Mid-trimester Amniotic Fluid Research Articles

Influence of the polymorphic fetal interleukin-1 receptor antagonist (IL-1RA) genotype on pregnancy history

To determine whether fetal carriage of the different IL-1ra genotypes is associated with past pregnancy outcomes. Fetal cells in midtrimester amniotic fluid samples from 139 women were blindly tested for IL-1ra intron 2 length polymorphisms. Pregnancy data were subsequently obtained. A higher prevalence of the 1,2 genotype was detected in fetuses from women whose prior pregnancies resulted in term births (17/41, 41.5%), or who had a combination of term births and spontaneous abortions (10/22, 45.5%), as compared to fetuses from women whose only pregnancies resulted in a spontaneous abortion (1/15, 6.7%) (P = 0.01). A similar relationship was observed when only the fetuses from the 81 white women in the study were analyzed (45.8% 1,2 genotype in term births only vs 11.1% in spontaneous abortions only, P = 0.04). Median midtrimester amniotic fluid concentrations of interleukin-1β (IL-1β) and IL-1ra were reduced in women with only prior spontaneous abortions as compared to those with only prior term births (IL-1β: 7.0 pg/mL vs 18.2 pg/mL, P = 0.02; IL-1ra: 4.1 ng/mL vs 6.6 ng/mL, P = 0.03). Lastly, the fetal IL-1ra genotype was related to the amniotic fluid IL-1β and IL-1ra levels. Fetal carriage of the 1,2 genotype was associated with elevated median IL-1β (18.3 pg/mL) as compared to fetal carriage of the 1,1 genotype (10.1 pg/mL, P = 0.01). Similarly, homozygous carriage of the 2 allele was associated with elevated median IL-1ra levels (7.0 ng/mL vs 4.7 ng/mL in allele 1 homozygotes). In contrast to the results with IL-1β, there was no relationship between amniotic fluid concentrations of tumor necrosis factor-α, interleukin-4, or interleukin-6 and pregnancy history. Elevated fetal production of IL-1β, related at least in part to carriage of the IL-1ra 1,2 genotype, is associated with a reduced occurrence of spontaneous abortion and an increased likelihood of a term birth.

Midtrimester amniotic fluid vitamin C levels inversely correlate with interleukin-6 levels

CORRELATE WITH INTERLEUKIN-6 LEVELS EVA PRESSMAN, DEB PITTINARO, JAMES WOODS, University of Rochester, Obstetrics and Gynecology, Rochester, NY OBJECTIVE: To compare levels of vitamin C and interleukin-6 (IL-6) in pregnancies undergoing midtrimester amniocentesis for genetic indications. STUDY DESIGN: Women undergoing midtrimester genetic amniocentesis were prospectively enrolled. The initial 1 cc of amniotic fluid obtained was immediately refrigerated and processed within 24 hours for vitamin C and IL-6 analysis. Data were collected onmaternal age, gestational age, and indication for amniocentesis. Demographic data, vitamin C levels, and IL-6 levels were analyzed with Pearson’s correlation and chi-square analysis. RESULTS: Data were available for 37 patients with normal karyotypes between the gestational ages of 16 and 21 weeks (mean 18.1 weeks). Mean maternal age was 35.1 years, with 66%of patients undergoing amniocentesis due to advanced maternal age. Vitamin C levels were noted to increase with advancing gestational age (r = 0.47, P = 0.003) but did not vary with maternal age or indication for amniocentesis. IL-6 values were not affected by these factors. A negative correlation was noted between vitamin C and IL-6 (r = -0.34, P = 0.034). CONCLUSION: Elevated levels of IL-6 in midtrimester amniotic fluid indicate intra-amniotic inflammation and have been associated with adverse pregnancy outcome. The inverse correlation between IL-6 and vitamin C suggests that consumption of vitamin C may occur as part of a chronic inflammatory process. Amniotic fluid vitamin C may also serve as a defense against chronic inflammation. Evaluation of the outcomes of these pregnancies may further clarify the potential role of vitamin C in the amniotic fluid.

Early mid-trimester amniotic fluid leptin concentration: indicator of fetal growth?

Early mid-trimester amniotic fluid leptin concentration: indicator of fetal growth?

Polymorphism in intron 2 of the fetal interleukin-1 receptor antagonist genotype influences midtrimester amniotic fluid concentrations of interleukin-1β and interleukin-1 receptor antagonist and pregnancy outcome

Objective Preterm labor in experimental models is initiated by intra-amniotic interleukin-1β (IL-1β) and inhibited by interleukin-1 receptor antagonist (IL-1ra). The IL-1ra gene is polymorphic and the different alleles are associated with variations in IL-1β and IL-1ra production. The relationship among the IL-1ra genotype of the fetus, concentrations of IL-1β and IL-1ra in second-trimester amniotic fluid, and pregnancy outcome was determined. Study design Amniotic fluids from 291 consecutive women with singleton pregnancies, obtained at 15 to 17 weeks' gestation, were tested for IL-1β and IL-1ra concentrations by enzyme-linked immunosorbent assay. DNA from fetal cells was analyzed for a length polymorphism in intron 2 of the IL-1ra gene by polymerase chain reaction. Pregnancy outcomes were obtained after completion of testing. Results The distribution of fetal IL-1ra genotypes was similar to that found in other populations: 50.9% (148) were homozygous for allele 1 (IL1RN ∗1), 39.5% (115) were IL1RN ∗1/allele 2 (IL1RN ∗2) heterozygotes, 6.9% (20) were IL1RN ∗2 homozygotes, whereas 2.7% (8) had combinations of other alleles. Fetal possession of IL1RN ∗2 was associated with a greater than 50% increase in midtrimester intra-amniotic IL-1β levels ( P = .006) and a smaller increase in IL-1ra levels ( P = .01) compared with fetuses who were IL1RN ∗1 homozygotes. Despite the low sample size, IL1RN ∗2 homozygosity, but not midtrimester intraamniotic levels of IL-1β and IL-1ra, was related to an increased rate of preterm birth ( P<.0001). In the 11 pregnancies that were subsequently terminated because of major malformations, there was a decreased frequency of IL1RN ∗1 homozygosity ( P = .04). Birth weight was unrelated to IL-1ra genotype. Conclusion Possession by the fetus of the IL1RN ∗2 allele is associated with enhanced intraamniotic IL-1β production. Induction of an intra-amniotic proinflammatory immune response might be more likely to lead to preterm labor in fetuses carrying the IL1RN ∗2 allele.

Differences in glycosylation and sperm-egg binding inhibition of pregnancy-related glycodelin.

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.

Amniotic levels of vascular endothelial growth factor and nitric oxide at the second trimester in Down's syndrome

Objective: Since placentae in trisomy 21 show trophoblastic hypoplasia and hypovascularity, we investigated amniotic fluid vascular endothelial growth factor (VEGF) and nitric oxide (NO) in normalpregnancy and pregnancy complicated by trisomy 21. Furthermore, we investigated a possible role of NO in neurodegeneration of the brain in Down's syndrome.Methods: We retrospectively assessedNO and VEGF on mid-trimester amniotic fluid from 15 women who had fetal Down's syndrome, and compared the results with those of 15 controls matched for age and gestation.Results: In pregnanciescomplicated by trisomy 21, NO levels were significantly higher than in healthy controls (p < 0.001), whereas VEGF levels were significantly lower than in healthy controls (p < 0.05).Conclusions: Our results suggest that the high levels of NO and the low levels of VEGF observed in the amniotic fluid of fetuses with Down's syndrome may be a sign of an imbalance of placentalvascularization and altered endothelial function. Overproduction of NO could contribute to pathological cell death in the central nervous system, a process that has been demonstrated in many neurodegenerativediseases.

Mid-trimester amniotic fluid angiogenin, lactate dehydrogenase and fibronectin in the prediction of preterm delivery

Objective: Our purpose was to compare angiogenin, lactate dehydrogenase (LDH) and fibronectin levels in mid-trimester amniotic fluid of patients with preterm and term deliveries and to find out their predictive values for preterm birth. Study Design: A prospective cohort study was conducted in 55 pregnancies with singleton gestations that underwent amniocentesis at 15–20 weeks for standard genetic indications. Amniotic fluid angiogenin, lactate dehydrogenase and fibronectin levels were measured by enzyme-linked immunosorbent assay (ELISA), radial immundiffusion technic and automated analyzer, respectively. Results: Five patients delivered preterm, five developed signs or symptoms of threatened preterm labor and 45 had term delivery after an uneventful pregnancy. Demographic data were not significantly different. Amniotic fluid angiogenin and lactate dehydrogenase levels were significantly higher in patients with preterm than term deliveries ( P<0.001 and 0.02, respectively). Receiver–operator characteristic curve analysis showed that the amniotic fluid angiogenin had the best screening efficiency in predicting preterm delivery. An angiogenin level of 35 ng/ml was the optimal cut-off value for the prediction of preterm delivery, with a sensitivity of 100% and specificity of 91%. Conclusion: Second-trimester angiogenin is found to be quite effective in the prediction of preterm delivery. Preexisting intrauterine ischemia may be an important risk factor for preterm delivery and already be present in the early mid-trimester.

Elevated Monocyte Chemoattractant Protein 1 in Midtrimester Amniotic Fluid

Elevated Monocyte Chemoattractant Protein 1 in Midtrimester Amniotic Fluid

Elevated monocyte chemoattractant protein 1 in midtrimester amniotic fluid: a risk for spontaneous abortion

Elevated monocyte chemoattractant protein 1 in midtrimester amniotic fluid: a risk for spontaneous abortion

Elevated amniotic fluid C-reactive protein at the time of genetic amniocentesis is a marker for preterm delivery

Objective: A pre-existing intrauterine inflammation in the first half of gestation has been proposed as a possible condition that leads to preterm delivery. Indeed, elevated levels of inflammatory mediators (eg, interleukin-6, tumor necrosis factor) in midtrimester amniotic fluid have been found in cases of preterm delivery and/or spontaneous abortion. The objective of this study was to investigate whether the amniotic fluid C-reactive protein level at the time of genetic amniocentesis is a marker for spontaneous preterm delivery before 34 and 37 weeks of gestation. Study Design: Women who underwent genetic amniocentesis between 15 and 18 weeks of gestation with (1) singleton gestation, (2) uneventful pregnancy course before the amniocentesis, and (3) absence of fetal abnormalities were included in the study. Patients with abnormal karyotype were excluded. C-reactive protein concentration was measured in amniotic fluid and in maternal blood immediately after genetic amniocentesis. All patients were followed until delivery for the occurrence of pregnancy complications. Nonparametric tests and receiver-operating characteristic curve analysis were used for statistical purposes. Results: The prevalence of spontaneous preterm delivery before 34 and 37 weeks was 3.3% (10 of 306 pregnancies) and 8.5% (26 of 306 pregnancies), respectively. Women with preterm delivery at <37 weeks had a higher median (range) of amniotic fluid C-reactive protein concentration than those women who delivered at term (median, 113.3 ng/mL [range, 16-623 ng/mL] vs median, 57.8 ng/mL [range, 0-808.9 ng/mL]; P <.005). Women with preterm delivery at <34 weeks had a higher median (range) amniotic fluid C-reactive protein concentration than those women who delivered at term (median, 183.8 ng/mL [range, 46.5-447 ng/mL] vs median, 57.8 ng/mL [range, 0-808.9 ng/mL]; P <.005]. No correlation was found between amniotic fluid C-reactive protein and maternal blood C-reactive protein concentrations. No relationship was found between maternal blood C-reactive protein concentration and preterm delivery before either 34 or 37 weeks. Amniotic fluid C-reactive protein concentration of >110 ng/mL had a sensitivity of 80.8% and a specificity of 69.5% in the prediction of spontaneous preterm delivery at <34 weeks. Conclusion: This study supports the theory that a subclinical intrauterine/fetal inflammatory process early in gestation may be important for the occurrence of preterm delivery in the second half of gestation. (Am J Obstet Gynecol 2002;186:·268-73.)

Cytokine Levels in Midtrimester Amniotic Fluid in Normal Pregnancy and in the Prediction of Pre‐eclampsia

Midtrimester amniotic fluid cytokines may reflect the function of the maternal immune system in the maternal-fetal interface and thus be predictive of pre-eclampsia. We determined the concentrations of interleukin (IL)-6, IL-8, IL-10, IL-11, IL-12, IL-15, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in amniotic fluid at 14-16 weeks of gestation from women with normal pregnancies and from those who subsequently developed severe pre-eclampsia. The concentrations of the cytokines in amniotic fluid did not significantly differ between patients and normal controls. The median concentration of IL-6 was 950 pg/ml in normal pregnant women and 578 pg/ml in the patient group. The median concentration of IL-8 was 606 pg/ml in normal controls and 294 pg/ml in the patient group. The levels of IL-6, IL-8 and TGF-beta correlated positively with each other. TNF-alpha concentrations were low and similar in both groups. IL-10 and IL-12 were detected at very low levels in 37 and 7% of the samples, respectively. No difference was found in IL-15 concentrations between the groups. IL-11 was found only at low levels in both groups. Although none of the cytokines measured was predictive of pre-eclampsia, this study provides information of cytokines in amniotic fluid during the period when the spiral arteries are remodelled.

Abnormal TGFbeta levels in the amniotic fluid of Down syndrome pregnancies.

The status of cytokines in amniotic fluid (AF) from chromosomally abnormal pregnancy is largely undefined. TGFbeta plays a key role in fetal growth and differentiation and is responsible for the immunoregulatory activity of AF in normal pregnancy, but its status in Down syndrome (DS) pregnancies is unknown. In addition we investigated the IL-2 status of AF from DS pregnancies. Midtrimester AF from chromosomally normal (n = 25) and abnormal pregnancies with DS (n = 15) were assayed for bioactive and latent TGFbeta levels using the mink lung epithelial cell growth inhibition bioassay and for IL-2 activity by the CTLL-2 cell proliferation bioassay and by ELISA. Levels of bioactive TGFbeta (mean 4.6+/-0.6 U/mL) were significantly increased in DS AF compared with the normal samples (mean 2.8+/-0.3 U/mL, P<0.003) but latent TGFbeta levels did not differ between DS and normal groups. In addition DS AFs showed reduced IL-2 like bioactivity compared with normal samples (P = 0.0006) but IL-2 immunoactivity was undetectable in DS and normal AFs by ELISA. DS AFs display increased TGFbeta activity and lacked IL-2 immunoactivity. The reduced ability of DS AFs to stimulate CTLL-2 cell proliferation is unrelated to the IL-2 status of AF. Altered TGFbeta levels may prove useful as an additional biochemical index for the detection of DS pregnancies.

▪ An Assessment of the Effects on Coagulation of Midtrimester and Final-Trimester Amniotic Fluid on Whole Blood by Thrombelastograph® Analysis

▪ An Assessment of the Effects on Coagulation of Midtrimester and Final-Trimester Amniotic Fluid on Whole Blood by Thrombelastograph® Analysis

Human amniotic fluid lacks interleukin-2 and interleukin-15 but can interact with the β-chain of the interleukin-2 receptor

The present study investigated whether an explanation for the conflicting reports on the interleukin-2 (IL-2) status of amniotic fluid is due to the presence of IL-15 which shares biological activities with IL-2 and utilizes the IL-2 receptor beta-chain. Amniotic fluids from 45 normally progressing pregnancies between 14 and 16 weeks after the last menstrual period were assayed for IL-2 and IL-15 by bioassay and enzyme-linked immunosorbent assay (ELISA). The ability of amniotic fluids to induce cytotoxic T lymphoblastoid line-2 (CTLL-2) cell proliferation was demonstrated to be dependent upon bioassay culture conditions. In serum-free medium each amniotic fluid stimulated CTLL-2 proliferation with a mean level of IL-2-like bioactivity of 14.7 +/- 2.3 ng/ml but amniotic fluids failed to induce CTLL-2 proliferation in serum-supplemented medium. Treatment with neutralizing anti-IL-2 or anti-IL-15 antibodies failed to inhibit amniotic fluid-induced CTLL cell proliferation in serum-free medium, indicating a lack of IL-2 and IL-15 bioactivity. In contrast, treatment with anti-IL-2 receptor beta-chain antibody significantly reduced amniotic fluid-induced proliferation. The lack of IL-2 and IL-15 activity in amniotic fluids was confirmed using ELISA. Although high levels of IL-15 immunoactivity were detected in all samples, specificity controls showed a lack of specific IL-15 immunoactivity in amniotic fluid. Pretreatment of amniotic fluids with 100-500 ng/ml mouse immunoglobulin G abrogated IL-15 immunoactivity, indicating that amniotic fluid contains molecules binding to Fc regions of immunoglobulins and responsible for false ELISA positivity. These studies unequivocally show that amniotic fluid lacks IL-2 and IL-15 but can stimulate CTLL-2 cell proliferation via the IL-2 receptor beta-chain. The absence of IL-2 and IL-15 in normal mid-trimester amniotic fluid suggests that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto-placental unit.

An Assessment of the Effects on Coagulation of Midtrimester and Final-Trimester Amniotic Fluid on Whole Blood by Thrombelastograph® Analysis

The Thrombelastograph® test (TEG; Haemoscope Corporation, Skokie, IL) was used to assess the effects of midtrimester and final-trimester amniotic fluid (AF) on whole blood coagulation. Different volumes of midtrimester and final-trimester AF were added to whole blood from nonpregnant volunteers in a series of TEG tests. The addition of both midtrimester and final-trimester AF resulted in significant decreases in reaction time (P < 0.001) and time from reaction to a fixed level of clot firmness (P < 0.05) and significant increases in angle (P < 0.05) and coagulation index (P < 0.05) values. This reflects accelerated clot initiation and propagation. There was no significant change in the maximal amplitude or % lysis at 30 and 60 min with the addition of either midtrimester or final-trimester AF. There was no significant difference between the effects of midtrimester and final-trimester AF on whole blood TEG. TEG may be an additional useful tool in the treatment of coagulopathy in AF embolism. Implications We used the Thrombelastograph® test (Haemoscope Corporation, Skokie, IL) to assess the effects of midtrimester and final-trimester amniotic fluid (AF) on whole blood coagulation. Results demonstrate that AF accelerates clot initiation and propagation. The Thrombelastograph® test may be useful in assessing coagulopathy in patients with AF embolism.

Involvement of fibrinolytic factors in mid trimester amniotic fluid with development of severe early-onset preeclampsia.

Impaired placental development is a well-known pathogenesis in preeclampsia. The present study was undertaken to elucidate the involvement of fibrinolytic factors in amniotic fluid in midtrimester with development of severe early-onset preeclampsia. Amniotic fluid was obtained by amniocentesis at 15 to 18 weeks of gestation. All specimens were retrospectively identified according to the hospital records as coming from gestations that later had severe early-onset preeclampsia (severe preeclamptic group, n = 9) or gestations with normal outcomes (control group, n = 73). Fibrinolytic factors such as tissue plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and t-PA-PAI-1 complex (PAI-C) of specimens were measured by enzyme-linked immunoassay. In the control group, concentrations of t-PA as well as PAI-1 in amniotic fluid remained at similar levels from 15 to 18 weeks, although PAI-1 levels were more than 10 times higher compared with t-PA levels. Levels of t-PA and PAI-1 in the severe preeclamptic group were not different from those of the control group. PAI-C levels gradually decreased from 15 through 18 weeks of gestation in the control group. PAI-C levels of the severe preeclamptic group were significantly lower than those of the control group (55.5 +/- 18.0% versus control; mean +/- standard deviation [SD], p <0.001). PAI-C, as the most specific indicator of the early stage of fibrinolytic activities, showed lower levels in midtrimester amniotic fluid in the severe preeclamptic group, suggesting fibrinolytic activities of amniotic fluid may have a significant role in the development of severe early-onset preeclampsia via impaired placental development in the latent stage of preeclampsia.

Cytogenetic aspects of the Canadian early and mid-trimester amniotic fluid trial (CEMAT)

Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0-12+6 weeks (EA) and 1999 fluids at 15+0-16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA. Maternal cell contamination was not significantly different between the groups, but maternal cells only were analysed from one bloody EA fluid. The number of repeat amniocenteses because of cytogenetic problems was 2.2 per cent in the EA group compared with only 0.3 per cent in the MA group. On average, culture of EA fluids required one day more than MA fluids. Although both culture success (97.7 per cent) and accuracy (99.8 per cent) were high for patients randomized to the EA group, routine amniocentesis prior to 13 weeks' gestation is not recommended for clinical reasons including an increased risk of fetal loss and talipes equinovarus.

Increased midtrimester amniotic fluid activin A: A risk factor for subsequent fetal death

Objective: The objective of this study was to determine whether concentrations of activin A and corticotropin-releasing factor in amniotic fluid can identify patients at risk of fetal death. Study Design: A retrospective case-control study of women who have had a midtrimester amniocentesis was designed. Case subjects consisted of patients who had a spontaneous fetal death after the procedure, whereas the control group consisted of patients who had a normal pregnancy outcome after midtrimester amniocentesis. Dimeric activin A was measured by a specific 2-site enzyme immunoassay, and corticotropin-releasing factor was measured by a specific and sensitive radioimmunoassay after acidic extraction. Statistical analysis was performed with Mann-Whitney U test, Fisher's exact test, and χ 2 tests and regression analysis. Results: First, activin A was detectable in all amniotic fluid samples. Second, the concentration of activin A in amniotic fluid increased with advancing gestational age. Third, patients who subsequently had a fetal death had a higher median concentration of activin A than those with a normal pregnancy outcome ( P < .01). Fourth, an amniotic fluid concentration of activin A greater than the 95th confidence interval for gestational age was found in 40% of patients who subsequently had a fetal death (odds ratio: 21.6; P < .005). Finally, the median concentration of corticotropin-releasing factor in amniotic fluid was not different in case subjects and control subjects. Conclusions: An elevated concentration of activin A in amniotic fluid identifies women at risk of fetal death. (Am J Obstet Gynecol 1999;180:194-7.)

Elevated IL-6 in midtrimester amniotic fluid is involved with the onset of preeclampsia.

The primary defect of placental development in preeclampsia is speculated to occur at midtrimester gestation. Abnormal feto-maternal immune reactions have been considered as factors in such defective placentation. Midtrimester amniotic fluid specimens were retrospectively identified as coming from gestations that later had severe preeclampsia develop, gestations with normal outcomes, and gestations measured for cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL-1 beta, IL-6, and IL-8). The effect of each cytokine on thrombomodulin levels was tested in cultured trophoblast cells. Among the measured cytokines, IL-6 and IL-8 were significantly elevated in the midtrimester amniotic fluid of the future preeclamptic group. Trophoblasts stimulated with TNF-alpha plus IL-6 had significantly decreased levels of cellular thrombomodulin compared to those without cytokine addition. Elevated cytokines in midtrimester amniotic fluid suggest an abnormal fetomaternal immune response occurring before the clinical manifestation of preeclampsia. Cytokine-induced suppression of thrombomodulin in trophoblasts may be directly involved in the pathogenesis of preeclampsia.

Are the cytokines interleukin-6 and angiogenin stable in frozen amniotic fluid?

OBJECTIVE: To determine the stability of cytokines in frozen stored amniotic fluid samples, we measured angiogenin, a potent inducer of neovascularization and interleukin-6, an inflammatory cytokine, in the same sample of midtrimester amniotic fluid 1 year apart. STUDY DESIGN: In this study paired aliquots of amniotic fluid kept at –70° C were immunoassayed for angiogenin and interleukin-6 at two different time periods 1 year apart. Inclusion criteria were (1) samples with clearly identifiable numbers, (2) no evidence of breaks in sealing of samples, and (3) singleton gestation. Amniotic fluid was immunoassayed for angiogenin and interleukin-6 in July 1995 and 1996. Angiogenin sensitivities were 0.078 and 0.026 ng/ml, interassay coefficients of variation were 3.8% and 4.6%, and intraassay coefficients of variation were 2.7% and 2.9%, respectively, in 1995 and 1996. Interleukin-6 sensitivities were 1.74 and 2.37 pg/ml, interassay coefficients of variation were 8.9% and 2.6%, and intraassay coefficients of variation were 3.5% and 1.9%, respectively, in 1995 and 1996. Statistical analysis included paired t test, Wilcoxon signed-rank test, and regression with p < 0.05 significant. Angiogenin and interleukin-6 values were normalized with natural log transformation for statistical analysis. RESULTS: Paired amniotic fluid samples from 30 patients were immunoassayed from 1993 to 1995. The values of angiogenin were significantly lower in the 1996 assay compared with the 1995 assay (median 12.4 [range 5.6 to 61.3] vs 26.7 [range 13.6 to 159.2] ng/ml, p < 0.001). A significant correlation was found between the change in angiogenin levels and the year of the sample, with older samples having the greatest change in values ( r = 0.5, p = 0.008). The values of interleukin-6 were significantly lower in the 1996 assay compared with the 1995 assay (median 230.8 [range 40.9 to 3711.3] vs 289.2 [range 53.7 to 19100.0] pg/ml, p < 0.001). CONCLUSIONS: Angiogenin and interleukin-6 values in amniotic fluid appear to decrease with time despite optimal freezing conditions. The year of sampling and length of storage should be taken into consideration when evaluating amniotic fluid cytokine levels from stored samples. (Am J Obstet Gynecol 1998;178:783-6.)