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Particulate matter in the amniotic fluid (AF) is present in about 4% of pregnancies during transvaginal ultrasound in the first and early second trimester1. The prevalence of this sonographic finding increases with gestational age, reaching 88% by 35 weeks2. Particulate matter in the first two trimesters of pregnancy has been associated with intra-amniotic bleeding3, 4 and the acrania–anencephaly sequence5, 6, and has been observed in women with high concentrations of maternal serum alpha-fetoprotein7. In contrast, in the last trimester of pregnancy, particulate matter and ‘echogenic amniotic fluid’ have been attributed to the presence of vernix caseosa and/or meconium8–11, and with a lecithin : sphingomyelin ratio indicative of lung maturity12, 13. Congenital anomalies associated with particulate matter in the AF include harlequin ichthyosis14 and epidermolysis bullosa letalis15.
Dense aggregates of particulate matter giving the ultrasound appearance of AF ‘sludge’ are frequently seen, in the proximity of the internal cervical os, in patients with preterm labor and intact membranes. However, the prevalence and clinical significance of this ultrasound finding have not been determined.
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Our results indicate that AF ‘sludge’ during transvaginal examination of the cervix is present in 22.6% of patients with preterm labor and intact membranes, and is a risk factor for MIAC, histological chorioamnionitis and impending preterm delivery.
Aggregates of hyperechogenic particulate matter in the gallbladder of adult patients have been described as biliary ‘sludge’18, 19. This sonographic finding is characterized by the presence of low-level echoes that layer in the dependent portion of the gallbladder without acoustic shadowing19, and is associated with ascending microbial invasion of the gallbladder20. Indeed, biliary ‘sludge’ is considered a risk factor for positive bile cultures obtained by percutaneous aspiration of the gallbladder21. Moreover, the presence of bacterial biofilms in the gallbladder has been proposed to be a key event in the formation of biliary ‘sludge’22.
Microbial invasion of the amniotic cavity is generally prevented by components of the innate and adaptive immune system, including the cervical epithelium23 and mucus plug24–27, chorioamniotic membranes28, 29 and cells of the decidua, amnion and chorion, including neutrophils, macrophages, natural killer cells and throphoblast30, 31. However, the integrity of the chorioamniotic membranes is not sufficient to prevent MIAC. Indeed, micro-organisms have been isolated from the AF of asymptomatic patients at the time of genetic amniocentesis32–34 in 12.8% (379/2963)35 of patients with preterm labor and intact membranes and in 18.8% (17/90)36 of patients in labor at term. Thus, these micro-organisms can penetrate intact membranes37.
The observation that patients with preterm labor and intact membranes with ‘sludge’ are more likely to have microbiological and histological evidence of MIAC and impending preterm delivery is novel. During MIAC, microbial proliferation may be prevented by a local inflammatory response elicited by cytokines38–40, chemokines41, matrix degrading enzymes42–46, antimicrobial peptides47, 48 and cells of the innate immune system49, 50. Micro-organisms may protect themselves from these host defense mechanisms by producing and embedding themselves in matrices of polymeric compounds, also known as bacterial biofilms51. Evidence in favor of this view includes: (1) bacteria can remain viable within a biofilm despite elevated concentrations of proinflammatory cytokines, including IL-1β, IL-12 and interferon-γ51, and (2) human leukocytes can penetrate bacterial biofilms in vitro but are not able to phagocyte the embedded bacteria51. Thus, the possibility that AF ‘sludge’ may represent clusters of bacterial biofilms and inflammatory cells should be considered.
It is possible that aggregates of exfoliated cells from the fetal digestive, respiratory and urinary tracts, amniotic membranes, fetal skin and umbilical cord52 may also contribute to the presence of AF ‘sludge’ and participate in the host response during MIAC. Indeed, it has been proposed that exfoliated fetal skin cells account for the antimicrobial properties of the vernix caseosa, where the presence of several antimicrobial peptides, including α-defensins, cathelicidin (LL-37), psoriasin and ubiquitin, has been described53. In contrast, our results indicate that debris from intra-amniotic bleeding in the index pregnancy may not contribute to the AF ‘sludge’. Indeed, the proportion of discolored AF, an index of earlier intra-amniotic bleeding54–56, was not different between the study groups. Moreover, vaginal bleeding, also associated with discolored amniotic fluid57, was included as a confounding variable in the logistic regression analysis, which indicated that AF ‘sludge’ is an independent explanatory variable for the occurrence of intra-amniotic infection and histological chorioamnionitis.
An inherent limitation of this study is its retrospective nature. However, 68% (57/84) of cases were examined with 3D ultrasound, and thus we were able to examine not only the pictures taken by the sonographer at the time of the examination but also the volume dataset. We were not able to identify cases in which AF ‘sludge’ could be seen in a parasagittal scan that did not include the endocervical canal. However, this possibility remains and only further studies will clarify this issue.
Collectively, our observations indicate that the sonographic finding of AF ‘sludge’ is associated with microbiological and histological evidence of intra-amniotic infection and impending preterm delivery. We propose that this sonographic sign may identify patients at risk for MIAC, who in turn are at risk for preterm delivery and short- and long-term complications such as cerebral palsy and chronic lung disease.