Characterization of amniotic fluid sludge in preterm and term gestations

Abstract

Objective:

To describe the characteristics of amniotic fluid sludge obtained from patients in term and preterm gestations.

Methods:

This cross-sectional study included patients with dense aggregates of particulate matter detected in amniotic fluid, observed with transvaginal sonography. All patients were in labor and had an impending delivery, either preterm or at term. Echogenic material contained within amniotic fluid was retrieved transvaginally by needle amniotomy under direct visualization. Amniotic fluid analysis consisted of a Gram stain, cultures for aerobic/anaerobic bacteria and genital mycoplasmas, and a white blood cell count.

Results:

Twenty-five patients ranging from 18 to 41 weeks of gestation were included in the study. We observed the following: (1) the appearance of amniotic fluid was consistent with pus-like material, vernix, or meconium by naked eye examination; (2) samples collected before 33 weeks of gestation (n=13) had a pus-like appearance; however, after this gestational age, most of the samples [83% (10/12)] appeared to be consistent with vernix; (3) amniotic fluid cultures were positive for microorganisms in 13 patients, of which 10 were preterm gestations before 33 weeks; (4) the most frequent microorganisms retrieved by culture were genital mycoplasmas (Ureaplasma urealyticum [46% (6/13)], followed by Mycoplasma hominis [31% (4/13)] and Candida albicans [15% (2/13)]; and (5) patients with sonographic particulate matter in preterm gestations frequently presented acute histologic chorioamnionitis and funisitis, but these conditions were rare in patients at term.

Conclusion:

The nature of amniotic fluid particulate material varies as a function of gestational age. The material obtained in preterm gestations is frequently related to an inflammatory process, while that obtained at term is often consistent with vernix and appears to represent a maturational process.

INTRODUCTION

Sonographic particulate matter in amniotic fluid can correspond to vernix [1–6], meconium [7,8], blood [9], and inflammatory material associated with intraamniotic infection or intraamniotic inflammation [10–13], or to desquamated skin cells in the case of congenital ichthyosis [14,15]. We use the term ‘sludge’ to describe particulate matter in amniotic fluid, identifying patients who present with preterm labor [13,16], a short cervix [17], and cervical insufficiency (also referred to as advanced cervical dilatation) [10,18,19]. Amniotic fluid sludge in the mid-trimester is a risk factor for intraamniotic infection and intraamniotic inflammation [10–13], spontaneous preterm delivery [17,20–22], acute histologic chorioamnionitis, and funisitis [10,13,16,17,20,22–32]. By contrast, particulate matter at term often reflects fetal maturation and has been reported as representing either vernix [1–6] or meconium [7,8], largely based on case reports.

This study was undertaken to characterize the appearance, microbiology, and inflammatory status of amniotic fluid, as well as the placental/extraplacental membranes and umbilical cord, in patients with particulate matter in amniotic fluid in term and preterm gestations.

MATERIALS AND METHODS

Study population

This cross-sectional study included patients with particulate matter, detected with transvaginal sonography, who had an impending delivery, either preterm or at term. Amniotic fluid was retrieved transvaginally by needle amniotomy under direct visualization during labor. Women who agreed to undergo this procedure were asked to donate additional amniotic fluid and to allow the collection of clinical information for research purposes. All patients provided written informed consent prior to the procedure and the collection of samples. The use of biological specimens as well as clinical and ultrasound data for research purposes was approved by the Human Investigation Committee of Wayne State University (Detroit, MI, USA).

Sample collection

Under sterile speculum examination, the membranes were cleansed with saline and 10% povidone-iodine (Scrub Care^®, Cardinal Health, Aurora, IL, USA). A needle amniotomy was performed and the sonographic particulate matter was immediately aspirated under direct visualization. Samples of amniotic fluid were transported to the laboratory in a sterile, capped syringe and cultured for aerobic/anaerobic bacteria as well as genital mycoplasmas. The white blood cell count [33], glucose concentration [34], and Gram stain [35] were also performed in amniotic fluid shortly after collection, as previously described. Shortly after amniocentesis, amniotic fluid not required for clinical assessment was centrifuged at 1,300×g for 10 min at 4 °C, and the supernatant was aliquoted and stored at −80 °C until analysis.

Determination of interleukin-6 in amniotic fluid

Interleukin (IL)-6 concentrations were determined to assess the presence and magnitude of the intraamniotic inflammatory response, by using a sensitive and specific enzyme immunoassay obtained from R&D Systems (Minneapolis, MN, USA). The details of the assay, including sensitivity and the inter- and intra-assay coefficients of variation, have been previously reported [36,37]. The cut-off value of 2.6 ng/mL has been previously reported for the diagnosis of intraamniotic inflammation [36–39].

Placental histopathologic examination

Placentas were collected in the Labor and Delivery Unit or Operating Room at Hutzel Women’s Hospital of the Detroit Medical Center and transferred to the Perinatology Research Branch laboratory (Detroit, MI, USA). A sampling of the placentas was conducted according to protocols of the Perinatology Research Branch, as previously described [40–45]. A minimum of five full-thickness sections of the chorionic plate, three sections of the umbilical cord, and three chorioamniotic membrane rolls from each case were examined by placental pathologists who were blinded to the clinical histories and additional testing results. Acute inflammatory lesions of the placenta (maternal inflammatory response and fetal inflammatory response) were diagnosed according to established criteria, including staging and grading [43,45,46]. Severe acute placental inflammatory lesions were defined as stage 3 and/or grade 2 [43,45].

Statistical analysis

Comparisons between proportions were performed with the Fisher’s exact test.

RESULTS

Twenty-five patients ranging from 18 to 41 weeks of gestation were included in the current study. Table 1 illustrates the clinical characteristics, appearance of the particulate material in amniotic fluid, amniotic fluid white blood cell count, glucose concentration, Gram stain, microbiology by culture, and the presence or absence of acute inflammatory lesions of the placenta.

Table 1.

Clinical characteristics, appearance of particulate material in amniotic fluid, amniotic fluid white blood cell count, glucose concentration, Gram stain, microbiology by culture, and the presence or absence of acute inflammatory lesions of the placenta

Patient number	Clinical presentation	GA at amniocentesis (weeks)	Amniotic fluid results						GA at delivery (weeks)	Acute chorioamnionitis		Acute funisitis
			Appearance	Color	Glucose (mg/dL)	WBC (cells/mm3)	Gram stain	Microorganisms detected by cultivation		Present	Severe lesions	Present	Severe lesions
1	Cervical insufficiency	18.7	Pus	Yellow	N/A	560	Positive	Sthaphylococcus simulans	18.9	Yes	Yes	Yes	Yes
2	Preterm labor	20.7	Pus	Yellow	22	54	Negative	Ureaplasma urealyticum	20.7	Yes	No	No	No
3	Preterm labor	21.3	Pus	Yellow	10	10400	Negative	Ureaplasma urealyticum, Prevotell a species	21.3	Yes	Yes	Yes	No
4	Preterm labor	21.6	Pus	Straw	10	800	Positive	Candida albicans, Proteus mirabilis, Corynebacterium spp, Ureaplasma urealyticum	21.6	Yes	Yes	Yes	Yes
5	Preterm labor	21.7	Pus	Yellow	<20	1564	Positive	Prevotell a species, Fusobacterium nucleatum	21.9	Yes	Yes	Yes	Yes
6	Preterm labor	21.7	Pus	Yellow	30	310	Negative	Negative	21.7	No	No	No	No
7	Preterm labor	23.7	Pus	Hazy	10	6	Negative	Streptoccocus viridans	23.7	Yes	No	No	No
8	Fetal death	26	Pus	Yellow	10	2520	Positive	Gemella moribillorum, Peptostre ptococcus spp, Prevotell a melaninogenica Ureaplasma urealyticum, Mycoplasma hominis	26	Yes	Yes	Yes	No
9	Preterm labor	26.4	Pus	Yellow	15	0	Positive	Lactobacillus gensenii, Streptococcus anginosus, Ureaplasma urealyticum, Mycoplasma hominis	26.4	Yes	No	Yes	No
10	Preterm labor	27.4	Pus	Yellow	36	1	Negative	Negative	28	Yes	No	No	No
11	Preterm labor	28.7	Pus	Milky	9	19650	Positive	Streptococcus mutans, Aspergill us flavus, Mycoplasma hominis	28.7	Yes	Yes	Yes	No
12	Preterm labor	32.1	Pus	Yellow	25	15	Negative	Negative	32.1	Yes	No	No	No
13	Preterm labor	32.6	Pus	Yellow	10	100	Positive	Candida albicans	32.6	No	No	Yes	No
14	Preterm labor	33	Vernix	Straw	10	650	Negative	Negative	33	Yes	No	No	No
15	Preterm labor	33.9	Vernix	White	17	13	Negative	Negative	33.9	No	No	No	No
16	Preterm labor	36.9	Vernix	Yellow	10	0	Negative	Negative	36.9	No	No	No	No
17	Term labor	38.9	Vernix	White	20	7	Negative	Negative	38.9	No	No	No	No
18	Term labor	39.1	Vernix	Str aw	10	0	Negative	Negative	39.1	No	No	No	No
19	Term labor	39.3	Vernix	Straw	10	0	Negative	Negative	39.4	No	No	No	No
20	Term labor	39.7	Vernix	White	30	5560	Negative	Ureaplasma urealyticum	39.7	No	No	Yes	No
21	Term labor	39.9	Pus	Yellow	N/A	N/A	Negative	Enterococcus faecalis, Lactobacillus species, Mycoplasma hominis	39.9	No	No	No	No
22	Term labor	40.7	Vernix	Milky	10	900	Positive	Clostridium innocuum, Mobiluncus mulieris	40.7	No	No	No	No
23	Term labor	40.9	Meconium	Green	14	0	Negative	Negative	40.9	No	No	No	No
24	Term labor	41	Vernix	Clear	10	0	Negative	Negative	41	Yes	No	No	No
25	Term labor	41.6	Vernix	Straw	10	30	Negative	Negative	41.7	No	No	No	No

GA; gestational age, WBC; white blood cell

The appearance of the material by the naked eye was identified as pus-like, vernix, or meconium (Figure 1). All samples of early preterm gestations (<33 weeks, n=13) contained pus-like material. After 33 weeks of gestation and, in particular, at term, most of the cases [83% (10/12)] were consistent with vernix. Pus-like material was only present in one patient at term. A positive amniotic fluid culture for bacteria was more frequent in early preterm samples than in those collected closer to term [77% (10/13) vs. 25% (3/12); p=0.02]. The most frequent microorganisms isolated were Ureaplasma urealyticum [46% (6/13)], followed by Mycoplasma hominis [31% (4/13)] and Candida albicans [15%, (2/13)]. In 54% (7/13) of these cases, infections were polymicrobial. The frequency of acute histologic chorioamnionitis [85% (11/13) vs. 17% (2/12); p=0.0012], as well as funisitis [62% (8/13) vs. 0% (0/12); p=0.0016], was higher in patients who were early preterm than in those close to term gestation. None of the patients with particulate matter at term presented severe acute placental inflammatory lesions (Table 1).

Figure 1.

Sonographic particular matter in amniotic fluid was identified as pus-like, vernix, or meconium.

Figure 2 displays an illustrative case of spontaneous preterm labor and delivery at 32.6 weeks of gestation (patient number 13), showing the presence of dense, irregular aggregates of particulate matter at the bulging membranes during transvaginal ultrasound (Figure 2a). Particulate matter in amniotic fluid was collected through transvaginal amniocentesis and macroscopically appeared as pus-like material (Figure 2b). The amniotic fluid glucose concentration was 10 mg/dL, the white blood cell count was 100 cells/mm³, and the Gram stain was positive for bacteria. The concentration of IL-6 in amniotic fluid was extremely elevated (79.6 ng/mL), consistent with intraamniotic inflammation. The amniotic fluid culture was positive for Candida albicans (Figure 2c). The fetus presented systemic fetal inflammatory response syndrome, determined by an elevated cord blood IL-6 concentration (110 pg/ml) and the presence of funisitis. The neonate had an erythematous maculopapular rash on the back and head, considered the result of a Candida infection (Figure 2d).

Figure 2.

A case of spontaneous preterm labor and delivery at 32.6 weeks of gestation (patient number 13) (a) Two-dimensional transvaginal ultrasound shows bulging membranes in the vagina containing a dense, irregular, and hyperechogenic aggregate of particulate matter. (b) The particular matter in amniotic fluid was collected by transvaginal amniocentesis and macroscopically appeared as pus-like material. (c) The amniotic fluid culture was positive for Candida albicans. (d) The neonate had an erythematous maculopapular rash on the back and head, considered as a Candida infection.

Figure 3 displays a case of spontaneous preterm labor and delivery at 33.9 weeks of gestation (patient number 15), showing homogeneous particulate matter close to the cervix (Figure 3a). Particulate matter in amniotic fluid was collected through transvaginal amniocentesis. This matter was opaque with a whitish appearance resembling vernix (Figure 3b) and had only flat epithelial cells without bacteria (Figure 3c); the results of amniotic fluid analysis and histopathologic examination of the placenta did not show any evidence of inflammation.

Figure 3.

A case of spontaneous preterm labor and delivery at 33.9 weeks of gestation (patient number 15) (a) Two-dimensional transvaginal ultrasound shows free-floating particles and an aggregate of particulate matter close to the uterine cervix. (b) The amniotic fluid was opaque with a whitish aspect, resembling vernix. (c) Only epithelial cells were observed in the Gram stain.

DISCUSSION

Principal findings of the study

The main finding of our study indicates that, in preterm gestations, sonographic amniotic fluid particulate matter often corresponds to an inflammatory process, while in term gestations, it is associated with either vernix or meconium. Therefore, the clinical significance of particulate matter is related to gestational age at diagnosis.

Sonographic particulate matter in amniotic fluid

Particulate matter observed during the second trimester of pregnancy has been attributed to intraamniotic inflammation or intraamniotic infection [10–13], bleeding [9], desquamated material such as in the case of an acrania-anencephaly sequence [47–49], or congenital ichthyosis [14,15]. Patients with an elevated maternal serum alpha-fetoprotein result have also been reported to have echogenic amniotic fluid [50], and this finding has been interpreted as reflecting intraamniotic bleeding.

A question that often emerges in clinical practice is whether the diagnosis of particulate matter requires amniocentesis to ascertain the presence or absence of intraamniotic infection or intraamniotic sterile inflammation and whether the sonographic characteristics of the particulate matter can provide insight as to the nature of the material responsible for the sonographic image.

Amniotic fluid sludge as a sonographic sign of intra-amniotic infection/inflammation in preterm gestation

The term amniotic fluid ‘sludge’ was introduced to refer to the presence of dense aggregates of particulate matter in close proximity to the internal cervical os in patients with spontaneous preterm labor and intact membranes [16]. We subsequently found that sludge corresponds to a microbial biofilm in some cases [11].

Table 2 displays a summary of previous studies in which amniotic fluid sludge has been detected in patients with preterm labor and intact membranes, a short cervix, and cervical insufficiency [16,17,29,51]. Sludge was present in 18–23% of patients with preterm labor and intact membranes and was associated with intraamniotic infection (defined as a positive amniotic fluid culture for microorganisms) in 33% of cases [16,51]. The majority of patients had histologic chorioamnionitis (77.8%) and delivered within 7 days (71.4%) among those who presented with sludge [16]. Genital mycoplasmas were the most frequent microorganisms reported in cases with amniotic fluid sludge [16,51]. Among patients with a sonographic short cervix between 13 and 29 weeks of gestation, those with sludge had a higher rate of positive amniotic fluid culture for bacteria (21.7% vs. 0%; p=0.01), intraamniotic inflammation (27.3% vs. 3.5%; p=0.03), and a greater risk of preterm delivery at <28 weeks (46.5% vs. 5.8%; p <0.001) than those without sludge [17]. Moreover, several reports indicate that amniotic fluid sludge is a risk factor for intraamniotic infection in patients with cervical insufficiency [29,52].

Table 2.

Summary of studies reporting frequency of intra-amniotic infection/inflammation, amniotic fluid microbiology in patients with amniotic fluid ‘sludge’ in preterm gestations

Study	Clinical presentat ion	Gestational age, weeks	Amniotic fluid ‘sludg e’ % (n/N)	Amniocentesis in patients with amniotic fluid ‘sludge’, N	Intra-amnio tic infection % (n/N)	Methods for the detection of microorganisms	Detected Microorganisms	Intra-amniotic inflammation* % (n/N)
Espinoza et al [16]	Preterm labor and intact membranes	20 – 35	22.6% (19/84)	18	33.3% (6/18)	Cultivation	Ureaplasma urealyticum, Fusobacterium nucleatum, Candida albicans, Peptostreptococcus spp., Group B streptococci, Gardnerella vaginalis	N/A
Yoneda et al [51]	Preterm labor and intact membranes	20 – 29	18.1% (19/105)	19	31.6% (6/19)	Cultivation and PCR	Group B streptococci, Ureaplasma parvum, Flavobacterium succinicans	N/A
Kusanovic et al [17]	Short cervix	13 – 29	23.1% (28/121)	23	21.7% (5/23)	Cultivation	Ureaplasma urealyticum, Staphylococcus aureus, Fusobacterium nucleatum	27.3% (6/22)
Paules et al [29]	Cervical insufficiency	21–24	N/A	3	66.7% (2/3)	Cultivation	Fusobacterium nucleatum	100% (3/3)
Kusanovic et al (current study)	Preterm labor	20 – 34	N/A	13	61.5% (8/13)	Cultivation	Ureaplasma urealyticum, Candida albicans, Fusobacteriu m nucleatum, Mycoplasma hominis, etc.	61.5% (8/13)

^*Intra-amniotic inflammation was defined as an elevated amniotic fluid white blood cell count >50 cells/mm³.

N/A : not applicable

The observations reported herein are consistent with previous studies, thus indicating that particulate matter in the mid and early third trimester in patients with spontaneous preterm labor is often related to intraamniotic inflammation and intraamniotic infection. Indeed, most of the patients with early preterm labor and acute cervical insufficiency had microorganisms in the amniotic fluid as well as acute histologic chorioamnionitis.

Amniotic fluid particulate matter close to term

Echogenic amniotic fluid close to term is most often due to vernix and infrequently due to meconium. Table 3 displays a summary of previous studies in which echogenic amniotic fluid has been detected in patients close to term. Among patients with echogenic amniotic fluid in the third trimester, the frequency of vernix ranges from 95–100%, whereas the frequency of meconium ranges from 5–10% [2,5,8]. In the current study, we observed that 83% of particulate material at near-term was vernix and 8% was meconium. None of cases with echogenic amniotic fluid at term had severe acute histologic chorioamnionitis and funisitis.

Table 3.

Summary of studies reporting characteristics of echogenic amniotic fluid close to term determined by amniocentesis

Study	Gestational age, weeks	Amniocentesis,N	Appearance of amniotic fluid, % (n/N)
Parulekar et al [2]	35 – 40	9	Vernix [100% (9/9)]
Brown et al [5]	32 – 39	19	Vernix [95% (18/19)] Meconium [5% (1/19)]
Sherer et al [8]	>37	20	Clear [90% (18/20)] Meconium [10% (2/20)]
Kusanovic et al (current study)	35 – 42	10	Vernix [80% (8/10)] Meconium [10% (1/10)]

Collectively, the nature of amniotic fluid particulate material obtained at term is often consistent with vernix and appears to represent a phenomenon related to fetal maturation at term.

Amniotic fluid sludge and biofilms

Microbial biofilms have been identified in cases of intraamniotic infection with amniotic fluid sludge by scanning electron microscopy, which allowed visualization of bacteria embedded in amorphous material and in inflammatory cells [11]. By forming a biofilm, bacteria protect themselves from host defense and antibiotics [53]. The recognition that bacteria can form biofilms within the amniotic cavity is important for both diagnostic and therapeutic reasons. First, the detection of microorganisms in the presence of a biofilm is difficult to isolate by conventional culture methods, which detect only viable bacteria. In the current study, we obtained particulate matter, or sludge, under direct visualization to identify bacteria specific to such material. In some cases, bacteria were not detected by culture, even though there was a suspicion of intraamniotic infection (e.g., pus material, intraamniotic inflammation, and acute placental inflammatory lesions). The frequency of intraamniotic infection may be underestimated in cases with sludge, and molecular microbiologic techniques will be required to identify pathogenic bacteria in amniotic fluid. Second, the optimal treatment of biofilm-related infections represents a challenge given recent evidence that maternal administration of antibiotics can eradicate intraamniotic infection [54–59]. Antimicrobial agents appear to be inactivated or fail to reach bacteria within a biofilm. Interestingly, bacteria within biofilms show increased resistance to antimicrobial agents even though the bacteria can be sensitive to the same agents if grown under standard conditions [60–62]. Biofilms in the amniotic fluid can be formed by multiple organisms [11]. In the current study, more than one-half of the cases had polymicrobial infection, and genital mycoplasmas (Ureaplasmas species and Mycoplasma hominis) are mainly associated with other microorganisms. This has implications for the selection of optimal antimicrobial agents. Third, biofilms in the amniotic fluid may represent a unique form of these structures that can be dislodged by fetal breathing or swallowing, resulting in the seeding of planktonic bacteria and eliciting the fetal systemic inflammatory response [63,64]. Further research is required to characterize, with specific probes, the microbial constituents of amniotic fluid biofilms.

CONCLUSION

The nature of amniotic fluid particulate material varies as a function of gestational age. The material obtained in preterm gestations was frequently related to an inflammatory process, while that obtained at term was often consistent with vernix and appears to represent a phenomenon related to fetal maturation at term.