Maternal and fetal inflammatory responses in unexplained fetal death

The Journal of Maternal–Fetal and Neonatal Medicine 2003;14:151– 157

Maternal and fetal inflammatory responses in unexplained fetal death S. Blackwell1, R. Romero2, T. Chaiworapongsa 2, Y. M. Kim 2, E. Bujold 1, J. Espinoza 2, N. Camacho 1 , S. Hassan 1, B. H. Yoon 3 and J. S. Refuerzo1 1

Department of Obstetrics and Gynecology, Wayne State University/Hutzel Hospital, Detroit, Michigan, USA Perinatology Research Branch, National Institute of Child Health and Human Development, NIH/DHHS, Bethesda, Maryland, USA 3 Department of Obstetrics and Gynecology, Seoul National University, Seoul, Korea J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by 180.254.160.204 on 05/20/14 For personal use only.

2

Objective: The role of intra-amniotic infection in the etiology of fetal death has been proposed.

This study was conducted to determine the prevalence of microbial invasion of the amniotic cavity (MIAC) and the frequency of maternal and/or fetal inflammation in patients presenting with a fetal death. Methods: A prospective study was conducted in patients with a fetal death. Amniocenteses were performed for clinical indications (karyotype), as well as to assess the microbiological and cytological state of the amniotic cavity. Fluid was cultured for aerobic and anaerobic bacteria and genital mycoplasmas. An amniotic fluid white blood cell count and glucose determinations were also performed. Histological examination of the placenta was conducted to identify a maternal inflammatory response (acute chorioamnionitis) or a fetal inflammatory response (funisitis). Results: This study included 44 patients with intrauterine fetal death. The median gestational age at diagnosis was 30.1 weeks (range 16.3–40.4 weeks). One patient had documented MIAC (1/44). Acute histological chorioamnionitis was found in 20.9% (9/43), but a fetal inflammatory response was observed in only 2.3% (1/43) of cases. One patient had a positive amniotic fluid culture for Streptococcus agalactiae (group B streptococcus). Conclusion: Histological chorioamnionitis was present in 20.9% of cases, but MIAC could be demonstrated with conventional microbiological techniques in only one case. A fetal inflammatory response was nine times less frequent than a maternal inflammatory response (maternal 20.9% vs. fetal 2.3%, p = 0.008) in cases of fetal death. Key words: FETAL DEATH; INTRAUTERINE INFECTION ; MICROBIAL INVASION OF THE AMNIOTIC CAVITY ; CHORIOAMNIONITIS ; FUNISITIS

INTRODUCTION

Intrauterine infection has been implicated in the genesis of a wide range of adverse perinatal outcomes, including preterm labor1–3, preterm premature rupture of membranes4–6, congenital anomalies7,8, fetal anemia9,10, fetal growth restriction11–14 and fetal death15,16. Moreover, some studies have implicated intrauterine infection as a risk factor for the development of adult neurological disease, such as schizophrenia17,18, cerebral palsy19–24 and other neurodevelopmental disabilities25–28. The precise role of intrauterine infection and inflammation in the genesis of fetal death, however, remains

unclear15,16,29. A recent National Institute of Child Health and Development (NICHD)-sponsored conference on the subject of fetal death concluded that further studies are required to elucidate the specific contribution of intrauterine infections in fetal death30. The purpose of this study was to examine the frequency of proven microbiologic intra-amniotic infection, as well as that of a fetal and maternal inflammatory response, as determined by examination of the placental membranes and umbilical cord in patients with unexplained fetal death.

Correspondence: Dr R. Romero, Perinatology Research Branch, NICHD, Wayne State University/ Hutzel Hospital, Department of Obstetrics and Gynecology, 4707 St. Antoine Boulevard, Detroit, MI 48201, USA ã 2003 The Parthenon Publishing Group

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Received 17–04–03 Accepted 07–05–03

Fetomaternal inflammation and fetal death

J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by 180.254.160.204 on 05/20/14 For personal use only.

METHODS A prospective study, performed at Hutzel Hospital from September 2000 to August 2002, included patients who presented with an intrauterine fetal death. This study was part of a larger investigation of the frequency of fetomaternal inflammation in the ‘great obstetrical syndromes’31. Patients diagnosed with a fetal death by ultrasound examination were offered amniocentesis for karyotype determination and assessment of the possible presence of intra-amniotic infection. Amniocentesis for karyotype determination was offered in view of evidence that the likelihood of a successful karyotype determination is higher by studying amniotic fluid cells rather than fetal tissues in fetal death32–34. An amniotic fluid white blood cell (WBC) count and glucose concentration were determined. The amniotic fluid was cultured for aerobic and anaerobic bacteria, as well as genital mycoplasmas, including Mycoplasma hominis and Ureaplasma urealyticum. Histological examination of the placenta was conducted to identify a maternal inflammatory response (acute chorioamnionitis) or a fetal inflammatory response (funisitis). Microbial invasion of the amniotic cavity (MIAC) was defined as a positive amniotic fluid culture. An elevated amniotic WBC count was defined35 as a WBC count ³ 50 cells/mm3. The presence of neutrophils within the extraplacental membranes led to the diagnosis of acute histological chorioamnionitis. Acute funisitis was diagnosed by the detection of an inflammatory reaction in the umbilical cord. The criteria for the diagnosis of histological chorioamnionitis and funisitis have been previously described36. The rate of the maternal inflammatory response was compared to the fetal rate using the McNemar test for correlated samples. Spearman’s rank correlation was used to examine the correlation between amniotic fluid WBC count and amniotic fluid glucose concentration. A p value of < 0.05 was considered significant.

RESULTS

This study included 44 patients with a fetal death. The median gestational age was 30.1 weeks (range 16.3–40.4 weeks) and the median birth weight was 1283 g (range 140–3620 g). An elevated amniotic WBC count was detected in 38.6% (17/44) of patients. A glucose concentration of 14 mg/ml or less was found in 66.7% (28/42) of patients (Table 1). There was no correlation between the amniotic fluid WBC count and the glucose concentration (Spearman’s rho = -0.1; p = 0.5). MIAC was documented in one patient (1/44). The organism identified in the amniotic fluid was Streptococcus agalactiae. This patient had no evidence of clinical chorioamnionitis. On histological 152 Journal of Maternal–Fetal and Neonatal Medicine

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examination, acute chorioamnionitis, but not funisitis, was detected. Acute inflammatory lesions of the extraplacental membranes were found in 20.9% (9/43) of patients, but funisitis was observed in only 2.3% (1/43) (Table 2). Acute chorioamnionitis was nine times more frequent than acute funisitis (maternal 20.9% vs. fetal 2.3%; McNemar test: p = 0.008) (Table 2). Among patients with acute chorioamnionitis, 22.2% (2/9) had an elevated amniotic fluid WBC count, but 77.8% (7/9) had a low glucose concentration. Only 11.8% (2/17) of patients with an elevated amniotic fluid WBC count had evidence of histological chorioamnionitis. One patient with acute funisitis presented with preterm premature rupture of membranes at 20 weeks of gestation. Placental examination also revealed acute chorioamnionitis. The amniotic fluid WBC count was 20 cells/mm3. The patient’s glucose level was 9 mg/dl, and the amniotic fluid culture was negative for microorganisms. Clinical chorioamnionitis was documented in two patients (2/44; 4.5%). One patient showed evidence of Table 1

Clinical characteristics and results of the study Value

Maternal age (years) Gestational age at admission (weeks) Clinical chorioamnionitis Birth weight (g)

25 (17–41) 30.1 (16.3–40.4) 4.5% (2/44) 1283 (140–3620)

Amniotic fluid Culture positive for micro-organisms WBC count ³ 50 cells/mm3 Glucose £ 14 mg/dl

1 38.6% (17/44) 66.7% (28/42)*

Histopathology of the placenta Chorioamnionitis† Funisitis†

20.9% (9/43) 2.3% (1/43)

*No amniotic fluid glucose concentration results were available for two patients † No extraplacental membrane or umbilical cord tissue was available for pathological examination for one patient WBC, white blood cell Table 2 funisitis

Frequency of acute histological chorioamnionitis and Funisitis

Chorioamnionitis Yes No Total

Yes

No

Total

1 0 1 (2.3%)

8 34 42 (97.7%)

9 (20.9%) 34 (79.1%) 43 (100%)

No placental tissue was available for pathological examination for one patient


elevated amniotic fluid WBC count (670 cells/mm3) and acute inflammatory lesions in the extraplacental membranes without acute funisitis. The other had neither an elevated amniotic fluid WBC count (45 cells/m3) nor histologic chorioamnionitis.


COMMENT The findings of this study indicate that intrauterine inflammation was present in one-fifth of cases of intrauterine fetal death and that a fetal inflammatory response (i.e. funisitis) was seven times less frequent than a maternal inflammatory response (i.e. histological chorioamnionitis). Despite the significantly higher maternal inflammatory response, most patients lacked clinical signs of chorioamnionitis and only one had microbiological evidence of bacterial infection with standard cultivation techniques. The limitations of cultivation-dependent methods in the identification of micro-organisms have been demonstrated by environmental surveys based upon the detection of conserved microbial sequences such as the 16S rRNA gene (16S rDNA). Recent estimations indicate that 99% of micro-organisms resist cultivation in the laboratory37. It should be stressed that the techniques employed in this study were those of a standard clinical microbiologic laboratory and that cultures for viruses were not included. We have recently reported that amniotic fluid culture detects only 40% of patients with evidence of microbial footprints, as determined by polymerase chain reaction (PCR) with specific primers for Ureaplasma urealyticum in the context of preterm premature rupture of membranes38. Moreover, this study found that patients with a negative amniotic fluid culture but positive PCR had a shorter interval to delivery, a higher amniotic fluid WBC count and interleukin (IL)-6, and worse neonatal outcome than patients with a negative culture and negative PCR. These observations indicate that organisms that resist cultivation but can be detected with molecular microbiological techniques have clinical significance. Similar observations have recently been made in the context of preterm labor with intact membranes39. Different molecular microbiological techniques need to be employed in this setting in order to establish microbial diversity. This approach may be particularly useful for the detection of fastidious microorganisms. The possibility of discovering micro-organisms implicated in perinatal disease that have not yet been identified should also be considered, as has been the case in other conditions such as Whipple disease40 and bacillary angiomatosis41. Other than case reports, the frequency of intrauterine infection in cases of fetal death has been ascertained by means of microbial culture of fluid retrieved by transabdominal amniocentesis in four case series42–45. The observation that only one of our patients (2.3%) with

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fetal death had a positive amniotic fluid culture for microorganisms contrasts with other studies, which found rates of positive cultures ranging from 5.9% to 43%42–45. For example, a study in Lithuania43 in which amniocenteses were performed in patients with fetal death reported that the presence of a positive amniotic fluid culture for micro-organisms was 43% (25/58). The most common isolates were coagulase-negative Staphylococcus, Escherichia coli, Enterococcus and Streptococcus agalactiae. A positive blood culture obtained from the stillbirth (by cardiac puncture) was observed in 36% of cases, and the most common micro-organism isolated was Escherichia coli, followed by coagulase-negative Staphylococcus, Streptococcus agalactiae, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter and a-hemolytic Streptococcus. A strong relationship was found between inflammation of the extrachorionic membranes and chorionic vasculitis and the isolation of micro-organisms from the blood of the stillbirth43. Other studies conducted in Zimbabwe42 and Mozambique44,46 have also demonstrated a high rate of fetal bacteremia in blood obtained by cardiac puncture of the stillbirth. The substantial difference in the rate of documented cultureproven intra-amniotic infection among these studies and the present report suggests that the prevalence of infection in association with fetal death varies markedly among populations around the world. Whether this reflects the role of infection in fetal death, the period of time that the dead fetus remained in utero, or intrapartum infection during induction has not been determined. A comprehensive and up-to-date review of the relationship between infection (including specific micro-organisms) and fetal death has been recently published by Goldenberg and Thompson15. An elevated amniotic fluid WBC count was found in 38.6% of cases (17/44). Traditionally, this finding is considered an index of intra-amniotic inflammation35. However, only 11.8% (2/17) of patients with an elevated amniotic fluid WBC count showed evidence of histological chorioamnionitis. The reasons for this discrepancy are not clear. It is possible that a new population of nucleated cells, such as macrophages derived from fetal tissues, may gain access to the amniotic cavity and be responsible for the increased WBC count observed in this study. We conclude that an elevated number of nucleated cells in cases of fetal death may not have the same significance in the context of a live fetus35,47. Further studies to characterize the cell population of the amniotic cavity in fetal death are warranted. There was also a discrepancy between the rate of low amniotic fluid concentration of glucose and histological chorioamnionitis (66.7% vs. 20.9%, respectively). Amniotic fluid glucose is believed to be decreased in intra-amniotic infection47–51, because of glucose consumption by micro-organisms and the presence of activated 153 Journal of Maternal–Fetal and Neonatal Medicine

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Journal of Maternal–Fetal and Neonatal Medicine 243 89 103 99 54 58 111 117 39 28 66 58

Cases (n) FD > 26 weeks FD > 20 weeks FD > 25 weeks FD > 13 weeks FD > 28 weeks UFD > 26 weeks FD > 28 weeks FD > 28 weeks ND UFD > 20 weeks UFD > 22 weeks FD > 28 weeks

Definition of stillbirth

ND, not described; FD, fetal death; UFD, unexplained fetal death

Helsinki, Finland Lansing, Michigan Morristown, New Jersey Aarhus, Denmark Cincinnati, Ohio Kaunas, Lithuania Uppsala, Sweden Lund, Sweden Maputo, Mozambique Toronto, Canada Harare, Zimbabwe Maputo, Mozambique

Population 6.7% (12/180) 7.9% (7/89) 12.6 % (13/103) 21.2% (21/99) 35.2% (19/54) 48.3% (28/58) 70.3% (78/111) 70.7% (82/116) 77.1% (27/35) 77.3% 78.8% (52/66) 96.4% (54/56)

Chorioamnionitis ND ND ND ND ND ND ND 19.7% (23/117) ND ND ND ND

Funisitis

Summary of studies reporting on frequency of inflammatory lesions of the placenta in cases of stillbirth

Hovatta52 Rayburn53 Ogunyemi54 Rudbeck55 Ornoy56 Maleckiene43 Olding57 Tolockiene58 Axemo44 Quinn59 Moyo60 Folgosa46

Author

Table 3


ND 7.9% (7/89) ND ND ND 27.6% (16/58) 9% (10/111) ND ND ND 9.1% (6/66) 21.4% (12/56)

Chorionic vasculitis

Placental lesions

ND 4.5% (4/89) 5.8% (6/103) ND 13% (7/54) ND ND ND ND ND 7.6% (5/66) 1.8% (1/56)

Chronic villitis

Fetomaternal inflammation and fetal death Blackwell et al.



leukocytes in the amniotic fluid. We could not find a relationship between the amniotic fluid WBC count and the amniotic fluid glucose concentration in this study, as we have reported in patients with preterm labor and preterm premature rupture of membranes and intraamniotic inflammation47–51. It is possible that fetal death changes amniotic fluid glucose metabolism and disposition, and this in turn alters the significance of amniotic fluid glucose as a marker of intra-amniotic inflammation and/or infection. The frequency of histological chorioamnionitis in our study was 20.9% (9/43), and only one patient had funisitis. Previous studies indicated a wide range of histological chorioamnionitis in fetal death, ranging from 4.9% to 96%43,44,46,52–60 (Table 3). The most interesting observation in our study was that the rate of maternal inflammation (histological chorioamnionitis) was nine times more frequent than that of fetal inflammation (funisitis). Two explanations may be considered. First, it is possible that fetal infection occurs, but fails to trigger a fetal inflammatory response and the onset of preterm labor. In this case, in utero fetal death would represent failure of the host response mechanisms dealing with intrauterine infection. This concept is supported by evidence that, in some cases of intrauterine death due to group B streptococci (with intact membranes), there may be absence of both a maternal and a fetal inflammatory response despite widespread fetal infection61–63. Similar observations can be derived from the study of Tafari and colleagues with intrauterine infection by Ureaplasma urealyticum64. Despite the fact that intrauterine infection (across intact membranes) was documented by culture of micro-organisms from fetal lung tissue at postmortem examination, fetal death could occur before the onset of labor. A recent genetic association study further supports this hypothesis65. In cases of unexplained stillbirth, there was an association between homozygosity for the IL-1 receptor antagonist (IL-1ra) allele 2 and the risk of fetal death65. Carriage of this allele is associated with increased production of IL-1ra66,67. An excess of IL-1ra in the fetal compartment may limit the ability of the fetus to deploy a pro-inflammatory response and thus limit the repertoire of mechanisms available for host defense, including the ability to exit a hostile intrauterine environment by initiating the onset of labor68–70. However, further studies are required to test this hypothesis. The second possible explanation for the discrepant rate of maternal and fetal inflammatory responses is that inflammation of the placental membranes (histological chorioamnionitis) may occur after fetal death and be etiologically unrelated to the fetal demise. Induction of labor after stillbirth was undertaken in our institution by administering misoprostol in most cases. Whether this compound can increase the rate of histological

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chorioamnionitis is unknown. However, a recent study by Jacques and co-workers addressed this question71. They studied the placentas after second-trimester therapeutic abortions where labor induction was undertaken with prostaglandins. Despite a prolonged labor induction, none of the placentas showed inflammatory lesions (chorioamnionitis, funisitis or villitis). In conclusion, histological chorioamnionitis is found in one-fifth of patients with unexplained fetal death. The application of molecular microbiological techniques to amniotic fluid, fetal blood and other tissues is required to assess the association between microbial invasion and fetal death. We propose that hematogenous or ascending fetal infections that do not elicit a fetal inflammatory response sufficient to signal the onset of labor are responsible for fetal death in some cases. Failure to mount an inflammatory response may be due to genetic factors72 that control susceptibility to infection, pro-inflammatory/ anti-inflammatory processes or the mechanisms determining activation of the common pathway of parturition (uterine contractility, cervical ripening and/or rupture of membranes)73. ACKNOWLEDGEMENTS The authors wish to acknowledge the contributions of the nursing staff of the Detroit Medical Center: Ms Sandy Field, Ms Vicki Ineson, Ms Mabubeh Mahoudieh, Ms Milagros Kitchen, Ms Cozette Kelly-Roberts, Ms De Andrea Duncan-Tolbert, Ms Audrey Milliken, Ms Leandra Ga-Pinlac and Ms Kathy Firchau; and the contributions of Ms Lisa Palmer for expert and timely data entry and verification of accuracy. REFERENCES 1. Romero R, Sirtori M, Oyarzun E, et al. Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989;161:817–24 2. Locksmith G, Duff P. Infection, antibiotics, and preterm delivery. Semin Perinatol 2001;25:295–309 3. Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med 2000;342:1500–7 4. Parry S, Strauss JF III. Premature rupture of the fetal membranes. N Engl J Med 1998;338:663–70 5. Arias F, Gonzalez-Ruiz AR, Jacobson RL. Recent advances in the pathophysiology and management of preterm premature rupture of the fetal membranes. Curr Opin Obstet Gynecol 1999;11:141–7 6. Carroll SG, Philpott-Howard J, Nicolaides KH. Amniotic fluid gram stain and leukocyte count in the prediction of intrauterine infection in preterm prelabour amniorrhexis. Fetal Diagn Ther 1996;11:1–5 7. Monif GR. Viruses as teratogens. Clin Obstet Gynecol 1975;18:209–20 155 Journal of Maternal–Fetal and Neonatal Medicine



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