Mid-trimester Amniotic Fluids Research Articles

New insight into the analysis of amniotic fluid microflora using 16S rRNA gene sequencing

Introduction: Intra-uterine infection is a major cause of spontaneous pre-term birth before 30 weeks of gestation. In addition, intra-amniotic inflammation is present in approximately 80 % of patients with acute cervical insufficiency. The microbiome is known to cause intra-amniotic infection causing pre-term birth. Recently, surveys of the microbial communities in the vagina or in amniotic fluid using sequencing of the 16s rRNA gene have been carried out. Here, we reported the microbiome compositions in mid-trimester amniotic fluids in two pregnant women with cervical insufficiency identified using 16S rRNA sequencing. Case presentation: A 35-year-old primipara and a 37-year-old nullipara were admitted to Hallym University Medical Center, Korea. They were negative for nitrazine and Actim PROM (Medix Biochemica) tests, and their amniotic membranes had prolapsed beyond the external os. A physical examination indicated that cerclage using a uniconcave balloon should be carried out, and amnioreduction was performed. Thereafter, they delivered at very early pre-term birth of below 24 weeks and the birth outcomes were pre-natal death. Analysis of mid-trimester amniotic fluids in these two pregnant women with cervical insufficiency identified Sneathia sanguinegens and Fusobacterium nucleatum. These bacteria were not identified using vaginal or amniotic cultures. Conclusion: These results suggest that 16S rRNA gene sequencing can be used to identify the predominant microbiome causing pre-term birth in pregnant women with cervical insufficiency. Further studies are needed in large samples to improve our understanding of microbes causing pre-term birth and to prevent pre-term birth in pregnant women with cervical insufficiency.

Protein Microarrays and Midtrimester Amniotic Fluids: A Novel Approach for the Diagnosis of Early Intrauterine Inflammation Related to Preterm Delivery

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, MCP-1, IFN-gamma and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-alpha, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

336 IP-10 and IL-1Ra elevation in cytomegalovirus-infected vs normal midtrimester amniotic fluids

336 IP-10 and IL-1Ra elevation in cytomegalovirus-infected vs normal midtrimester amniotic fluids

Heat shock protein-containing exosomes in mid-trimester amniotic fluids

Exosomes are multivesicular bodies formed by inverse membrane budding into the lumen of an endocytic compartment. Fusion with the plasma membrane leads to their release into the external milieu. The incorporation of heat shock proteins into exosomes has been associated with immune regulatory activity. We have examined whether heat shock protein-containing exosomes are present in mid-trimester amniotic fluid. Exosomes were isolated from mid-trimester amniotic fluids by sequential low-speed and high-speed centrifugation followed by sucrose density gradient centrifugation. Biochemical characterization included floatation pattern in sucrose gradients, acetylcholinesterase (AChE) activity and Western blot analysis for exosome-containing proteins. Exosomes were present in each of 23 amniotic fluids tested. They banded at a density of 1.17 g/ml in sucrose gradients, were positive for AChE activity and contained tubulin, the inducible 72 kDa heat shock protein, Hsp72 and the constitutively expressed heat shock protein, Hsc73; they were negative for calnexin. Exosome concentrations correlated positively with the number of pregnancies. Heat shock protein-containing exosomes are constituents of mid-trimester amniotic fluids and may contribute to immune regulation within the amniotic cavity.

Detection of bacteria in mid-trimester amniotic fluids by gene amplification of bacterial 16s ribosomal DNA sequences, cloning and sequence analysis

Detection of bacteria in mid-trimester amniotic fluids by gene amplification of bacterial 16s ribosomal DNA sequences, cloning and sequence analysis

Clara cell protein 16 concentration in mid-trimester amniotic fluid: Association with fetal gender, fetal G > A +38 CC16 gene polymorphism and pregnancy outcome

Clara cell protein 16 (CC16) is a major immunomodulatory protein produced in the fetal lung. We hypothesized that the mid-trimester amniotic fluid concentration of CC16 would vary according to a +38 CC16 polymorphism in the fetal genome and that increased levels would be an early indicator of subsequent adverse pregnancy outcome. Mid-trimester singleton amniotic fluids from 244 women were assayed for CC16 by ELISA. DNA from fetal cells in 179 amniotic fluids were tested for the A > G polymorphism at position +38 in exon 1 by PCR. Outcome data were obtained from 233 women after completion of laboratory testing. Median CC16 levels were higher in amniotic fluids containing male fetuses than in those with females ( p = 0.0005). Median amniotic fluid CC16 levels were higher in Hispanics than in Whites and Asians ( p < 0.05). CC16*G homozygosity was associated with elevated amniotic fluid CC16 concentrations compared to CC16*A homozygotes ( p = 0.02). Intraamniotic CC16 levels were highest in pregnancies that subsequently resulted in preterm premature rupture of membranes (PPROM) ( p = 0.01). We conclude that mid-trimester intraamniotic CC16 concentrations vary by gender, ethnicity and fetal CC16 gene polymorphism. Elevated CC16 levels are predictive of subsequent development of PPROM.

Amniotic fluid folate, vitamin B12 and transcobalamins in neural tube defects

Levels of folate, vitamin B12, the vitamin B12 binding proteins, apotranscobalamin I, II and III (TC I, II and III) and the unsaturated vitamin B12 binding capacity (UBBC) were measured in mid-trimester amniotic fluids from normal pregnancies, and from those where the fetus had open spina bifida, anencephaly or omphalocoele, and where the fetus was normal but the mother had had a previous neural tube defect pregnancy. At 15-19 weeks' gestation, vitamin B12 levels were low in the fluids of all the types of abnormal fetuses, and also of normal fetuses where there had been a previous NTD sib. In contradistinction, TC I, II and III and UBBC levels were generally abnormally high in all these groups. Low vitamin B12 levels in the face of high carrier protein levels suggest deranged vitamin B12 production or transport. Since these abnormalities are present in fluids from normal sibs of NTD individuals as well as from those with midline lesions, an inherited defect is implied. We propose that at least part of the genetic predisposition to NTD, and possibly other midline defects, could reside in an abnormality connected with vitamin B12 production, transport or metabolism, and a mechanism is suggested.

The association of congenital skin disorders with acetylcholinesterase in amniotic fluid

We describe a fetus with epidermolysis bullosa dystrophica and a fetus with aplasia cutis congenita who were normal by careful ultrasound examination but whose midtrimester amniotic fluids exhibited elevated concentrations of alpha-fetoprotein and presence of acetylcholinesterase. These cases show that serious fetal skin pathology can be a source of amniotic fluid acetylcholinesterase and elevated alpha-fetoprotein concentration and should be considered as part of the differential diagnosis of these amniotic fluid findings.

A prospective study of amniotic fluid cholinesterases: Comparison of quantitative and qualitative methods for the detection of open neural tube defects

The value of quantitative and qualitative methods of cholinesterase (ChE) analysis in the detection of open neural tube defect (NTD) has been assessed in a prospective survey of 1495 mid-trimester amniotic fluids. Using a quantitative method the mean ChE values were much lower in fluids from pregnancies of normal outcome but it was not possible to discriminate these fluids completely from those associated with NTD pregnancies, particularly when the specimens were contaminated with blood. Similarly, measurement of acetylcholinesterase (AChE) activity alone by three different methods also failed to eliminate the overlap between the two groups. In contrast, polyacrylamide gel electrophoresis revealed only a single band of ChE activity in 1408 out of 1410 fluids from pregnancies with a normal outcome whilst amniotic fluids from all 60 cases of open NTD, 6 out of 7 cases of exomphalos and 3 out of 4 cases of intra-uterine death gave the characteristic second faster-running AChE band. A qualitative gel method which requires the same amount of ChE activity to be loaded from each amniotic fluid is an effective method for pre-natal diagnosis of NTDs.

Immunological and Microbiological Studies of Midtrimester Amniotic Fluid

Midtrimester amniotic fluids (MAF) from 73 women undergoing an amniocentesis were studied. The mean immunoglobin G (IgG) level was 34 +/- 14 mg/dl, which is higher than that reported in the third trimester. The maternal serum:MAF IgG ratio was 29 suggesting the blood:MAF barrier is less restrictive than others like the blood:cerebrospinal fluid barrier. Antibodies to herpes simplex virus (HSV) type 1 were detected in 78% of the MAF and the maternal serum:MAF HSV antibody titer ratio was 33. Antibodies to cytomegalovirus (CMV) were found in 84% of the MAF and the maternal serum:MAF CMV antibody titer ration was 40. Whenever either antibody was detected in the maternal serum it was also found in the MAF. No viruses, bacteria, mycoplasma, fungi and chlamyidae were isolated from MAF.