Female-bearing Pregnancies Research Articles

Placental Inflammasome mRNA Levels Differ by Mode of Delivery and Fetal Sex.

Parturition signals the end of immune tolerance in pregnancy. Term labour is usually a sterile inflammatory process triggered by damage associated molecular patterns (DAMPs) as a consequence of functional progesterone withdrawal. Activation of DAMPs recruits leukocytes and inflammatory cytokine responses in the myometrium, decidua, cervix and fetal membranes. Emerging evidence shows components of the inflammasome are detectable in both maternal decidua and placenta. However, the activation of the placental inflammasome with respect to mode of delivery has not been profiled. Placental chorionic villus samples from women delivering at term via unassisted vaginal (UV) birth, labouring lower segment caesarean section (LLSCS, emergency caesarean section) and prelabour lower segment caesarean section (PLSCS, elective caesarean section) underwent high throughput RNA sequencing (NextSeq Illumina) and bioinformatic analyses to identify differentially expressed inflammatory (DE) genes. DE genes (IL1RL1, STAT1, STAT2, IL2RB, IL17RE, IL18BP, TNFAIP2, TNFSF10 and TNFRSF8), as well as common inflammasome genes (IL1B, IL1R1, IL1R2, IL6, IL18, IL18R1, IL18R1, IL10, and IL33), were targets for further qPCR analyses and Western blotting to quantify protein expression. There was no specific sensor molecule-activated inflammasome which dominated expression when stratified by mode of delivery, implying that multiple inflammasomes may function synergistically during parturition. Whilst placentae from women who had UV births overall expressed pro-inflammatory mediators, placentae from LLSCS births demonstrated a much greater pro-inflammatory response, with additional interplay of pro- and anti-inflammatory mediators. As expected, inflammasome activation was very low in placentae from women who had PLSCS births. Sex-specific differences were also detected. Placentae from male-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with PLSCS, and placentae from female-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with UV. In conclusion, placental inflammasome activation differs with respect to mode of delivery and neonatal sex. Its assessment may identify babies who have been exposed to aberrant inflammation at birth that may compromise their development and long-term health and wellbeing.

Circulating Total Cell-Free DNA Levels Are Increased in Hypertensive Disorders of Pregnancy and Associated with Prohypertensive Factors and Adverse Clinical Outcomes.

Previous studies have described increased circulating cell-free DNA (cfDNA) in hypertensive disorders of pregnancy (HDP). Here, we aimed first to confirm this information using a simple, but sensible fluorescent assay, and second to investigate whether total cfDNA is associated with circulating factors known to be linked to the pathophysiology of HDP as well as with poor maternal-fetal outcomes. We studied 98 women with healthy pregnancies (HP), 88 with gestational hypertension (GH), and 91 with preeclampsia (PE). Total DNA was extracted from plasma using the QIAamp DNA blood mini kit and quantified using Quant-iT™ PicoGreen® dsDNA fluorescent detection kit. We found higher total cfDNA levels in GH and PE (197.0 and 174.2 ng/mL, respectively) than in HP (140.5 ng/mL; both p < 0.0001). Interestingly, total cfDNA levels were elevated in both male and female-bearing pregnancies diagnosed with either HDP, and in more severe versus less severe HDP cases, as classified according to responsiveness to antihypertensive therapy. In addition, total cfDNA was independently associated with HDP, and a cutoff concentration of 160 ng/mL provided appropriate sensitivity and specificity values for diagnosing GH and PE compared to HP (70–85%, both p < 0.0001). Moreover, high total cfDNA was associated with adverse clinical outcomes (high blood pressure, low platelet count, preterm delivery, fetal growth restriction) and high prohypertensive factors (sFLT-1, sEndoglin, MMP-2). These findings represent a step towards to the establishment of cfDNA as a diagnostic tool and the need to understand its role in HDP.

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

The Effects of Fetal Gender on Serum Human Chorionic Gonadotropin and Testosterone in Normotensive and Preeclamptic Pregnancies

Introduction. The aim of the present study was to evaluate the effects of fetal sex on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. Materials and Methods. This is a cross-sectional study and 139 women with singleton pregnancies in the third trimester were studied. Seventy-one pregnancies were uncomplicated; among those were 35 male and 36 female fetuses. Sixty-eight pregnancies were complicated by preeclampsia; among those were 35 male and 33 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. Data analyzed by SPSS software. Results. In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001 and P < 0.001, resp.) in female-bearing pregnancies testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001). Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in mlae-bearing than in female-bearing pregnancies. Conclusion. According to our results, there is a correlation between maternal serum hCG and testosterone levels and preeclampsia. Therefore these tests can be used as routine during 30–38 weeks of gestation. High maternal serum concentrations of these markers can predict preeclampsia.

Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study

PurposeTo prospectively validate a protocol for noninvasive fetal sex determination in maternal plasma and demonstrate its applicability to clinical practice. MethodsPeripheral blood from 404 pregnant women undergoing prenatal invasive testing was collected from 6 to 23 weeks of gestation. Real-time PCR was performed for the SRY gene and multicopy DYS14 marker sequence located within the TSPY gene by the TaqMan minor groove binder probe assay as a first-line test. Owing to a false-positive result, amplification of repetitive motifs of the DAZ gene region was also tested as a second-line test performed in the last 232 patients enrolled in our series. A diagnostic algorithm was designed using a combination of these three markers. Fetal gender determined by noninvasive prenatal diagnosis (NIPD) was compared with that diagnosed by quantitative fluorescent PCR after invasive testing or ultrasound. ResultsA single false-positive result was obtained in the first 172 pregnancies. Reporting criteria were modified in the subsequent 232 pregnancies, giving an overall sensitivity and specificity of 100% (95% CI 99.8–100%) and 99.5% (95% CI 98.1–100%), respectively. Pregnancy outcome was obtained in all cases, including 221 male-bearing and 183 female-bearing pregnancies. ConclusionNIPD for fetal sex determination in maternal plasma is highly accurate and clinically applicable if robust reporting criteria are applied.

Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA: A Systematic Review and Meta-Analysis

Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997-April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%-96.1%) and specificity, 98.6% (95% CI, 98.1%-99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989-0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979-0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993-0.998) for real-time quantitative PCR (RTQ-PCR) (P = .02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965-0.998) ( 20 weeks) (P = .02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96.0%; specificity, 99.0%) outperformed conventional PCR (sensitivity, 94.0%; specificity, 97.3%). Testing after 20 weeks (sensitivity, 99.0%; specificity, 99.6%) outperformed testing prior to 7 weeks (sensitivity, 74.5%; specificity, 99.1%), testing at 7 through 12 weeks (sensitivity, 94.8%; specificity, 98.9%), and 13 through 20 weeks (sensitivity, 95.5%; specificity, 99.1%). Conclusions Despite interstudy variability, performance was high using maternal blood. Sensitivity and specificity for detection of Y chromosome sequences was greatest using RTQ-PCR after 20 weeks' gestation. Tests using urine and tests performed before 7 weeks' gestation were unreliable.

Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Systematic review and meta-analysis with search of PubMed (January 1, 1997-April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%-96.1%) and specificity, 98.6% (95% CI, 98.1%-99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989-0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979-0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993-0.998) for real-time quantitative PCR (RTQ-PCR) (P = .02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965-0.998) (<7 weeks); AUC, 0.994 (95% CI, 0.987-0.997) (7-12 weeks); AUC, 0.992 (95% CI, 0.983-0.996) (13-20 weeks); and AUC, 0.998 (95% CI, 0.990-0.999) (>20 weeks) (P = .02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96.0%; specificity, 99.0%) outperformed conventional PCR (sensitivity, 94.0%; specificity, 97.3%). Testing after 20 weeks (sensitivity, 99.0%; specificity, 99.6%) outperformed testing prior to 7 weeks (sensitivity, 74.5%; specificity, 99.1%), testing at 7 through 12 weeks (sensitivity, 94.8%; specificity, 98.9%), and 13 through 20 weeks (sensitivity, 95.5%; specificity, 99.1%). Despite interstudy variability, performance was high using maternal blood. Sensitivity and specificity for detection of Y chromosome sequences was greatest using RTQ-PCR after 20 weeks' gestation. Tests using urine and tests performed before 7 weeks' gestation were unreliable.

P23.02: The effects of fetal gender on serum HCG and testestron in normotensive and preeclamtic pregnancies

Objective: The aim of the present study was to evaluate the effects of fetal gender on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. Materials and Methods: The study consisted of 139 women with singleton pregnancies in the third trimester. Seventy-one pregnancies were uncomplicated; among those were 35 male and 36 female fetuses. Sixty-eight pregnancies were complicated by preeclampsia; among those were 35 male and 33 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. Detailed ultrasonographic structural examinations of the fetuses were performed. Results: In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001 and P < 0.001, respectively) in female-bearing pregnancies testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001) whereas the hCG levels were not significantly different. Male-bearing preeclamptic women had significantly higher testosterone levels than female-bearing preeclamptic women (P < 0.001) whereas the hCG levels were not significantly different. In uncomplicated pregnancies the hCG levels were significantly higher in female-bearing than in male bearing mothers (P < 0.001). Whereas the testosterone levels were not significantly different. Discussion and Conclusion: In preeclamptic pregnancies with male fetuses, the maternal serum hCG levels were significantly higher than in uncomplicated pregnancies. Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in male-bearing than in female-bearing pregnancies.

Human Chorionic Gonadotropin and Testosterone in Normal and Preeclamptic Pregnancies in Relation to Fetal Sex

In Brief OBJECTIVE The aim of the present study was to evaluate the effects of fetal gender on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. METHODS The study consisted of 137 women with singleton pregnancies in the third trimester. Seventy-three pregnancies were uncomplicated; among those were 35 male and 38 female fetuses. Sixty-four pregnancies were complicated by preeclampsia; among those were 33 male and 31 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. RESULTS In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < .001). In female-bearing pregnancies, testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < .001), whereas the hCG levels were not significantly different. Male-bearing preeclamptic women had significantly higher testosterone levels than female-bearing preeclamptic women (P < .02), whereas the hCG levels were not significantly different. In uncomplicated pregnancies the hCG levels were significantly higher in female-bearing than in male-bearing mothers (P < .005), whereas the testosterone levels were not significantly different. CONCLUSION In preeclamptic pregnancies with male fetuses, the maternal serum hCG levels were significantly higher than in uncomplicated pregnancies. Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in male-bearing than in female-bearing pregnancies. This may indicate an androgen influence on the pathophysiologic mechanism of preeclampsia. Human chorionic gonadotropin and testosterone levels were elevated in preeclamptic women, particularly in those with male fetuses.

Human chorionic gonadotropin and testosterone in normal and preeclamptic pregnancies in relation to fetal sex

OBJECTIVE: The aim of the present study was to evaluate the effects of fetal gender on serum human chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. METHODS: The study consisted of 137 women with singleton pregnancies in the third trimester. Seventy-three pregnancies were uncomplicated; among those were 35 male and 38 female fetuses. Sixty-four pregnancies were complicated by preeclampsia; among those were 33 male and 31 female fetuses. Human chorionic gonadotropin and total testosterone were measured in maternal peripheral blood. RESULTS: In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers ( P < .001). In female-bearing pregnancies, testosterone levels were significantly higher in preeclamptic than normotensive mothers ( P < .001), whereas the hCG levels were not significantly different. Male-bearing preeclamptic women had significantly higher testosterone levels than female-bearing preeclamptic women ( P < .02), whereas the hCG levels were not significantly different. In uncomplicated pregnancies the hCG levels were significantly higher in female-bearing than in male-bearing mothers ( P < .005), whereas the testosterone levels were not significantly different. CONCLUSION: In preeclamptic pregnancies with male fetuses, the maternal serum hCG levels were significantly higher than in uncomplicated pregnancies. Total testosterone levels were significantly higher in pregnancies with either gender and significantly higher in male-bearing than in female-bearing pregnancies. This may indicate an androgen influence on the pathophysiologic mechanism of preeclampsia.

Evaluation of Different Approaches for Fetal DNA Analysis from Maternal Plasma and Nucleated Blood Cells

One potential noninvasive approach for antenatal diagnosis is the use of fetal cells in maternal circulation as a source of fetal DNA (1). To date, fetal DNA has been extracted from maternal nucleated blood cells and amplified by nested PCR of Y-chromosome sequences from male fetuses (2)(3). A recent study (4) demonstrated the presence of fetal DNA in maternal plasma. After a single PCR with a high number of cycles, detection of fetal DNA was possible in 24 (80%) of the 30 maternal plasma samples, using 10 μL of boiled plasma (4). When DNA extracted from a similar volume of whole blood was used, only 17% of the samples gave a Y-positive signal, suggesting an enrichment of fetal DNA in plasma compared with whole blood cells (4). The mechanism responsible for fetal DNA release in maternal circulation is unclear. It has been postulated that fetal cell lysis after physical and immunological damage, as well as developmentally regulated apoptosis of certain fetal tissues, may be responsible for this phenomenon (5). Furthermore, the release and clearance of fetal DNA from maternal plasma may be influenced by pathophysiological factors that are still to be determined. The efficacy of the single PCR protocol on maternal plasma DNA has not yet been compared to established methodologies of nested amplification of DNA prepared from intact cells. The present work compares single PCR on plasma DNA with nested PCR on DNA extracted from plasma or nucleated blood cells. We collected 27 maternal blood samples at gestational ages ranging from the 7th to the 32nd weeks (16 male- and 11 female-bearing pregnancies). Nine, 14, and 4 samples were from the first (7–12 weeks), second (14–20 weeks), and third (27–32 weeks) trimesters. Fetal gender was unknown at the time of sampling and analysis and was verified …

Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction.

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test.

Effects of fetal sex and dexamethasone on preterm maternal serum concentrations of human chorionic gonadotropin, progesterone, estrone, estradiol, and estriol

The serum human chorionic gonadotropin concentration for the 43 female-bearing gestations was significantly higher (geometric mean 15,603 mIU/ml; 95% confidence interval 12,337 to 19,733) than that for 26 male-bearing pregnancies (geometric mean 8902 mIU/ml; 95% confidence interval 6288 to 12,596), p = 0.0087. The mean gestational age was 32 +/- 2.4 weeks (mean +/- SD). There was no significant difference between male- and female-bearing pregnancies for progesterone, cortisol, estrone, estradiol, estriol, or gestational age. In 19 placebo-treated pregnancies and 14 dexamethasone-treated pregnancies with a mean gestational age of 31.5 +/- 2.7 weeks, there was no significant change from the baseline value observed for either maternal serum human chorionic gonadotropin level or maternal serum progesterone level at 42 hours after institution of treatment. In the dexamethasone-treated subjects the geometric mean was reduced to 20% of baseline for cortisol; 42% of baseline for estrone; 30% of baseline for estradiol; and 41% of baseline for estriol (all p less than 0.001).