) were at low risk, while 13 (6.5%) cases revealed high risk for aneuploidy. Among these chromosomal abnormalities, 7 (3.5%) cases of Down’s syndrome, 5 (2.5%) Edwards’ Syndrome, and only 1 case of (0.5%) Patau’s syndrome was observed. Out of the 13 high-risk cases, 2 (15.3%) were found in women below the age of 30.Conclusion: This is the first study reporting the successful implementation of an in-house NIPT screening service in Saudi Arabia. Our data showed high accuracy and sensitivity to detect high-risk cases indicating the usefulness of such a technique as an alternative to invasive testing and (hopefully) will change the common screening practice for pregnant women in Saudi Arabia.

Background: Non-invasive prenatal testing (NIPT) for aneuploidy in pregnant women screening has been recently established in Saudi Arabia. We aim from this study to report our experience in the implementation of this new technology in clinical practice and to assess factors influencing cell-free fetal (cffDNA) fraction and successful NIPT reporting. Methods: In total, 200 pregnant women were subjected to the NIPT test using standard methods. Next-generation sequencing (NGS) was used to analyze cffDNA in maternal plasma. Results: Out of the 200 NIPT cases, the average age of pregnant women was 35 ± 6 years (range: 21–48 years). The average cffDNA fraction of reported cases was 13.72% (range: 3–31%). Out of these 200 cases, 187 (93.5%) were at low risk, while 13 (6.5%) cases revealed high risk for aneuploidy. Among these chromosomal abnormalities, 7 (3.5%) cases of Down's syndrome, 5 (2.5%) Edwards' Syndrome, and only 1 case of (0.5%) Patau's syndrome was observed. Out of the 13 high-risk cases, 2 (15.3%) were found in women below the age of 30. Conclusion: This is the first study reporting the successful implementation of an in-house NIPT screening service in Saudi Arabia. Our data showed high accuracy and sensitivity to detect high-risk cases indicating the usefulness of such a technique as an alternative to invasive testing and (hopefully) will change the common screening practice for pregnant women in Saudi Arabia. [ABSTRACT FROM AUTHOR]

Background and objective: Optimal sample storage conditions are essential for non‐invasive prenatal testing of cell‐free fetal and total DNA. We investigated the effect of long‐term storage of plasma samples and extracted cfDNA using qPCR. Materials and methods: Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0–6 years at −25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18 months and 3 years of storage at −25°C. Results: We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2‐3 years, and fetal fraction was slightly decreased after 3 years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2 years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA. Conclusion: Fetal and total cell‐free DNA is readily detectable in plasma after long‐term storage at −25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes. [ABSTRACT FROM AUTHOR]

Chromosomal abnormalities effect 0.4 % of pregnancies. 50 % of these abnormalities are Trisomy 21, 15% Trisomy 18, 5% Trisomy 13 respectively. The major purpose of using prenatal screening tests is; to determine the pregnants at high risk for chromosomal aneuploidy at early gestational weeks and to give information about the risks associated with each pregnant woman. In recent years, prenatal screening tests are divided into two as traditional prenatal screening tests and cell free fetal DNA (cffDNA, fetal DNA, NIPT). The traditional prenatal screening tests which use certain maternal serum biomarkers that is combined with ultrasonographic findings are used as first line screening test. From the beginning of 10th week of pregnancy, fetal DNA fragments in maternal blood are analyzed using new generation sequencing techniques. In which population fetal DNA would be recommended, its usefulness in clinical use, and its cost effectivity, limitations and advantages are still controversial. [ABSTRACT FROM AUTHOR]

Background The performance of noninvasive prenatal testing (NIPT) assays is critically determined by the proportion of fetal DNA or fetal fraction (FF). Fetomaternal differential methylation of certain genomic regions has been proposed as a universal marker of fetal origin, and previous reports have suggested the use of methylation-sensitive restriction enzyme (MSRE) assays to estimate FF. Methods We analyzed the performance of FF estimation using an MSRE assay with duplex quantitative polymerase chain reaction (qPCR). Mixtures of genomic DNA from placental cells and from adult women were digested with 2 MSRE and FF estimates obtained, for a total of 221 pairwise treatment/control comparisons. Results The coefficient of variance (CV) of the MSRE assays was high, ranging from 24% to 60%. An alternative in silico FF estimation algorithm, SeqFF, displayed slightly lower variability, with a CV of 22%. Conclusion These results cast doubts on the usefulness of the MSRE-based assay of differentially methylated markers for FF estimation. The lack of a universal method capable of precisely estimating FF remains an incompletely solved issue. [ABSTRACT FROM AUTHOR]

The object of that investigation was detect the sex of fetuses in Arabian pregnant she camel plasma, through identifying SRY gene in the 10th to 15th weeks of pregnancy in the cell-free fetal DNA (cffDNA) by using the polymerase chain reaction (PCR). Blood samples were collected from 34 Arabian pregnant she camels. The positive PCR product bands that obtained 20/34 (58.82%) samples were positive ranges for the SRY and GABDH genes, which indicate male sex, and 14/34 samples (41.18 %) were positive ranges for only GABDH gene, which clearly showed female sex. The findings show that the sex of the fetus can be identified at a very early stage of gestation in Arabian she camel with high accuracy investigated were congruent with the actual gender of the offspring without need to re-examine again, as well as showed that fetal DNA can be reliably identified in pregnant female blood and can be used to identify certain genetic disorders. [ABSTRACT FROM AUTHOR]

Abstract Genetic disease can occur due to imbalance of whole chromosomes, smaller chromosome microdeletions or duplications, or at the single-gene level where even a single base change can cause significant disease. This review focuses on the methods available to achieve genetic diagnosis of a fetus in pregnancy, both in the context of a family history of a known disease-causing mutation and where there is clinical suspicion of a genetic disorder based on ultrasound findings. Until recently, genetic testing of a fetus invariably required invasive procedures to sample fetal tissue, with associated risk of miscarriage. However, non-invasive methods of achieving prenatal diagnosis by sampling fetal DNA present in maternal blood have undergone considerable development. Current applications and future utility of these techniques are discussed. [ABSTRACT FROM AUTHOR]

Objectives Non-invasive prenatal aneuploidy testing (NIPT) by next-generation sequencing of circulating cell-free DNA in maternal plasma relies on chromosomal ratio (chr ratio ) measurements to detect aneuploid values that depart from euploid ratios. Diagnostic performances are known to depend on the fraction of fetal DNA ( FF ) present in maternal plasma, although how this translates into specific quantitative changes in specificity/positive predictive values and which other variables might also be important is not well understood. Design & methods To explore this issue, theoretical relationships between FF and various measures of diagnostic performances were assessed for a range of parameter values. Empirical data from three NIPT assays were then used to validate theoretical calculations. Results For a given positivity threshold, dramatic changes in specificity and positive predictive values (PPV) as a function of both FF and the coefficient of variation (CV) of the chr ratio measurement were observed. Theoretically predicted and observed chr ratio z-scores agreed closely, confirming the determinant impact of small changes in both FF and chr ratio CV. Conclusions Evaluation of NIPT assay performances therefore requires knowledge of the FF distribution in the population in which the test is intended to be used and, in particular, of the precise value of the assay chr ratio CV for each chromosome or genomic region of interest. Laboratories offering NIPT testing should carefully measure these parameters to ensure test reliability and clinical usefulness in interpreting individual patients' results. [ABSTRACT FROM AUTHOR]

Abstract: Aim: Detection of cell‐free fetal DNA in maternal blood is a type of noninvasive prenatal diagnosis test (NIPT), which has already been known for some time but has not yet been introduced in most of public hospitals in Spain. How the implementation of cell‐free fetal DNA (cffDNA) in a contingent protocol has influenced the aneuploidy screening in our hospital is described. Methods: Two cohorts of patients with positive combined screening were compared: the first one (years 2012–2013, 5747 patients) from a period of time in which the protocol valid until March 2016 – that included the use of invasive procedures – was applied; and the second one in which the current protocol – that included NIPT versus invasive procedures – was applied (first 7 months after protocol implementation, 898 patients). Results: Comparison of both periods resulted in a 60.5% reduction of invasive procedures (P < 0,001) preserving the same chromosomopathy detection rate. The ratio of positive invasive procedures–indicated invasive procedures was improved by 15% in the first period to 50% in the second period (P = 0.01). Conclusion: NIPT introduction has caused a significant reduction of 60.5% of IP in high chromosomopathy risk patients after combined screening without modifying detection rate. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the effectiveness of placental-specific markers, extracellular fetal DNA (sexdetermining region Y and hypermethylated RASSF1A sequences) and circulating C19MC microRNAs (miR-516-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, miR-525, and miR-526a) for the diagnosis and consecutive follow-up of gestational trophoblastic disease/neoplasia. Increased levels of extracellular fetal DNA and C19MC microRNAs were detected in patients with active disease when compared with the period when the patients reached remission of the disease. The positive correlation between plasma levels of hypermethylated RASSF1A sequence, C19MC microRNAs, and human chorionic gonadotropin serum levels was found. MiR-520a-5p had the best performance to detect patients with active disease (a positive predictive value of 100% at a null false positive ratio (FPR)). MiR-516-5p and miR-525 were able to diagnose 100% of women with active disease at the FPR 3.9%/7.7%. The overall predictive capacity of single miR-526a (81.8% at null FPR), miR-517-5p (90.9% at 15.4% FPR), miR-518b (100% at 38.5% FPR), and miR-520h (90.9% at 26.9% FPR) biomarkers to detect active disease cases was slightly lower. Transient increase in C19MC microRNA plasma levels after the first cycle of chemotherapy indicated the decay of placental trophoblast residual tissue. The increased levels of extracellular fetal DNA and placental-specific C19MC microRNAs are associated with gestational trophoblastic disease/neoplasia. Screening of extracellular placental-specific biomarkers may represent an additional option to identify a significant proportion of women with active disease and to monitor the therapy response. Non-invasive follow-up of the decomposing residual tissue in the form of extracellular nucleic acids of placental origin packed into apoptotic bodies derived from placental trophoblasts is available. [ABSTRACT FROM AUTHOR]

Fetal development can be altered by DNA damage caused by maternal exposure to chemical, physical, or biological agents during gestation. One method of assessing genotoxicity is to detect micronuclei (MNs) and/or nuclear abnormalities. This can be performed in vivo and requires only frequently dividing tissues, such as amniotic tissue (AT), which is in contact with the fetal environment and is composed of very thin layers of cells. This study evaluated the presence of MNs, nucleoplasmic bridges, and nuclear buds (NBs) in the fetal AT following maternal exposure to cyclophosphamide (CP) during pregnancy. Pregnant Wistar rats were divided into a negative control group and an experimental group that was orally administered CP (10 mg/kg). Daily blood smears were obtained from pregnant rats on days 14–19 of gestation. The rats were dissected, and fetal ATs were obtained on the 19th day of gestation. The MN and NB frequencies in AT cells were analyzed using a fluorescence microscope (100 ×). Micronucleated erythrocytes in the peripheral blood of the control rats were also assessed. Micronucleated polychromatic erythrocyte frequencies were significantly higher than those in the controls. Polychromatic erythrocyte frequencies were lower in CP-treated rats than in controls at 48–120 h. Fetuses in the CP-treated group also showed a significant increase in MNs and NBs in AT cells. In conclusion, AT could be used for analyzing MNs and NBs in rats following maternal exposure to a genotoxic agent and as a viable alternative for analyzing the integrity of fetal DNA during gestation. • Rat amniotic tissue is useful for genotoxicity assays. • Nuclear abnormalities were identified in rat amniotic tissue. • Cyclophosphamide induces genotoxicity in rat amniotic tissue. • Amniotic tissue analysis allows evaluation of the integrity of fetal DNA. [ABSTRACT FROM AUTHOR]

Since the discovery of the presence of fetal DNA in maternal blood, non-invasive fetal sex determination has been the test most widely translated into clinical practice. To date there is no agreement between the different laboratories performing such tests in relation to which is the best protocol. As a consequence there are almost as many protocols as laboratories offering the service, using different methodologies and thus obtaining different diagnostic accuracies. By the end of 2007, after a validation study performed in 316 maternal samples collected between the 5th and 12th week of gestation, the fetal sex determination was incorporated into clinical practice in our Service. The test is performed in the first trimester of pregnancy, and it is offered as part of the genetic counseling process for couples at risk of X-linked disorders. As a general rule and in order to avoid misdiagnosis, two samples at different gestational ages are tested per patient. The analysis is performed by the study of the SRY gene by RT-PCR. Two hundred and twenty six pregnancies have been tested so far in these 5 years. Neither false positives nor false negatives diagnoses have been registered, thus giving a diagnostic accuracy of 100%.

Background: Chorionic villus sampling and amniocentesis as definitive diagnostic procedures represent a gold standard for prenatal diagnosis of chromosomal abnormalities. The methods are invasive and lead to a miscarriage and fetal loss in approximately 0.5–1 %. Non-invasive prenatal DNA testing (NIPT) is based on the analysis of cell-free fetal DNA from maternal blood. It represents a highly accurate screening test for detecting the most common fetal chromosomal abnormalities. In our study we present the results of NIPT testing in the Diagnostic Center Strah, Slovenia, over the last 3 years.Methods: In our study, 123 pregnant women from 11th to 18th week of pregnancy were included. All of them had First trimester assessment of risk for trisomy 21, done before NIPT testing.Results: 5 of total 6 high-risk NIPT cases (including 3 cases of Down syndrome and 2 cases of Klinefelter’s syndrome) were confirmed by fetal karyotyping. One case–Edwards syndrome was false positive. Patau syndrome, triple X syndrome or Turner syndrome were not observed in any of the cases. Furthermore, there were no false negative cases reported. In general, NIPT testing had 100 % sensitivity (95 % confidence interval: 46.29 %–100.00 %) and 98.95 % specificity (95 % confidence interval: 93.44 %–99.95 %). In determining Down syndrome alone, specificity (95 % confidence interval: 95.25 %- 100.00 %) and sensitivity (95 % confidence interval: 31.00 %–100.00 %) turned out to be 100 %. In 2015, the average turnaround time for analysis was 8.3 days from the day when the sample was taken. Repeated blood sampling was required in 2 cases (redraw rate = 1.6 %).Conclusions: Our results confirm that NIPT represents a fast, safe and highly accurate advanced screening test for most common chromosomal abnormalities. In current clinical practice, NIPT would significantly decrease the number of unnecessary invasive procedures and the rate of fetal loss caused by invasive diagnostics.

Nowadays we are witnessing the rapid penetration of non-invasive prenatal diagnostics into routine practice. In the past two years many types of cfDNA tests both commercial kits and „homemade“ tests have appeared on the market. The aim of our contribution is to bring your attention to the significant differences in quality of available non-invasive tests (NIPT) from molecular and clinical perspective. [ABSTRACT FROM AUTHOR]

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher ( P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 104 copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model. [ABSTRACT FROM AUTHOR]

Circulating fetal DNA in maternal plasma can be used for both fetal genetic testing and evaluation of complications of pregnancy. As a prenatal genetic test, the greatest advantage of using circulating fetal DNA is the lack of risk. This approach has been applied to the diagnosis of fetal gender, fetal Rhesus D (RhD) blood type, and fetal single-gene disorders. However, it only allows examination of disorders where the gene of interest is present in the fetal genome but absent from maternal DNA. Since most fetal DNA probably originates from villous trophoblasts, concentrations can be used to evaluate damage to trophoblasts, particularly in pathologic complications of pregnancy such as pre-eclampsia, invasive placenta, hyperemesis gravidarum, and trisomy 21. Fetal DNA in the plasma of pregnant women thus offers a new source of data that can be used in various clinical settings.

In this paper, we review the major highlights in the development of clinical noninvasive prenatal diagnostic approaches analyzing fetal genetic material recovered from maternal circulation.

Clinical observations and animal experiments have shown that higher fetal DNA in maternal plasma could participate on the pathogenesis of preeclampsia. The understudied factor that could participate in interindividual variability in cell-free DNA is enzymatic activity of deoxyribonuclease (DNase). We have found that healthy pregnant animals have higher plasma DNase activity than healthy pregnant women. Injection of cell-free fetal DNA into pregnant animals had no effect on DNase activity. Interspecies differences in DNase activity should be considered in animal experiments focusing on the role of fetal DNA in preeclampsia and cell-free DNA in other disease models. [ABSTRACT FROM AUTHOR]