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Preterm labor and bacterial intraamniotic infection: Arachidonic acid liberation by phospholipase A2 of Fusobacterium nucleatum
Preterm labor and bacterial intraamniotic infection: Arachidonic acid liberation by phospholipase A2 of Fusobacterium nucleatum Hiroshige Mikamo, MD, PhD, Kyoko Kawazoe, MD, Yasumasa Sato, MD, Atsushi Imai, MD, PhD, and Teruhiko Tamaya, MD, PhD Gifu, Japan OBJECTIVE: The studies presented in this report were undertaken to evaluate whether Fusobacterium nucleatum, a common anaerobic isolate in intrauterine infection, stimulates arachidonic acid metabolism, a rate-limiting step for prostaglandin synthesis, in the human uterine endometrium. STUDY DESIGN: Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells and of F nucleatum extract on lysophosphatidylcholine production in human uterine endometrial cells were investigated. RESULTS: When human uterine endometrial cells labeled with tritiated arachidonic acid to an isotopically steady state were exposed to an extract of F nucleatum, arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Similar stimulatory effects on phospholipid degradation were also observed in the experiment with bacterially conditioned media. CONCLUSIONS: These results suggest that F nucleatum stimulates endometrial phospholipid metabolism, related to activity of phospholipase A2, which might induce the onset of labor associated with intraamniotic infection. (Am J Obstet Gynecol 1998;179:1579-82.)
Key words: Arachidonic acid, Fusobacterium nucleatum, phospholipase A2, preterm labor
A growing body of evidence suggests that infection plays a key role in the pathogenesis of preterm labor and delivery. In particular the evidence supports an etiologic role for intrauterine infection in the onset of preterm labor. In preterm premature rupture of membranes, aerobic gram-positive cocci and anaerobic gram-negative bacilli are commonly isolated.1, 2 Aerobic bacteria include Streptococcus agalactiae, Enterococcus faecalis, Staphylococcus haemolyticus, and Gardnerella vaginalis. Anaerobes include Bacteroides fragilis, Prevotella bivia, and Fusobacterium nucleatum. The precise roles of bacteria or bacterial products in the onset of preterm labor remain to be clarified; however, cellular and biochemical mechanisms have been proposed to mediate preterm labor in the setting of infection. A growing body of evidence is available to support a role for the metabolites of arachidonic acid in the context of infection-mediated preterm labor.3 B fragilis
From the Department of Obstetrics and Gynecology, School of Medicine, Gifu University. Received for publication November 11, 1997; revised May 29 1998; accepted May 29, 1998. Reprint requests: Hiroshige Mikamo, MD, PhD, Department of Obstetrics and Gynecology, School of Medicine, Gifu University, 40, Tsukasa-machi, Gifu-city, Gifu 500-8705, Japan. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/92097
and F nucleatum both possess high phospholipase A2 activity, which directly induces arachidonic acid metabolism.4-9 F nucleatum, an anaerobic gram-negative rod, is frequently isolated in preterm premature rupture of membranes and produces endotoxin, a stimulant for arachidonic acid metabolism.1 The objective of this study was therefore to determine the effects of F nucleatum–derived phospholipase A2 on human uterine endometrium. Material and methods Material. Tritiated arachidonic acid (78 Ci/mmol) was obtained from New England Nuclear (New England Nuclear Nuclides and Sources, Du Pont Diagnostic Imaging Division, Wilmington, Del). Silica gel 60 plates and collagenase were purchased from Merck (Darmstadt, Germany) and Sigma Chemical Company (St Louis, Mo), respectively. All other chemicals and reagents were of experimental grade. Preparation of tritiated arachidonic acid–labeled human endometrial cells. Agreement for this study was obtained from 6 patients (35-45 years old) in the department of obstetrics and gynecology of the School of Medicine, Gifu University, and from the ethics committee. Specimens of uterine endometrium were obtained from the patients undergoing hysterectomy for myoma uteri from June 1996 to October 1996. The tissues were 1579
1580 Mikamo et al
Fig 1. Incorporation of tritiated arachidonic acid into human uterine endometrial cell phospholipids. Tritiated arachidonic acid (10 µCi) was added to a 1-mL aliquot of endometrial cell suspension (1 × 107 cells) at 37°C. Lipid was extracted and its radioactivity was counted. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. dpm, Disintegrations per minute.
trimmed and minced under a laminar-flow hood in Hanks’ balanced salt solution. The endometrial tissue was digested for 1.5 hours at 37°C with 0.25% type I collagenase dissolved in Hanks’ balanced salt solution. Stroma and cellular debris were removed by filtration through a nylon mesh. The cellular suspension was then centrifuged at 500g for 5 minutes. The resulting pellet was resuspended in Hanks’ balanced salt solution and labeled with tritiated arachidonic acid (10 µCi/mL) at 37°C for 2 to 3 hours, a time that was almost sufficient to attain an isotopic steady state.5 The tritiated arachidonic acid–labeled endometrial cells were then washed and resuspended in appropriate concentration in Hanks’ balanced salt solution. Bacterial preparation. Stock isolate of F nucleatum cultured on Brucella hemin and vitamin K1 agar (Kyokuto Pharmaceutical Co, Tokyo, Japan) was inoculated into 50 mL Gifu anaerobic medium broth (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) with 106 organisms/mL medium. The isolate was incubated until there were 108 organisms/mL medium and the organisms were then separated by centrifugation and filtration. F nucleatum was homogenized by sonication and centrifuged at 100,000g for 1 hour. The supernatant was used as the bacterial extract. Protein was determined by the method of Lowry et al,10 with bovine serum albumin as a standard. The extract and the conditioned medium were stored in aliquots at –20°C. Examination of F nucleatum–induced arachidonic acid liberation from endometrial cells. To examine effects of F nucleatum on the liberation of arachidonic acid, the triti-
December 1998 Am J Obstet Gynecol
Fig 2. Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells. Tritiated arachidonic acid–labeled endometrial cells (1.0 × 107 cells/0.2 mL) were incubated with bacterial extracts in protein of 0.3 mg/mL, medium conditioned with F nucleatum, control medium, or normal saline solution for 1 hour at 37°C. The radioactivity of liberated arachidonic acid and metabolites was determined. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. Asterisk, P < .001, versus control medium; two asterisks, P < .005, between F nucleatum extract and conditioned medium. dpm, Disintegrations per minute.
ated arachidonic acid–labeled endometrial cell suspensions (0.5-1.0 × 107 cells/0.2 mL) were exposed to bacterial extracts in protein of 0.3 mg/mL, medium conditioned with F nucleatum, control medium, or normal saline solution at 37°C. The reaction was terminated by adding 1 mL chloroform/methanol (1:2, volume/volume), followed by 0.2 mL 10 mmol/L ethylenediaminetetraacetic acid with 0.01 N hydrochloric acid and 0.2 mL chloroform. After the 2 phases were separated, the lower phase (chloroform layer) was washed with chloroform-saturated water. The extracted lipid was analyzed with thin-layer chromatography as described previously.9 The areas corresponding to free fatty acids and arachidonic acid metabolites formed a single spot in this system, and each phospholipid was scraped into vials and its radioactivity was counted in a liquid scintilation counter. In some experiments lipid phosphorus was determined by the procedure of Rouser et al.12 Statistical analysis. Values were reported as mean ± SD. All results were analyzed with the Student t test, and significance was assigned at P < .05.
Volume 179, Number 6, Part 1 Am J Obstet Gynecol
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Results The uptake of tritiated arachidonic acid into phospholipids by human endometrial cells increased in time-dependent manner and reached the plateau level in approximately 6 hours (Fig 1). As illustrated in Fig 2, exposure of the tritiated arachidonic acid–labeled human endometrial cells to the Gifu anaerobic medium conditioned with F nucleatum induced an increase in the radioactivity of free fatty acid and arachidonate metabolite fractions. Control Gifu anaerobic medium showed no significant effects. A similar stimulatory effect on the rate of free arachidonate and metabolite production was observed in the experiments with bacterial extract. A significant difference in free arachidonic acid generation by endometrial cells was noted between F nucleatum extract and the conditioned medium treatments. Fig 3 shows the effect of F nucleatum extract on production of lysophospholipid, another metabolite of phospholipase A2. When the endometrial cells were challenged with the extract, lysophosphatidylcholine was observed to increase significantly (P < .01). Stimulated degradation of phosphatidylcholine to its lyso derivative might be observed in endometrial cells when exposed to the component secreted from F nucleatum. Even when heated at 100°C for 30 minutes, the extract increased the degradation at a similar level. Comment This and previous studies suggest that there is an increase in arachidonic acid metabolism seen with preterm or term labor that is associated with an increase in the production and release of prostaglandin E2. The statement made in the article regarding prostaglandins should be considered only as a justifiable assumption. Others have found that arachidonic acid metabolites play a major role in the initiation and maintenance of labor.13-15 We studied how this might occur in the presence of F nucleatum. We found that the uptake of tritiated arachidonic acid into phospholipids by human endometrial cells increased in a time-dependent manner and reached the plateau level in approximately 6 hours. This means that the phospholipids were labeled to almost constant specific radioactivity, and the changes in the levels of tritiated phospholipids and tritiated arachidonate therefore accurately reflected those in lipid mass. Exposure of tritiated arachidonic acid–labeled human endometrial cells to the Gifu anaerobic medium conditioned with F nucleatum induced an increase in the radioactivity of free fatty acid and arachidonate metabolite fractions. These findings suggest that F nucleatum yields and secretes phospholipase, which hydrolyzes endometrial phospholipids to liberate arachidonic acid and subsequently prostaglandins.
Fig 3. Effects of F nucleatum extract on lysophosphatidylcholine (Lyso PC) production in human uterine endometrial cells. Experimental conditions were described in Methods section. The phosphorus of lysophosphatidylcholine was determined and expressed as percentage of total phospholipid. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. Heating was at 100°C for 30 minutes. Asterisk, P < .05, versus control. PC, Phosphatidylcholine.
The mechanisms through which arachidonic acid was released from endometrial phospholipids on incubation with bacterial component was then investigated. Because the obligatory precursor of prostaglandins, arachidonic acid, is present as an ester of glycerophospholipids, the action of phospholipase is likely to liberate arachidonic acid. In particular, phospholipase A2, which is found in bacterial lysosomes and also in a mammalian cell membrane–bound form, plays a major role in the hydrolysis of phospholipids. When human endometrial cells were exposed to an extract from F nucleatum, arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Further, stimulatory effects on phospholipid degradation were observed in the experiments with the bacterial extract. These results show that phospholipase A2 is secreted from F nucleatum, acting on endometrial phospholipids, or its product stimulates endometrial phospholipase A2, leading to phospholipid breakdown, or both. Heating of the extract did not influence the degradation of phosphatidylcholine. Endotoxin, a stimulant of cell surface receptor, increases in amniotic fluid in the setting of preterm labor.8 F nucleatum could therefore produce both phospholipase and endotoxin, doubly stimulating arachidonic acid metabolism with subsequent prostaglandin formation. Asymptomatic or subclinical intrauterine infection with growth of F nucleatum thus might easily induce labor.
1582 Mikamo et al
Finally, our study demonstrates that F nucleatum products can stimulate endometrial phospholipid hydrolysis through phospholipase A2 activity. We speculate that some subset of these mechanisms may be operative in mediating preterm labor in association with clinical or subclinical intrauterine infection in the presence of F nucleatum. REFERENCES
1. Mikamo H, Ito K, Tamaya T. Studies on the clinical implication of anaerobes, especially Prevotella bivia, in obstetrics and gynecology. Acta Sch Med Univ Gifu 1994;42:230-48. 2. Mikamo H, Izumi K, Ito K, Sawamura H, Sawa K, Watanabe K, et al. Bacterial flora detected in the uterine endometrial cavity of normal puerperae on the puerperal first day and on the puerperal fifth day after incidental use of cefpodoxime proxetil (CPDX-PR). Jpn J Antibiot 1993;46:269-73. 3. Romero R, Avila C, Sepulveda W. The role of systemic and intrauterine infection in preterm labor. In: Fuchs AR, Fuchs F, Stubblefield PG, editors. Preterm birth. 2nd ed. New York: McGraw-Hill; 1993. p. 97-136. 4. Bejar R, Curbelo V, Davis C. Premature labor II: bacterial sources of phospholipases. Obstet Gynecol 1981;57:479-83. 5. Takahashi K, Imai A, Tamaya T. Preterm labor and bacterial intraamniotic infection: arachidonic acid liberation by the action of phospholipase A2. Arch Gynecol Obstet 1984;244:1-6. 6. Khan AA, Imai A, Tamaya T. Possible mechanism for preterm labor associated with bacterial infection I: stimulation of phos-
December 1998 Am J Obstet Gynecol
7.
8.
9.
10.
11. 12.
13.
14.
15.
phoinositide metabolism by endotoxin in endometrial fibroblasts. Res Commum Chem Pathol Pharmacol 1990;69:119-22. Khan AA, Imai A, Tamaya T. Possible mechanism for preterm labor associated with bacterial infection II: enhancement of endotoxin-stimulated phosphoinositide metabolism by sex steroids in endometrial fibroblasts. Res Commum Chem Pathol Pharmacol 1990;69:123-6. Romero R, Roslansky P, Oyarzun E, Wan M, Emamian M, Novitsky TJ, et al. Labor and infection II: bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol 1988;158:1044-9. Lamont RF, Rose M, Elder MG. Effects of bacterial products on prostaglandin E production by amnion cells. Lancet 1985;2:1331-3. Lowry OH, Rosenbrough N, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. Imai A, Gershengorn MC. Measurement of lipid turnover in response to TRH. Methods Enzymol 1986;141:100-11. Rouser G, Siakotos AN, Fleisher S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1966;1:85-6. MacDonald P, Schulz M, Duenhoelter J, Gant NF, Jimenez FJ, Pritchard JA, et al. Initiation of human parturition I: mechanism of action of arachidonic acid. Obstet Gynecol 1984;44:62936. Carsten ME. Prostaglandins and cellular calcium transport in the pregnant human uterus. Am J Obstet Gynecol 1973;117:82432. Bejar R, Curbello V, Davis C, Gluck L. Premature labor and bacterial sources of phospholipase. Obstet Gynecol 1981;57:473-82.
Key words: Arachidonic acid, Fusobacterium nucleatum, phospholipase A2, preterm labor
A growing body of evidence suggests that infection plays a key role in the pathogenesis of preterm labor and delivery. In particular the evidence supports an etiologic role for intrauterine infection in the onset of preterm labor. In preterm premature rupture of membranes, aerobic gram-positive cocci and anaerobic gram-negative bacilli are commonly isolated.1, 2 Aerobic bacteria include Streptococcus agalactiae, Enterococcus faecalis, Staphylococcus haemolyticus, and Gardnerella vaginalis. Anaerobes include Bacteroides fragilis, Prevotella bivia, and Fusobacterium nucleatum. The precise roles of bacteria or bacterial products in the onset of preterm labor remain to be clarified; however, cellular and biochemical mechanisms have been proposed to mediate preterm labor in the setting of infection. A growing body of evidence is available to support a role for the metabolites of arachidonic acid in the context of infection-mediated preterm labor.3 B fragilis
From the Department of Obstetrics and Gynecology, School of Medicine, Gifu University. Received for publication November 11, 1997; revised May 29 1998; accepted May 29, 1998. Reprint requests: Hiroshige Mikamo, MD, PhD, Department of Obstetrics and Gynecology, School of Medicine, Gifu University, 40, Tsukasa-machi, Gifu-city, Gifu 500-8705, Japan. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/92097
and F nucleatum both possess high phospholipase A2 activity, which directly induces arachidonic acid metabolism.4-9 F nucleatum, an anaerobic gram-negative rod, is frequently isolated in preterm premature rupture of membranes and produces endotoxin, a stimulant for arachidonic acid metabolism.1 The objective of this study was therefore to determine the effects of F nucleatum–derived phospholipase A2 on human uterine endometrium. Material and methods Material. Tritiated arachidonic acid (78 Ci/mmol) was obtained from New England Nuclear (New England Nuclear Nuclides and Sources, Du Pont Diagnostic Imaging Division, Wilmington, Del). Silica gel 60 plates and collagenase were purchased from Merck (Darmstadt, Germany) and Sigma Chemical Company (St Louis, Mo), respectively. All other chemicals and reagents were of experimental grade. Preparation of tritiated arachidonic acid–labeled human endometrial cells. Agreement for this study was obtained from 6 patients (35-45 years old) in the department of obstetrics and gynecology of the School of Medicine, Gifu University, and from the ethics committee. Specimens of uterine endometrium were obtained from the patients undergoing hysterectomy for myoma uteri from June 1996 to October 1996. The tissues were 1579
1580 Mikamo et al
Fig 1. Incorporation of tritiated arachidonic acid into human uterine endometrial cell phospholipids. Tritiated arachidonic acid (10 µCi) was added to a 1-mL aliquot of endometrial cell suspension (1 × 107 cells) at 37°C. Lipid was extracted and its radioactivity was counted. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. dpm, Disintegrations per minute.
trimmed and minced under a laminar-flow hood in Hanks’ balanced salt solution. The endometrial tissue was digested for 1.5 hours at 37°C with 0.25% type I collagenase dissolved in Hanks’ balanced salt solution. Stroma and cellular debris were removed by filtration through a nylon mesh. The cellular suspension was then centrifuged at 500g for 5 minutes. The resulting pellet was resuspended in Hanks’ balanced salt solution and labeled with tritiated arachidonic acid (10 µCi/mL) at 37°C for 2 to 3 hours, a time that was almost sufficient to attain an isotopic steady state.5 The tritiated arachidonic acid–labeled endometrial cells were then washed and resuspended in appropriate concentration in Hanks’ balanced salt solution. Bacterial preparation. Stock isolate of F nucleatum cultured on Brucella hemin and vitamin K1 agar (Kyokuto Pharmaceutical Co, Tokyo, Japan) was inoculated into 50 mL Gifu anaerobic medium broth (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) with 106 organisms/mL medium. The isolate was incubated until there were 108 organisms/mL medium and the organisms were then separated by centrifugation and filtration. F nucleatum was homogenized by sonication and centrifuged at 100,000g for 1 hour. The supernatant was used as the bacterial extract. Protein was determined by the method of Lowry et al,10 with bovine serum albumin as a standard. The extract and the conditioned medium were stored in aliquots at –20°C. Examination of F nucleatum–induced arachidonic acid liberation from endometrial cells. To examine effects of F nucleatum on the liberation of arachidonic acid, the triti-
December 1998 Am J Obstet Gynecol
Fig 2. Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells. Tritiated arachidonic acid–labeled endometrial cells (1.0 × 107 cells/0.2 mL) were incubated with bacterial extracts in protein of 0.3 mg/mL, medium conditioned with F nucleatum, control medium, or normal saline solution for 1 hour at 37°C. The radioactivity of liberated arachidonic acid and metabolites was determined. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. Asterisk, P < .001, versus control medium; two asterisks, P < .005, between F nucleatum extract and conditioned medium. dpm, Disintegrations per minute.
ated arachidonic acid–labeled endometrial cell suspensions (0.5-1.0 × 107 cells/0.2 mL) were exposed to bacterial extracts in protein of 0.3 mg/mL, medium conditioned with F nucleatum, control medium, or normal saline solution at 37°C. The reaction was terminated by adding 1 mL chloroform/methanol (1:2, volume/volume), followed by 0.2 mL 10 mmol/L ethylenediaminetetraacetic acid with 0.01 N hydrochloric acid and 0.2 mL chloroform. After the 2 phases were separated, the lower phase (chloroform layer) was washed with chloroform-saturated water. The extracted lipid was analyzed with thin-layer chromatography as described previously.9 The areas corresponding to free fatty acids and arachidonic acid metabolites formed a single spot in this system, and each phospholipid was scraped into vials and its radioactivity was counted in a liquid scintilation counter. In some experiments lipid phosphorus was determined by the procedure of Rouser et al.12 Statistical analysis. Values were reported as mean ± SD. All results were analyzed with the Student t test, and significance was assigned at P < .05.
Volume 179, Number 6, Part 1 Am J Obstet Gynecol
Mikamo et al 1581
Results The uptake of tritiated arachidonic acid into phospholipids by human endometrial cells increased in time-dependent manner and reached the plateau level in approximately 6 hours (Fig 1). As illustrated in Fig 2, exposure of the tritiated arachidonic acid–labeled human endometrial cells to the Gifu anaerobic medium conditioned with F nucleatum induced an increase in the radioactivity of free fatty acid and arachidonate metabolite fractions. Control Gifu anaerobic medium showed no significant effects. A similar stimulatory effect on the rate of free arachidonate and metabolite production was observed in the experiments with bacterial extract. A significant difference in free arachidonic acid generation by endometrial cells was noted between F nucleatum extract and the conditioned medium treatments. Fig 3 shows the effect of F nucleatum extract on production of lysophospholipid, another metabolite of phospholipase A2. When the endometrial cells were challenged with the extract, lysophosphatidylcholine was observed to increase significantly (P < .01). Stimulated degradation of phosphatidylcholine to its lyso derivative might be observed in endometrial cells when exposed to the component secreted from F nucleatum. Even when heated at 100°C for 30 minutes, the extract increased the degradation at a similar level. Comment This and previous studies suggest that there is an increase in arachidonic acid metabolism seen with preterm or term labor that is associated with an increase in the production and release of prostaglandin E2. The statement made in the article regarding prostaglandins should be considered only as a justifiable assumption. Others have found that arachidonic acid metabolites play a major role in the initiation and maintenance of labor.13-15 We studied how this might occur in the presence of F nucleatum. We found that the uptake of tritiated arachidonic acid into phospholipids by human endometrial cells increased in a time-dependent manner and reached the plateau level in approximately 6 hours. This means that the phospholipids were labeled to almost constant specific radioactivity, and the changes in the levels of tritiated phospholipids and tritiated arachidonate therefore accurately reflected those in lipid mass. Exposure of tritiated arachidonic acid–labeled human endometrial cells to the Gifu anaerobic medium conditioned with F nucleatum induced an increase in the radioactivity of free fatty acid and arachidonate metabolite fractions. These findings suggest that F nucleatum yields and secretes phospholipase, which hydrolyzes endometrial phospholipids to liberate arachidonic acid and subsequently prostaglandins.
Fig 3. Effects of F nucleatum extract on lysophosphatidylcholine (Lyso PC) production in human uterine endometrial cells. Experimental conditions were described in Methods section. The phosphorus of lysophosphatidylcholine was determined and expressed as percentage of total phospholipid. Each point represents the mean from triplicate determinations of a representative experiment; error bars represent SDs. Heating was at 100°C for 30 minutes. Asterisk, P < .05, versus control. PC, Phosphatidylcholine.
The mechanisms through which arachidonic acid was released from endometrial phospholipids on incubation with bacterial component was then investigated. Because the obligatory precursor of prostaglandins, arachidonic acid, is present as an ester of glycerophospholipids, the action of phospholipase is likely to liberate arachidonic acid. In particular, phospholipase A2, which is found in bacterial lysosomes and also in a mammalian cell membrane–bound form, plays a major role in the hydrolysis of phospholipids. When human endometrial cells were exposed to an extract from F nucleatum, arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Further, stimulatory effects on phospholipid degradation were observed in the experiments with the bacterial extract. These results show that phospholipase A2 is secreted from F nucleatum, acting on endometrial phospholipids, or its product stimulates endometrial phospholipase A2, leading to phospholipid breakdown, or both. Heating of the extract did not influence the degradation of phosphatidylcholine. Endotoxin, a stimulant of cell surface receptor, increases in amniotic fluid in the setting of preterm labor.8 F nucleatum could therefore produce both phospholipase and endotoxin, doubly stimulating arachidonic acid metabolism with subsequent prostaglandin formation. Asymptomatic or subclinical intrauterine infection with growth of F nucleatum thus might easily induce labor.
1582 Mikamo et al
Finally, our study demonstrates that F nucleatum products can stimulate endometrial phospholipid hydrolysis through phospholipase A2 activity. We speculate that some subset of these mechanisms may be operative in mediating preterm labor in association with clinical or subclinical intrauterine infection in the presence of F nucleatum. REFERENCES
1. Mikamo H, Ito K, Tamaya T. Studies on the clinical implication of anaerobes, especially Prevotella bivia, in obstetrics and gynecology. Acta Sch Med Univ Gifu 1994;42:230-48. 2. Mikamo H, Izumi K, Ito K, Sawamura H, Sawa K, Watanabe K, et al. Bacterial flora detected in the uterine endometrial cavity of normal puerperae on the puerperal first day and on the puerperal fifth day after incidental use of cefpodoxime proxetil (CPDX-PR). Jpn J Antibiot 1993;46:269-73. 3. Romero R, Avila C, Sepulveda W. The role of systemic and intrauterine infection in preterm labor. In: Fuchs AR, Fuchs F, Stubblefield PG, editors. Preterm birth. 2nd ed. New York: McGraw-Hill; 1993. p. 97-136. 4. Bejar R, Curbelo V, Davis C. Premature labor II: bacterial sources of phospholipases. Obstet Gynecol 1981;57:479-83. 5. Takahashi K, Imai A, Tamaya T. Preterm labor and bacterial intraamniotic infection: arachidonic acid liberation by the action of phospholipase A2. Arch Gynecol Obstet 1984;244:1-6. 6. Khan AA, Imai A, Tamaya T. Possible mechanism for preterm labor associated with bacterial infection I: stimulation of phos-
December 1998 Am J Obstet Gynecol
7.
8.
9.
10.
11. 12.
13.
14.
15.
phoinositide metabolism by endotoxin in endometrial fibroblasts. Res Commum Chem Pathol Pharmacol 1990;69:119-22. Khan AA, Imai A, Tamaya T. Possible mechanism for preterm labor associated with bacterial infection II: enhancement of endotoxin-stimulated phosphoinositide metabolism by sex steroids in endometrial fibroblasts. Res Commum Chem Pathol Pharmacol 1990;69:123-6. Romero R, Roslansky P, Oyarzun E, Wan M, Emamian M, Novitsky TJ, et al. Labor and infection II: bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. Am J Obstet Gynecol 1988;158:1044-9. Lamont RF, Rose M, Elder MG. Effects of bacterial products on prostaglandin E production by amnion cells. Lancet 1985;2:1331-3. Lowry OH, Rosenbrough N, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. Imai A, Gershengorn MC. Measurement of lipid turnover in response to TRH. Methods Enzymol 1986;141:100-11. Rouser G, Siakotos AN, Fleisher S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1966;1:85-6. MacDonald P, Schulz M, Duenhoelter J, Gant NF, Jimenez FJ, Pritchard JA, et al. Initiation of human parturition I: mechanism of action of arachidonic acid. Obstet Gynecol 1984;44:62936. Carsten ME. Prostaglandins and cellular calcium transport in the pregnant human uterus. Am J Obstet Gynecol 1973;117:82432. Bejar R, Curbello V, Davis C, Gluck L. Premature labor and bacterial sources of phospholipase. Obstet Gynecol 1981;57:473-82.