Midgestational Amniotic Fluid Research Articles

Characterisation of Mid-Gestation Amniotic Fluid Cytokine and Bacterial DNA Profiles in Relation to Pregnancy Outcome in a Small Australian Cohort.

A well-established association exists between intrauterine bacteria and preterm birth. This study aimed to explore this further through documenting bacterial and cytokine profiles in Australian mid-gestation amniotic fluid samples from preterm and term births. Samples were collected during amniocenteses. DNA was extracted and the full-length 16S rRNA gene was amplified and sequenced. Levels of the cytokines IL-1β, IL-6, IL-10, TNF-α and MCP-1 were determined using the Milliplex MAGPIX system. Bacterial DNA profiles were low in diversity and richness, with no significant differences observed between term and preterm samples. No differences in the relative abundance of individual OTUs between samples were identified. IL-1β and TNF-α levels were significantly higher in samples containing reads mapping to Sphingomonas sp.; however, this result should be interpreted with caution as similar reads were also identified in extraction controls. IL-6 levels were significantly increased in samples with reads that mapped to Pelomonas sp., whilst TNF-α levels were elevated in fluid samples from pregnancies that subsequently delivered preterm. Bacterial DNA unlikely to have originated from extraction controls was identified in 20/31 (64.5%) mid-gestation amniotic fluid samples. Bacterial DNA profiles, however, were not predictive of preterm birth, and although cytokine levels were elevated in the presence of certain genera, the biological relevance of this remains unknown.

Amniotic fluid biomarkers predict the severity of congenital cytomegalovirus infection.

BACKGROUNDCytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury.METHODSWe performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays.RESULTSUsing unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins — the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3–binding protein (Gal-3BP) — were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%–93.8% positive predictive value, and 92.9%–92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSIONAnalysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.FUNDINGIsrael Science Foundation (530/18 and IPMP 3432/19); Research Fund – Hadassah Medical Organization.

Comprehensive human amniotic fluid metagenomics supports the sterile womb hypothesis

As metagenomic approaches for detecting infectious agents have improved, each tissue that was once thought to be sterile has been found to harbor a variety of microorganisms. Controversy still exists over the status of amniotic fluid, which is part of an immunologically privileged zone that is required to prevent maternal immune system rejection of the fetus. Due to this privilege, the exclusion of microbes has been proposed to be mandatory, leading to the sterile womb hypothesis. Since nucleic acid yields from amniotic fluid are very low, contaminating nucleic acid found in water, reagents and the laboratory environment frequently confound attempts to address this hypothesis. Here we present metagenomic criteria for microorganism detection and a metagenomic method able to be performed with small volumes of starting material, while controlling for exogenous contamination, to circumvent these and other pitfalls. We use this method to show that human mid-gestational amniotic fluid has no detectable virome or microbiome, supporting the sterile womb hypothesis.

91: Human milk oligosaccharides are present in midgestation amniotic fluid & associated with a sparse microbiome

91: Human milk oligosaccharides are present in midgestation amniotic fluid & associated with a sparse microbiome

Natural History of Fetal Lower Urinary Tract Obstruction with Normal Amniotic Fluid Volume at Initial Diagnosis

Objective: The aim of this study was to define the natural history of lower urinary tract obstruction (LUTO) with normal midgestational amniotic fluid volumes. Materials and Methods: We performed a retrospective review of 32 consecutive patients with LUTO with normal midgestational amniotic fluid volume followed at 11 North American Fetal Therapy Network (NAFTNet) centers from August 2007 to May 2012. Normal amniotic fluid volume was defined as an amniotic fluid index (AFI) of ≥9 cm. Results: The mean gestational age (GA) and AFI at enrollment were 23.1 ± 2.1 weeks and 15.8 ± 3.9 cm, respectively. The mean GA at delivery was 37.3 ± 2.8 weeks. The mean creatinine level at discharge was 1.2 ± 0.8 mg/dL. Perinatal survival was 97%. Twenty-five patients returned for serial postnatal assessment. Renal replacement therapy (RRT) was required in 32%. Development of oligohydramnios and/or anhydramnios, development of cortical renal cysts, posterior urethral valves, prematurity, and prolonged neonatal intensive care unit stay were associated with need for RRT (p < 0.05) by univariate analysis. By multivariate analysis, preterm delivery remained predictive of need for RRT (p = 0.004). Conclusion: Prenatal diagnosis of LUTO with normal midgestational amniotic fluid volumes is associated with acceptable renal function in the majority of patients. Approximately one-third of these children require RRT. Surrogate markers of disease severity appear to be predictive of need for RRT.

Midtrimester amniotic fluid concentrations of angiogenic factors in relation to maternal, gestational and neonatal characteristics in normal pregnancies

Objective: To describe associations among maternal/gestational/neonatal characteristics and midpregnancy amniotic fluid concentrations of the main angiogenic markers vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Methods: In a cohort of 206 normal full-term pregnancies, midpregnancy amniotic fluid VEGF and PlGF reference values were recorded. Possible associations among the above concentrations and various parameters, such as maternal age and body mass index, race, parity, smoking, gestational age, delivery mode, birth-weight and fetal gender were investigated. Results: Midpregnancy amniotic fluid concentrations of both VEGF and PlGF increased with increasing gestational age (r = 0.173, p = 0.013 and r = 0.255, p < 0.001, respectively), whereas PlGF concentrations positively correlated with birth-weight (r = 0.154, p = 0.027). The effect of the other above-mentioned parameters on VEGF and PlGF concentrations was not significant. Conclusions: In normal pregnancies, midgestation amniotic fluid VEGF and PlGF concentrations positively correlate with gestational age. Furthermore, midgestation amniotic fluid PlGF concentrations may be a predictor of neonatal birth weight.

Prenatal dexamethasone treatment in pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: effect on midgestational amniotic fluid steroid levels.

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by amniotic fluid (AF) steroid analysis is not possible in those cases in which prenatal dexamethasone (DEX) therapy is initiated to prevent virilization of female CAH fetuses because AF steroid levels are suppressed if DEX therapy is continued beyond amniocentesis (AC). In order to use AF steroids for prenatal diagnosis, it is necessary to discontinue DEX therapy for 5 days before AC. To study the effects of this interruption on AF steroid levels, we measured levels of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone (170HP), 11-deoxycortisol, cortisol, and cortisone as well as androstenedione (delta 4-A) in AF samples (16-18 weeks) obtained from 25 pregnancies at risk for CAH treated with Dex (daily dosage: 1.0-1.5 mg). The prenatal diagnosis of 14 normal fetuses and 11 affected CAH fetuses was postnatally confirmed in all cases. Additionally, steroid levels were measured in AF samples (16-18 weeks) from 8 untreated CAH fetuses and in 19 AF samples (weeks 16-20) obtained in normal pregnancies. In 17/19 prenatally diagnosed CAH fetuses, the affected sibs had the salt wasting (SW)-form, in 2 cases the simple virilizing (SV)-form. All steroids were measured by RIA after extraction and Sephadex LH-20 chromatography. AF levels of aldosterone, corticosterone, deoxycorticosterone, progesterone, cortisol, cortisone, and 11-deoxycortisol were not different between CAH fetuses, prenatally DEX-treated normal fetuses and untreated controls. The 170HP-levels of the CAH-SW-fetuses (range: 19.9-59.8 mmol/L) were clearly above the normal range (3.74-11.6), but normal in the SV-fetuses (10.9, 11.5), whereas delta-4 A-levels (normal range: 0.87-5.13 mmol/L) were elevated both in the SW-(range: 6.53-37.6) and the SV-form (9.37,6.25) of CAH. 170HP and delta-4 A levels of prenatally DEX-treated pregnancies with normal fetuses were not different from levels found in normal pregnancies. Mean 170HP and delta-4 A AF steroid levels of prenatally DEX-treated CAH-pregnancies were slightly lower (NS) than levels of untreated CAH-pregnancies (170HP: 30.5 vs. 40.7; delta-4 A: 15.8 vs. 21.1). 170HP levels are elevated in the SW-form of CAH, but not in the SV-form. However, with the combination of 170HP and delta-4 A levels it is possible to diagnose prenatally both forms. There is no rebound phenomenon of AF steroid levels if DEX therapy is interrupted 5 days before amniocentesis.

Prenatal dexamethasone treatment in pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: effect on midgestational amniotic fluid steroid levels

Prenatal dexamethasone treatment in pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: effect on midgestational amniotic fluid steroid levels

Expression of transforming growth factor-α and epidermal growth factor receptor in human fetal kidneys

Beginning at the fifth week of fetal life, successive generations of individual nephrons are induced by contact between metanephric mesenchyme and ureteric bud. Following phenotypic transformation, cells of each primitive renal vesicle undergo a phase of rapid cell division. In order to identify genes which might regulate nephron development in man, we screened adult and fetal kidney RNA for expression of a panel of growth-related genes. Among the genes which were expressed at higher levels in fetal kidney was the epidermal growth factor (EGF) receptor. There is controversy as to the most likely physiologic EGF receptor ligand in fetal kidney; we were able to identify a transcript for transforming growth factor-α (TGF-α) but not EGF on Northern blots of fetal kidney RNA. Since the abundance of TGF-α mRNA is low, we confirmed its presence by polymerase chain reaction amplification. Using specific radioimmunoas-says, we also provide direct evidence for TGF-α but not EGF peptide in extracts of fetal kidney and mid-gestational amniotic fluid. We suggest that TGF-α/EGF receptor interactions may serve an important function in development of human fetal kidney.

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.

Midgestational amniotic fluid (AF) steroid levels in CAH-pregnancies: effect of prenatal dexamethasone (DEX) treatment

Midgestational amniotic fluid (AF) steroid levels in CAH-pregnancies: effect of prenatal dexamethasone (DEX) treatment

Midgestational Amniotic Fluid Human Atrial Natriuretic Polypeptide (HANP): Possible Predictor of Third-Trimester Pregnancy-Induced Hypertension

Atrial natriuretic polypeptide (ANP) comprises a group of potent diuretic and vasorelaxant substances synthesized and released by mammalian atrial cardiocytes. In late pregnancy, plasma ANP concentrations appear to be increased in gravidas with severe pregnancy-induced hypertension (PIH) versus normotensive parturients. Earlier gestational assessments of ANP in plasma and other fluid compartments are needed at various stages of gestation in gravidas who remain normotensive and those who become hypertensive. The present retrospective study was undertaken to determine if midgestational amniotic fluid concentrations of ANP in healthy controls and in gravidas destined to develop PIH would be significantly different and possibly predictive of later hypertensive disease. The findings in 14 gravidas who underwent midtrimester genetic amniocentesis and later developed PIH were compared with 44 matched control patients. Midgestational amniotic fluid ANP concentrations were determined to not be significantly differ...

Isolation of a somatomedin-binding protein from preterm amniotic fluid. Development of a radioimmunoassay.

Amniotic fluid binding protein (AFBP) is a heat and acid stable somatomedin (Sm)-binding protein with a mol wt of 35-40,000 and an isoelectric point of +/- 4.7. It is reactive in RRAs for Sm and inhibits Sm activity in Sm bioassays. AFBP was purified from midgestational human amniotic fluid (AF) using acid-ethanol extraction, Sephadex G-150 chromatography, high speed gel filtration chromatography, and disc gel-electrophoresis. Specific binding activity (microgram equivalents per mg protein) was quantitated by incubation with 125I-insulin-like growth factor II and dextran-coated charcoal separation. Protein recovery was less than 1%. AFBP antiserum was produced by immunizing rabbits with purified AFBP. The antiserum was cleared of human serum albumin antibodies by affinity chromatography. Immunoelectrophoresis of 20x concentrated preterm AF and fetal serum resulted in one precipitin line. AFBP was labeled by the chloramine-T method. The AFBP antiserum specifically bound +/- 35% of added 125I-AFBP at a final dilution of 1:5000. A double antibody RIA was developed. The AFBP level measured by RIA in midgestation AF (n = 30) was 148 +/- 18 (SEM) and in term AF (n = 12) 72 +/- 36 mu geq/ml. Insulin-like growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (less than 0.10 U/ml). When serum was chromatographed on Sephadex G-200 at pH 2.2, AFBP-RIA activity eluted in one peak corresponding to a mol wt of 35-40,000. Highest activity was found in fetal serum (gestational age +/- 20 weeks) and lowest in serum from adults. The development of the AFBP-RIA may contribute to further elucidation of the physiological importance of Sm and the Sm-binding proteins in pre- and postnatal growth.

Chapter 2. Morphological and Biochemical Heterogeneity of Amniotic Fluid Cells in Culture

This chapter describes the morphological and biochemical heterogeneity of amniotic fluid cells in culture. Midgestation amniotic fluid cells constitute a precious source of human cells for the purpose of cell biological investigation. Several properties contribute to the diagnostic and scientific value of these cells, the most important of which are the presence in a single amniotic fluid specimen of multiple, morphologically and biochemically distinct cell types that are all derived from the fetus or from fetal membranes and thus are isogenic. It is found that in addition to variable amounts of cellular debris and anucleate particles, second-trimester amniotic fluid contains between 103 and 105 nucleated cells per milliliter of fluid. All epithelial surfaces in direct or indirect contact with the amniotic cavity are possible sites of origin of these cells. According to their behavior in the culture environment, nucleated amniotic fluid cells can be operationally divided into three separate categories. The first category includes cells that remain afloat even after prolonged periods of incubation. It is found that amniotic fluid F-type cells have the greatest in vitro growth potential of all colony-forming cells.

Characterization of a Protein in Mid-Term Human Amniotic Fluid which Reacts in the Somatomedin-C Radioreceptor Assay*

Amniotic fluid contains materials other than insulin which react in a somatomedin C radioreceptor assay using human placental membranes. The material in mid-gestational amniotic fluid which reacted with the somatomedin C radioreceptor assay eluted slightly after albumin from a Sephadex G-150 column equilibrated with 0.1M NH4HCO3. Neither boiling nor treatment of this fraction with 1% formic acid yielded small molecular weight somatomedin-like peptides. Separation of the somatomedin reactive material (Sm RM) from albumin was achieved by gel filtration through Ultrogel AcA44 in 3.1M NH4HCO3. The active product had an apparent molecular weight of 33,000 to 35,000 Daltons; its isoelectric point determined by focusing was between 4.1 and 5.1. The purified amniotic fluid protein displaced somatomedin C in the somatomedin C radioreceptor assay but did not compete with insulin in the insulin radioreceptor assay. Sm RM produced only a slight stimulation of thymidine uptake in human fibroblasts but was inactive in stimulating sulfate uptake in hypox rat costal cartilage. In human fibroblast cultures Sm RM inhibited the stimulation of thymidine uptake induced by human serum and by purified rat somatomedin. When Sm RM was added to the 125I somatomedin C, some of the radioactivity eluted from gel filtration at pH 8.6 was converted to a molecular weight complex of about 43,000. We conclude that the material which we have isolated from mid-gestational amniotic fluid is a protein which may bind somatomedin and make it unavailable to the somatomedin receptor of human placenta and human fibroblasts.