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Elevated Amniotic Fluid Nucleosome Levels in Women With Intra-amniotic Infection

From the Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut.

Address reprint requests to: Chaur-Dong Hsu, MD, MPH, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520-8063

	Abstract

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Objective: To compare amniotic fluid (AF) soluble nucleo-some levels in pregnant women with and without intra-amniotic infection.

Methods: Amniocentesis was performed in 74 pregnant women with preterm contractions, labor, or premature rupture of membranes. Intra-amniotic infection was defined as a positive AF culture. Amniotic fluid tests for Gram stain, glucose, neutrophils, creatinine, pH, and specific gravity were performed. Amniotic fluid soluble nucleosome levels were determined by enzyme-linked immunosorbent assay and were normalized by AF creatinine levels.

Results: Twenty-eight patients had intra-amniotic infection and 46 did not. Amniotic fluid soluble nucleosome levels were significantly higher in pregnant women with intra-amniotic infection than in those without infection (48.1±21.3 compared with 0.0 ± 0.0 U/mg creatinine; P = .005). The AF nucleosome levels were positively correlated with AF neutrophil counts and negatively correlated with AF glucose concentrations.

Conclusion: Our data indicate that elevated AF nucleo-some levels are associated with intra-amniotic infection and may have potential as a clinical marker to detect intra-amniotic infection.

Intra-amniotic infection is a clinical or subclinical infection of the amniotic fluid (AF) and intrauterine contents during pregnancy. It may result in substantial perinatal mortality and morbidity.^1,2 The diagnosis is made from clinical symptoms and signs such as fever, maternal and fetal tachycardia, uterine tenderness, peripheral blood leukocytosis, and foul-smelling vaginal discharge.^1,2 However, these symptoms and signs are usually late and nonspecific,³ so direct examination of the AF is frequently necessary.

Nucleosomes are the end products of DNA fragmentation during apoptosis,⁴ a process by which single cells undergo death and deletion from a tissue (programmed cell death).⁵ The nucleosome consists of 180–200 base pairs of DNA around a histone core, which are protected conformationally from digestion by endonuclease. The formation of nucleosomes is recognized to be a hallmark of apoptosis.⁴ A linear correlation has been demonstrated between the amount of nucleosomes and the morphology of apoptosis.⁶ Nucleosomes can be released spontaneously from apoptotic bodies if they have not undergone phagocytosis, and they have important immunoproliferative effects in vitro.⁷

The relation between apoptosis and inflammation has been reported recently.⁸ Apoptosis acts as a proinflammatory event. Infection of cells by pathogenic microorganisms can induce cell apoptosis and then promote an inflammatory response that causes tissue damage or necrosis.⁸ To our knowledge, the relevance of apoptosis to intra-amniotic infection has not been investigated. We sought to determine the role of apoptosis in intra-amniotic infection by measuring the AF levels of nucleosomes in patients with and without intra-amniotic infection.

	Materials and Methods

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This investigation was performed at Yale-New Haven Hospital with the approval of the institutional Human Investigational Committee. Amniotic fluid samples were obtained from the Amniotic Fluid Bank at our institution. Women with singleton pregnancies who presented with preterm contractions, preterm labor, premature rupture of membranes (PROM), or other suspicious signs and symptoms were offered amniocentesis to rule out intra-amniotic infection. The exclusion criteria were multiple gestations and bloody taps. Preterm contractions were defined as two or more contractions every 10 minutes at a gestational age of less than 35 weeks. Preterm labor included cervical changes. The gestational age for PROM was limited to 35 weeks. Before we obtained the consent form, we explained the study protocol to obtain extra AF for the investigation of potential markers for intra-amniotic infection. Amniocentesis was performed transabdominally under ultrasound guidance by the private physician or a maternal-fetal medicine staff member. Intra-amniotic infection was defined as a positive AF culture.

Amniotic fluid was sent immediately to the clinical laboratory for microscopic cultures (aerobic and anaerobic bacteria and mycoplasma), Gram stain, neutrophil count, cell differentiation, glucose, pH, and specific gravity. MacConkey agar and Thayer-Martin medium were used for aerobic culture. Blood agar, Bacteroides-bile-esculin agar/laked kanamycin-vancomycin agar, and anaerobic agar were used for anaerobic culture. Mycoplasma hominis and Ureaplasma urealyticum were cultured with the Mycotrim-GU system (Irvine Scientific, Santa Ana, CA). The remaining AF was centrifuged immediately at 1500 rpm for 10 minutes at 4C. The AF supernatant was divided into 500-µL aliquots and stored in polypropylene tubes at -80C until assays were performed.

Seventy-four AF samples of 74 patients were obtained from the Amniotic Fluid Bank collected between July 1995 and December 1997. Twenty-eight patients had intra-amniotic infection and 46 did not. Among the samples showing intra-amniotic infection, 11 were U urealyticum, six were mixed microorganisms, four were Streptococcus viridans, three were Fusobacterium sp, two were Haemophilus sp, one was Candida albicans, and one was Enterobacter sp. According to our preliminary observation, the AF nucleosome concentration was undetectable in patients without intra-amniotic infection. Twenty-eight patients with intra-amniotic infection were needed to detect a 30% difference in AF nucleo-some levels with a power of 0.8 and an of .5 between patients with and without intra-amniotic infection.

Amniotic fluid soluble nucleosome levels were measured using an enzyme-linked immunosorbent assay (ELISA) (Oncogene Research Products, Cambridge, MA). The intra-assay and interassay coefficients of variation were both less than 10%. The lower limit of detection (sensitivity) was 0.2 U/mL. Samples were not subjected to freeze-thaw cycles before analysis.

Amniotic fluid creatinine levels were measured using the alkaline picrate method (Sigma Chemical Company, St. Louis, MO). Amniotic fluid soluble nucleosome levels were normalized by AF creatinine concentrations.

The two-tailed Student t test was used to analyze the AF levels of soluble nucleosomes, creatinine, neutrophil count, glucose level, pH, and specific gravity, as well as continuous variables of maternal characteristics. The Mann-Whitney U test was used to compare the intra-amniotic infection samples with detectable and undetectable nucleosome levels. Pearson correlation coefficient was used to analyze the correlations among nucleosomes, glucose, and neutrophil counts. P < .05 was considered statistically significant.

	Results

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There were no significant differences in maternal age, parity, gestational age, race, PROM, cervical dilatation at amniocentesis, AF pH, specific gravity, or creatinine levels between pregnant women with and without intra-amniotic infection (Table 1). Pregnant women with intra-amniotic infection had significantly higher rates of PROM, AF neutrophil counts (Table 1), and concentrations of nucleosomes (Figure 1), and significantly lower glucose concentrations, than those without intra-amniotic infection (Table 1). Patients with intra-amniotic infection also had significantly shorter amniocentesis-to-delivery intervals and lower gestational age at delivery (Table 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Amniotic fluid nucleosome levels were significantly higher in intra-amniotic infection (IAI) (48.1 ± 21.3 U/mg creatinine) than in women without intra-amniotic infection (non-IAI) (0.00 U/mg creatinine) (P = .005).

Within the intra-amniotic infection group, those with detectable nucleosome levels (n = 11) had significantly higher neutrophil counts than those with undetectable nucleosome levels (n = 17) (Table 2). However, there were no significant differences in maternal and neonatal outcomes between the groups.

	Discussion

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Our data demonstrate that pregnant women with intra-amniotic infection have significantly higher AF levels of nucleosomes than those without intra-amniotic infection. Amniotic fluid neutrophils and the fetal amnion are two possible sources for soluble nucleosomes. Neutrophils are the predominant immune cells that transmigrate through the epithelial lining of the amnion during infection.¹⁵ Further, neutrophils can undergo apoptosis and release abundant nucleosomes during infection.^11,16 This is supported by the finding of a positive correlation between AF nucleosome levels and neutrophil counts. As AF nucleosome levels become elevated in intra-amniotic infection, the negative correlation between AF nucleosomes and glucose levels may simply indicate an indirect correlation between increased glucose consumption and apoptosis.

The fetal amnion may be another site for the generation of elevated AF nucleosomes in intra-amniotic infection. We have recently localized apoptosis in the fetal amnion using immunohistochemistry in patients with intra-amniotic infection (data not shown). It is likely that nucleosomes can be released from these apoptotic amniocytes.

Many of the positive culture samples had nucleosome levels below the detection range. It is likely that our ELISA kit was not sensitive enough to detect these relatively low nucleosome concentrations. We speculate that these patients may have been in the early stage of infection or had relatively mild forms, so that the majority of apoptotic cells were still in the condemned phase (not in the execution phase)¹⁷ and only a few nucleosomes were released. We also noted significantly lower AF neutrophil counts than in those samples with detectable nucleosomes (Table 2). Further study with improved sensitivity for nucleosomes can prove this hypothesis and establish an adequate cutoff value to diagnose intra-amniotic infection. Although we did not observe any significant differences in demographic and clinical data between the groups with detectable versus undetectable nucleosomes, this analysis was limited by the small sample sizes in these two subgroups.

	Footnotes

 
PII S0029-7844(99)00250-1

Received August 17, 1998. Received in revised form December 18, 1998. Accepted January 7, 1999.

	References

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