Short-chain fatty acids (SCFA) in infants' plasma and corresponding mother's milk and plasma in relation to subsequent sensitisation and atopic disease.

BACKGROUND: Short-chain fatty acids (SCFAs) in intestinal contents may influence immune function, while less is known about SCFAs in blood plasma. The aims were to investigate the relation between infants' and maternal plasma SCFAs, as well as SCFAs in mother's milk, and relate SCFA concentrations in infant plasma to subsequent sensitisation and atopic disease. METHODS: Infant plasma (N = 148) and corresponding mother's milk and plasma were collected four months postpartum. Nine SCFA (formic, acetic, propionic, isobutyric, butyric, succinic, valeric, isovaleric, and caproic acid) were analysed by UPLC-MS. At 12 months of age, atopic disease was diagnosed by a pediatric allergologist, and sensitisation was measured by skin prick test. All families participated in the Swedish birth cohort NICE (Nutritional impact on Immunological maturation during Childhood in relation to the Environment). FINDINGS: Infants with sensitisation, atopic eczema, or food allergy had significantly lower concentrations of five, three, and two SCFAs, respectively, in plasma at four months. Logistic regressions models showed significant negative associations between formic, succinic, and caproic acid and sensitisation [ORadj (95% CI) per SD: 0.41 (0.19-0.91); 0.19 (0.05-0.75); 0.25 (0.09-0.66)], and between acetic acid and atopic eczema [0.42 (0.18-0.95)], after adjusting for maternal allergy. Infants' and maternal plasma SCFA concentrations correlated strongly, while milk SCFA concentrations were unrelated to both. Butyric and caproic acid concentrations were enriched around 100-fold, and iso-butyric and valeric acid around 3-5-fold in mother's milk, while other SCFAs were less prevalent in milk than in plasma. INTERPRETATION: Butyric and caproic acid might be actively transported into breast milk to meet the needs of the infant, although mechanistic studies are needed to confirm this. The negative associations between certain SCFAs on sensitisation and atopic disease adds to prior evidence regarding their immunoregulatory potential. FUNDING: Swedish Research Council (Nr. 2013-3145, 2019-0137 and 2023-02217 to A-S.S.), Swedish Research Council for Health, Working Life and Welfare FORTE, Nr 2018-00485 to A.W.), The Swedish Asthma and Allergy Association's Research Fund (2020-0020 to A.S.).

Trajectories of Human Milk Gangliosides during the First Four Hundred Days and Maternal-to-Offspring Transfer of Gangliosides: Results from a Chinese Cohort Study.

BACKGROUND: Gangliosides are crucial for early-life cognition and immunity development. However, limited data exist on gangliosides within the Chinese population, and maternal-to-fetal/infant ganglioside transport remains unclear. OBJECTIVES: This study aimed to investigate gangliosides concentrations and trajectories in Chinese human milk during the first 400 d of lactation, and seek to understand gangliosides transmission between mother and offspring. METHODS: This study involved 921 cross-sectional participants providing human milk samples across 0-400 d of lactation and 136 longitudinal participants offering maternal plasma, cord plasma, and human milk samples within the first 45 d postpartum. Ultrahigh-performance liquid chromatography-tandem mass spectrometry was used for the quantification of gangliosides. RESULTS: Human milk GM3 (Neu5Acα2-3Galß1-4GlcßCer) concentration increased from 2.29 ± 1.87 to 13.93 ± 4.82 µg/mL, whereas GD3 (Neu5Acα2-8Neu5Acα2-3Galß1-4GlcßCer) decreased from 17.94 ± 6.41 to 0.30 ± 0.50 µg/mL during the first 400 d postpartum (all P < 0.05). Consistent results were observed in cross-sectional and longitudinal participants. GD3 concentration gradually increased from maternal plasma (1.58 µg/mL) through cord plasma (2.05 µg/mL) to colostrum (21.35 µg/mL). Significant positive correlations were observed between maternal and cord plasma for both GM3 (r = 0.30, P < 0.001) and GD3 (r = 0.35, P < 0.001), and maternal plasma GD3 also correlated positively with colostrum concentrations (r = 0.21, P = 0.015). Additionally, in maternal and cord plasma, gangliosides were mainly linked with 16- and 18-carbon fatty acids. However, human milk GM3 showed a broad spectrum of fatty acid chain lengths, whereas GD3 was primarily tied to very long-chain fatty acids (≥20 carbon). CONCLUSIONS: We identified an increase in GM3 and a decrease in GD3 concentration in human milk, with GD3 notably more concentrated in cord plasma and colostrum. Importantly, ganglioside concentrations in maternal plasma positively correlated with those in cord plasma and colostrum. Our findings contribute to the existing Chinese data on gangliosides and enhance understanding of their transmission patterns from mother to offspring. This trial was registered at chictr.org.cn as ChiCTR1800015387.

Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood.

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100-1000 nm) and exosomes (30-150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25-2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Integrative profiling of extrachromosomal circular DNA in placenta and maternal plasma provides insights into the biology of fetal growth restriction and reveals potential biomarkers.

Introduction: Fetal growth restriction (FGR) is a placenta-mediated pregnancy complication that predisposes fetuses to perinatal complications. Maternal plasma cell-free DNA harbors DNA originating from placental trophoblasts, which is promising for the prenatal diagnosis and prediction of pregnancy complications. Extrachromosomal circular DNA (eccDNA) is emerging as an ideal biomarker and target for several diseases. Methods: We utilized eccDNA sequencing and bioinformatic pipeline to investigate the characteristics and associations of eccDNA in placenta and maternal plasma, the role of placental eccDNA in the pathogenesis of FGR, and potential plasma eccDNA biomarkers of FGR. Results: Using our bioinformatics pipelines, we identified multi-chromosomal-fragment and single-fragment eccDNA in placenta, but almost exclusively single-fragment eccDNA in maternal plasma. Relative to that in plasma, eccDNA in placenta was larger and substantially more abundant in exons, untranslated regions, promoters, repetitive elements [short interspersed nuclear elements (SINEs)/Alu, SINEs/mammalian-wide interspersed repeats, long terminal repeats/endogenous retrovirus-like elements, and single recognition particle RNA], and transcription factor binding motifs. Placental multi-chromosomal-fragment eccDNA was enriched in confident enhancer regions predicted to pertain to genes in apoptosis, energy, cell growth, and autophagy pathways. Placental eccDNA-associated genes whose abundance differed between the FGR and control groups were associated with immunity-related gene ontology (GO) terms. The combined analysis of plasma and placental eccDNA-associated genes in the FGR and control groups led to the identification of potential biomarkers that were assigned to the GO terms of the epigenetic regulation of gene expression and nutrient-related processes, respectively. Conclusion: Together, our results highlight links between placenta functions and multi-chromosomal-fragment and single-fragment eccDNA. The integrative analysis of placental and plasma eccDNA confirmed the potential of these molecules as disease-specific biomarkers of FGR.

The natural occurring RRR-α-Tocopherol and synthetic α-Tocopherol stereoisomers in maternal plasma, cord plasma and breast milk among six regions of China.

The study aimed to investigate RRR-α-Tocopherol and synthetic α-Tocopherol stereoisomers in maternal plasma, cord plasma and breast milk from different regions of China, providing a reference for further guidance on maternal diet and the potential need to supplement mothers with vitamin E. Two hundred and twenty-one sample sets from maternal plasma, cord plasma and three stages of milk (colostrum: 1-5 d after delivery; transitional milk: 10-15 d; mature milk: 40-45 d) were collected longitudinally in six regions of Shanghai, Guangzhou, Tianjin, Chengdu, Lanzhou and Changchun. α-Tocopherol and the stereoisomers were determined by HPLC with a fluorescence detector. The RRR configuration accounted for more than 80 % of α-Tocopherol in maternal plasma, cord plasma and breast milk. Overall, there were regional differences both in α-Tocopherol and RRR. There were significant correlations between α-Tocopherol and RRR in maternal plasma, cord plasma and milk (P < 0.001). As well as negative correlations among α-Tocopherol, RRR and weight-related indicators, which refer to pre-gestation weight and BMI, pre-delivery weight and BMI and pregnancy weight gain, in almost all of samples. This study suggested that RRR-α-Tocopherol was the dominant configuration of α-Tocopherol and the main active form of vitamin E in the early life, guiding the rational supplement of pregnant women and the addition of α-Tocopherol in infant formula milk powder.

Plasma activin A concentrations during late gestation in Thoroughbred mares with abnormal pregnancies.

Late-term fetal loss in horses is a major problem in the horse-breeding industry globally. Abnormal pregnancies should be diagnosed as early as possible to prevent abortions and other gestational problems. According to our previous longitudinal study in healthy pregnant mares, the plasma activin A concentration increases as pregnancy progresses. The aim of the present study was to compare plasma activin A concentrations in healthy pregnant Thoroughbred mares (n=40) with those in pregnant mares that suffered fetal loss or showed abnormal symptoms (n=30) during late gestation. This field study found that plasma activin A concentrations were higher in the abnormal group (pregnancy loss, red bag delivery, premature udder development, and vaginal discharge) than the normal group (P < 0.001; cutoff value: ≥ 138.2 pg/mL; sensitivity, 74.4%; specificity, 77.5%). More specifically, plasma activin A concentrations in the "symptom" and "abnormal delivery" subgroups were higher than those in gestational-age-matched normal groups (P < 0.001). Nevertheless, the plasma activin A concentration in the "normal delivery" subgroup was not different from that in the "abnormal delivery" subgroup in samples collected within 10 days before delivery. In conclusion, this study is the first to demonstrate a significantly earlier increase in plasma activin A concentration in abnormal pregnancies of Thoroughbred mares during late gestation.

An Evaluation of Exposure to 18 Toxic and/or Essential Trace Elements Exposure in Maternal and Cord Plasma during Pregnancy at Advanced Maternal Age.

Pregnant women of advanced maternal age (AMA) are vulnerable to exposure to the surrounding environment. Assessment of trace elements in pregnant women living in specific areas is important for biomonitoring. However, exposure levels and variation patterns during pregnancy remains controversial and attracts extensive public concern. Therefore, we aimed to evaluate exposure of 18 toxic and/or essential trace elements in maternal plasma and in paired cord plasma during pregnancy at AMA. A total of 48 pregnant women of AMA were recruited in Peking University Third Hospital from 2018 to 2021. Eighteen elements found in maternal plasma during the 1st, 2nd, or 3rd trimester of pregnancy and paired cord plasma were measured by 7700x ICP-MS (Agilent Technologies, Palo Alto, CA, USA) and Elan DRC type II ICP-MS (The Perkin-Elmer Corporation, Waltham, MA USA). Concentrations of Pb, Se, Fe, Zn, and Mo all decreased during pregnancy, while Cu increased. Interestingly, concentrations of Rb decreased initially but then increased. Elements as Al, Co, Se, Cu, and Ni showed significantly lower levels in cord than in maternal plasma, while elements as Sr, Fe, Rb, Mn and Zn displayed significantly higher levels in cord than in maternal plasma. Moreover, positively- interacted clusters were found in Ni-Co-Cu-Al-Rb-Zn and Zn-Mn-Al-Pb in maternal blood. Similar positively-interacted clusters were found in Zn-Ni-Co, Zn-Ni-Fe, Mn-Al-Pb, Fe-Pb-Mn, Fe-Ni-Cu, and Rb-Cu-Sb-Fe-Mn in cord plasma. Furthermore, correlations between paired maternal and cord blood samples for As, Sr, and Mo were statistically significant, indicating that the fetus burden may reflect maternal exposure to some extent. Admittedly, levels of toxic and essential elements in our cohort study were comparatively lower than those in the scientific literature.

Progressive trends in prenatal genetic screening / Tendencias progresivas en el cribado genético prenatal

According to the global report on birth defects in 2021, it is estimated that 8 million children are born with birth defects of genetic origin annually. These birth defects vary in their degree of severity; where some types are mild and do not require treatment but others may necessitate lifelong medications or even cause instant death just after birth. That is why prenatal screening is doubtless necessary to detect such genetic defects before birth aiming to drop the tragedy of these children off. Recently, this approach has been developing towards non-invasive techniques that reduce the risk of miscarriage, which was common in the old-fashioned invasive ones. Non-invasive Prenatal Tests (NIPTs) like Chromosomal Microarray Analysis (CMA) and cell-free fetal DNA (cffDNA) caused a breakthrough in the screening methods of chromosomal aneuploidies. Thanks to their benefits, NIPTs are considered a fundamental clinical approach for pregnant women’ screening in multiple countries. Thence, this paper gives prominence to the recentness of NIPTs along with each’s assets, liabilities, and prospective recommendations. In addition, it would demonstrate the importance of modern molecular technologies like next-generation sequencing (NGS) which are enforced for the appliance of NIPTs. (AU)

Según el informe mundial sobre anomalías congénitas de 2021, se estima que anualmente nacen 8 millones de niños con anomalías congénitas de origen genético. Estos defectos de nacimiento varían en su grado de severidad; donde algunos tipos son leves y no requieren tratamiento, pero otros pueden necesitar medicamentos de por vida o incluso causar la muerte instantánea justo después del nacimiento. Por eso es sin duda necesario el cribado prenatal para detectar tales defectos genéticos antes del nacimiento con el fin de acabar con la tragedia de estos niños. Recientemente, este enfoque se ha ido desarrollando hacia técnicas no invasivas que reducen el riesgo de aborto espontáneo, que era común en las antiguas invasivas. Las pruebas prenatales no invasivas (NIPT) como el análisis de micromatrices cromosómicas (CMA) y el ADN fetal libre de células (cffDNA) provocaron un gran avance en los métodos de detección de aneuploidías cromosómicas. Gracias a sus beneficios, las NIPT se consideran un enfoque clínico fundamental para la detección de mujeres embarazadas en múltiples países. Por lo tanto, este documento destaca la actualidad de los NIPT junto con los activos, pasivos y recomendaciones prospectivas de cada uno. Además, demostraría la importancia de las tecnologías moleculares modernas, como la secuenciación de próxima generación (NGS), que se aplican para la aplicación de NIPT. (AU)

Galectins-1, -3 and -9 Are Present in Breast Milk and Have a Role in Early Life Development.

Galectins (Gal) are a family of conserved soluble proteins with high affinity for ß-galactoside structures. They have been recognized as important proteins for successful pregnancy. However, little is known about their presence in breast milk and their role in early infancy. Gal-1, -3 and -9 concentrations were evaluated by Multiplex immunoassays in mother-infant pairs from the MAMI cohort in maternal plasma (MP) (n = 15) and umbilical cord plasma (UCP) (n = 15) at birth and in breast milk samples (n = 23) at days 7 and 15 postpartum. Data regarding mother and infant characteristics were collected. Gal-9 was present in a lower concentration range than Gal-1 and Gal-3 in plasma, specifically in UCP. A major finding in the current study is that Gal-1, -3 and -9 were detected for the first time in all the transitional breast milk samples and no differences were found when comparing the two breastfeeding time points. Finally, Gal levels were associated with some maternal and infant characteristics, such as gestational age, pregnancy weight gain, maternal diet, the gender, infant growth and infant infections. In conclusion, Gal levels seem to be involved in certain developmental aspects of early life.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Longitudinal Proteomic Analysis of Plasma across Healthy Pregnancies Reveals Indicators of Gestational Age.

Longitudinal changes in the blood proteome during gestation relate to fetal development and maternal homeostasis. Charting the maternal blood proteome in normal pregnancies is critical for establishing a baseline reference when assessing complications and disease. Using mass spectrometry-based shotgun proteomics, we surveyed the maternal plasma proteome across uncomplicated pregnancies. Results indicate a significant rise in proteins that govern placentation and are vital to the development and health of the fetus. Importantly, we uncovered proteome signatures that strongly correlated with gestational age. Fold increases and correlations between the plasma concentrations of ADAM12 (ρ = 0.973), PSG1 (ρ = 0.936), and/or CSH1/2 (ρ = 0.928) with gestational age were validated with ELISA. Proteomic and validation analyses demonstrate that the maternal plasma concentration of ADAM12, either independently or in combination with either PSG1 or CSH1/2, correlates with gestational age within ±8 days throughout pregnancy. These findings suggest that the gestational age in healthy pregnancies may be determined by referencing the concentration of select plasma proteins.

Noninvasive prenatal testing of beta-thalassemia for common Pakistani mutations: a comparative study using cell-free fetal DNA from maternal plasma and chorionic villus sampling.

BACKGROUND: The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders. OBJECTIVE: Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method. METHODS: An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR. RESULTS: Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT). CONCLUSION: NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT.

Human plasma pregnancy-associated miRNAs and their temporal variation within the first trimester of pregnancy.

BACKGROUND: During pregnancy, maternal metabolism undergoes substantial changes to support the developing fetus. Such changes are finely regulated by different mechanisms carried out by effectors such as microRNAs (miRNAs). These small non-coding RNAs regulate numerous biological functions, mostly through post-transcriptional repression of gene expression. miRNAs are also secreted in circulation by numerous organs, such as the placenta. However, the complete plasmatic microtranscriptome of pregnant women has still not been fully described, although some miRNA clusters from the chromosome 14 (C14MC) and the chromosome 19 (C19MC and miR-371-3 cluster) have been proposed as being specific to pregnancy. Our aims were thus to describe the plasma microtranscriptome during the first trimester of pregnancy, by assessing the differences with non-pregnant women, and how it varies between the 4th and the 16th week of pregnancy. METHODS: Plasmatic miRNAs from 436 pregnant (gestational week 4 to 16) and 15 non-pregnant women were quantified using Illumina HiSeq next-generation sequencing platform. Differentially abundant miRNAs were identified using DESeq2 package (FDR q-value ≤ 0.05) and their targeted biological pathways were assessed with DIANA-miRpath. RESULTS: A total of 2101 miRNAs were detected, of which 191 were differentially abundant (fold change < 0.05 or > 2, FDR q-value ≤ 0.05) between pregnant and non-pregnant women. Of these, 100 miRNAs were less and 91 miRNAs were more abundant in pregnant women. Additionally, the abundance of 57 miRNAs varied according to gestational age at first trimester, of which 47 were positively and 10 were negatively associated with advancing gestational age. miRNAs from the C19MC were positively associated with both pregnancy and gestational age variation during the first trimester. Biological pathway analysis revealed that these 191 (pregnancy-specific) and 57 (gestational age markers) miRNAs targeted genes involved in fatty acid metabolism, ECM-receptor interaction and TGF-beta signaling pathways. CONCLUSION: We have identified circulating miRNAs specific to pregnancy and/or that varied with gestational age in first trimester. These miRNAs target biological pathways involved in lipid metabolism as well as placenta and embryo development, suggesting a contribution to the maternal metabolic adaptation to pregnancy and fetal growth.

Analysis of essential, toxic, rare earth, and noble elements in maternal and umbilical cord blood.

Progressive industrialization in recent decades has contributed to the increase of metal levels in the environment, which has a dangerous impact on human health, primarily pregnant women. In this study, we aimed to compare levels of various elements in maternal and umbilical cord (UC) plasma samples collected from 125 healthy pregnant women, conduct correlation analysis among paired plasma samples, and compare our data with other populations worldwide. The study design included the following elements: essential (Mn, Co, Cu, Zn, Se, Mo), non-essential (Be, Al, Ni, As, Rb, Sr, Cd, Sb, Pb, U), rare earth (La, Pr, Ce, Nd, Sm, Eu, Gd, Dy, Ho, Er), and noble metals (Ru, Rh, Re, Pt). Levels of 30 elements were higher in maternal plasma than in UC plasma samples. However, no disparities at the statistically significant level were found for Be, Zn, Rb, Cd, Ce, and Ho. Correlation analysis among paired plasma samples revealed only positive/synergistic correlations of different strengths between most elements. Compared to other countries across the globe, our participants had considerably lower plasma levels of Zn and higher levels of Co, Ni, and As. This study provides not only a new and deeper comprehension, but also the first insight into the levels, correlation, distribution, and potential transplacental transfer of 30 elements.

Integrity of cell-free DNA in maternal plasma extracellular vesicles as a potential biomarker for non-invasive prenatal testing.

OBJECTIVE: Large proportions of cell-free DNA (cfDNA) in plasma are localized in extracellular vesicles (EVs), which are secreted from placental cells. This study was conducted to reveal the integrity pattern of cfDNA in maternal plasma EVs (evcfDI) across gestation, and explore if evcfDI could be a potential biomarker in screening for aneuploid fetus in non-invasive prenatal testing (NIPT). METHODS: A total of 180 maternal plasma samples were collected during NIPT. Both evcfDNA and fetal evcfDNA (evcffDNA) were measured by quantitative PCR of LINE1 and SRY gene amplicons with different sizes. The evcfDI was calculated as the ratio of long to short fragments. RESULTS: evcfDI is not affected by gestational age; whereas evcffDI has a mild decreasing trend with increasing gestational age (P = 0.048). evcfDI is significantly and negatively correlated with maternal body mass index (BMI; calculated as weight in kilograms divided by the square of height in meters: ≤18.5, 18.5-25, and ≥25) (P < 0.01) and age (<35 and ≥35 years) (P < 0.01). Mean evcfDI decreases from 2.113 in euploid controls to 0.681 in those with an aneuploid fetus in NIPT (P = 0.003). CONCLUSION: Maternal clinical characteristics such as BMI and age could be innovative biomarkers to calibrate evcfDI, which was shown to be a potential indicator of an aneuploid fetus. Analysis of evcfDI based on quantitative PCR could serve as a novel, rapid, and low-cost NIPT strategy, which might facilitate testing at earlier gestations.

Prenatal Identification of Confined Placental Mosaicism in Pregnant Women with Fetal Growth Restriction.

We examined the influence of confined placental mosaicism (CPM) as a cause of fetal growth restriction (FGR), and whether CPM can be screened using cell-free DNA (cfDNA) analysis of the maternal plasma. We analyzed cfDNA in the maternal plasma of 40 FGR cases with an estimated fetal weight of less than - 2.0 SD using massively parallel sequencing to detect chromosomal aberrations. Fetal and placental genotyping was performed to confirm CPM cases. cfDNA analyses of maternal plasma detected suspected CPM cases with chromosomal aneuploidy or copy number variations in 5 of 40 cases (12.5%). For 4 cases in which the entire placenta consisted of cells with chromosomal abnormalities, fetal growth was severely restricted. CPM can be screened by cfDNA analysis in maternal plasma, accounting for approximately 10% of the causes of moderate or severe FGR, and the higher the proportion of abnormal karyotype cells in the placenta, the more severe the placental dysfunction and FGR.

Protein Profile Changes in Circulating Placental Extracellular Vesicles in Term and Preterm Births: A Longitudinal Study.

Spontaneous preterm birth (PTB) is a major obstetrical problem around the globe and the mechanisms leading to PTB are unclear. Recently, changes in the circulating levels of placental extracellular vesicles (EVs) during pregnancy have been associated with various pregnancy complications. However, progress in the field is hindered by the inability to isolate placental EVs from the maternal circulation. A longitudinal study design was used to determine the protein cargo present in circulating placental EVs in maternal plasma of term and PTB across gestation (ie, first, second, and third trimester). Placental-derived EVs were enriched from the total EV population based on their expression of membrane-bound placental alkaline phosphatase (PLAP). A quantitative, information-independent acquisition (sequential windowed acquisition of all theoretical mass spectra [SWATH]) approach identified and quantified the placental EV protein contents. PLAP+ EVs did not change in characteristics (size, shape, and markers) but did differ in numbers across gestation with low levels in PTB. A comparison analysis between the PLAP+ EV proteome from term and PTB revealed 96 proteins differing significantly (P < 0.05, false discovery rate 1%) across gestation. Bioinformatics analysis of differentially expressed proteins revealed consistent upregulation of inflammatory pathways in both upregulation of epithelial mesenchymal transition pathways at term and downregulation of coagulation/complement activation in preterm. Characterization of the proteomic profile in PLAP+ EVs across gestation demonstrates dramatic changes, which might be used to understand the biological process associated with early parturition and develop biomarkers for predicting high-risk status for PTB.

Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Cadmium-binding proteins in human blood plasma.

Blood is the transmission medium for metal contaminants to and from bodily organs; as such, it can provide useful and reliable information about their bio-kinetics as they're distributed throughout the body. Metals can interact with endogenous proteins present in the blood, and these metal-protein complexes often dictate the fates of the introduced metals. The aim of this study was to investigate cadmium-binding protein characteristics in normal human plasma. Cadmium-binding plasma proteins in two different groups: normal human plasma (n = 29), and normal paired maternal and fetal umbilical cord plasmas (n = 3), were analyzed. In order to detect cadmium-binding plasma proteins present in low concentrations, blood plasma samples were first depleted of their two most abundant proteins - albumin and immunoglobulin G. Both the crude and depleted plasma samples were analyzed using column gel electrophoresis in conjunction with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). One cadmium-binding protein was detected in 11 of 29 normal plasma samples and all three paired maternal and cord plasma samples. This protein was further identified as apolipoprotein A-I by high-resolution mass spectrometry. To the best of our knowledge, this is the first study to reveal cadmium-binding proteins in real human blood plasma, which is extremely critical to our understanding of cadmium transportation and accumulation in human blood.

Non-invasive prenatal rhesus D genotyping using cell-free foetal DNA.

Background & objectives: Non-invasive prenatal diagnosis (NIPD) of rhesus D (RHD) genotype using cell-free foetal DNA is extensively used in many developed countries. Studies on NIPD from India are scarce. The aim of the present study was to evaluate the performance of non-invasive foetal RHD genotyping by targeting exon 10 of the RHD gene using cell-free DNA. Methods: DNA was extracted from the maternal plasma of alloimmunized and non-alloimmunized women between 7 and 34 wk of gestation. RHD sequence was determined by quantitative real time polymerase chain reaction (PCR). Results were compared with RhD phenotype obtained from cord blood samples of neonates. Results: A total of 135 samples from RhD-negative pregnant women were collected. The foetal RHD status was conclusive in all 135 (100%) cases. The highest number of cases reported for RHD genotyping were from Punjab (38.5%) followed by Haryana (24.4%), Himachal Pradesh (17.0%) and Chandigarh Union Territory (13.3%). The non-invasive test correctly predicted the foetal RhD phenotype in 133 of 135 cases, making the accuracy of the test as 98.51 per cent [95% confidence interval (CI): 97.90-99.50%]. The overall sensitivity and specificity of the test were 99.18 per cent (95% CI: 95.52-99.98%) and 92.31 per cent (95% CI: 63.97-99.81%), respectively, with negative and positive predictive values of 99.80 per cent (95% CI: 94.85-99.87%) and 96.31 per cent (95% CI: 62.87-98.84%), respectively. Interpretation & conclusions: Non-invasive foetal RHD determination by single-exon quantitative PCR exhibited high accuracy and could be used in routine clinical practice after confirmatory studies are done.