Endothelin-1,2 levels are increased in the amniotic fluid of women with preterm labor and microbial invasion of the amniotic cavity

Endothelin-l,2 levels are increased in the amniotic fluid of women with preterm labor and microbial invasion of the amniotic cavity Roberto Romero, MD:'c Cecilia Avila, MD: Samuel S. Edwin, MS,b and Murray D. Mitchell, DPhilb New Haven, Connecticut, Detroit, Michigan, and Salt Lake City, Utah The purpose of this study was to determine the effect of gestational age, labor, and microbial invasion of the amniotic cavity on amniotic fluid concentrations of endothelln-1,2. Amniotic fluid was retrieved by amniocentesis from 148 women : patients at term With and without labor, patients With preterm labor with and without intraamniotic infection, and women in the second trimester of pregnancy. Endothelin-1,2 was measured by a sensitive and specific radioimmunoassay. Immunoreactive endothelin-1,2 was detectable in all samples of human amniotic fluid. Advancing gestational age and spontaneous term labor did not result in changes in amniotic fluid concentrations of endothelin-1,2. Women with preterm labor and positive amniotic fluid cultures for microorganisms had higher amniotic fluid concentrations of endothelin-1,2 than did those without microbial invasion of the amniotic cavity (p < 0.05) . These results support a role for endothelins in the mechanisms responsible for preterm delivery associated with Intraamniotic tnfection. (AM J OSSTET GVNECOL 1992;166:95-9.)

Key words: Endothelins, intraamniotic infection, preterm labor, chorioamnionitis Endothelins are a family of small proteins that were originally isolated from the culture supernatants of porcine aortic endothelial cells. I The first member of this family to be characterized was endothelin-l. Expression of the endothelin-l gene is regulated by several vasoactive agents, and the peptide is a potent vasoconstrictor. Subsequently, this family of peptides has expanded to include endothelin-l (porcine/human) , endothelin-2, and endothelin-3 (rat en dothelin)."·4 These 21-residue peptides are formed from a preproendothelin (- 200 residues) by way of a 38/39-residue intermediate.' 2 It is thought that endothelins act as endogenous agonists of dihydropyridine-sensitive calcium channels. I Endothelins can induce two types of contractions of the rat uterus,S activate phospholipase A2,6 and activate phospholipase

From the Department of Obstetncs and C.~necology. Yale Umversity School of Medlcme," the Department of Obstetrics and Gynecology, Umverslty of Utah Medical Center.' and the Department uf Obstetncs and Gynecology, Wa.~ne State Umvenity.' Supported b.~ a grant fmm the Walter Scott FoundatIOn fur Medical Research. Dr. Rumero IS the renplent of a PhYSICIan Scientist Award from the Natzonal! nstitute of Chzld H eaith and Human Development. Drs. EdWin and Mitchell are supported by NatIOnal Instztutes of Health grant HD 20779. ReceIVed for publicatwn Octuber 25, 1990: reVIsed May 23, 1991; accepted May 29,1991. Repnnt requests: Roberto Romem, MD, Department ofObs/etnes and Gynecology, Wayne State Umvemty School of MedlClne, 4707 St. Antome Blvd., Detroit, M1 48201. 611 1)1438

C. 7.8 Endothelins have recently been found to be syn-

thesized not only by endothelial cells but also by kidney cells. 9 Moreover, it has been suggested that endothelin1 is wnthesi7ed hv amnion.lO· II A role for endotht'lins in the control of human parturition can be postulated on the basis of their effect on myometrial contractility and on phospholipase A2 and C. This study was undertaken to determine the effect of labor and microbial invasion of the amniotic cavity on amniotic fluid concentrations of endothelin-l,2.

Material and methods Patient population and study design. Amniotic fluid was collected from 155 women. Patients were subdivided into four groups: group I, women in the second trimester of pregnancy (gestational age of 16 to 18 weeks) undergoing amniocentesis for genetic indications (maternal age >35 years) (n = 22); group 2, women in the third trimester (gestational age of 38 to 39 weeks) who had amniocentesis for the assessment of fetal lung maturity before elective cesarean section ('II = 25); group 3, women with spontaneous active labor at term and negative amniotic fluid cultures (n = 44). Amniotic fluid was obtained at the time of repeat or primary cesarean section or by amniocentesis in patients who were thought to have preterm labor but who subsequently were delivered of babies weighing >2800 gm and considered term by pediatric examination. Group 4 consisted of women admitted with 95

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January 1992 Am J Obstet Gynecol

500

400

E

--E

(5

~

N. ~

C

200

Qi

.r::

(5

"'0

c

w 100 I

..loa 0,a.0 0

Mid Trimester n = 22 1 fmol

..... 0 0

.!. :1"10

of

Term No Labor n = 25

Term Labor n = 44

I

°0

= 2.5 pg

Fig. 1. Amniotic fluid endothelin-l,2 concentrations in women in midtrimester, at term WIthout labor, and at term in active labor. Midtrimester: median 57.5 fmol/ml, range 38 to 125 fmol/ml; term no labor: median 56 fmol/ml, range 26 to 450 fmol/ml; term labor: median 50.5 fmol/ml, range 32 to 110 fmol/m!. No differences among groups (p > 0.05).

preterm labor and intact membranes (n = 57). Amniocentesis was performed in this group for microbiologic assessment of the amniotic cavity and fetal lung maturity studies. To study the relationship between intraamniotic infection, pre term labor, and amniotic fluid endothelin-1,2 concentrations, a cross-sectional study of patients in group 4 (preterm labor) was constructed. Patients with preterm labor were subdivided into three subgroups according to their response to tocolysis and the results of their amniotic fluid culture: subgroup 4a, women with preterm labor and negative amniotic fluid cultures who were responsive to tocolysis (n = 21); subgroup 4b, women with preterm labor and negative amniotic fluid cultures who were unresponsive to toco lysis and were delivered of preterm neonates (n = 19); subgroup 4c, women with preterm labor and intraamniotic infection who were delivered of preterm neonates (n = 17). Preterm labor was defined as the presence of regular uterine contractions with a frequency of at least two every 10 minutes for at least 60 minutes' duration. Amniocentesis was performed before the initiation of toco lytic therapy with intravenous ritodrine or terbutaline. Failure of tocolysis was diagnosed when cervical

dilatation progressed beyond 5 cm and/or delivery occurred. Informed consent was obtained from all patients according to the guidelines of the Yale University Human Investigations Committee. Microbiologic studies. Amniotic fluid was transported to the laboratory in a capped plastic syringe immediately after collection and plated within 30 minutes of collection. Fluid was cultured for aerobic and anaerobic bacteria and for Mycoplasma hominis and Ureaplasma urealytlcum, as previously described. 12 Endothelin-I,2 determinations. Endothelin-l,2 was measured with radioimmunoassay kits from Amersham Corporation (Arlington Heights, Ill.). The assay was performed directly on duplicate 100 fl.1 amniotic fluid samples. We have found that purification ofliquor samples before assay has a limited effect on measured values, in marked contrast to its significant impact on plasma measurements. The sensitivity of the assay (2 SD of the zero point binding) is 1.03 fmol. Intraassay and interassay coefficients of variation are 4.1 % and 10.4%, respectively. Statistical analysis. Comparisons of endothelin-l ,2 concentrations in amniotic fluid samples from different groups were conducted with a Kruskal-Wallis one-way

Endothelin-1,2 in amniotic fluid

Volume 166 Number 1. Part 1

97

700

600

500

300

200

100

. ··fIII··

·

-.

....· 0\

·

.~.

o~----~----------~------------~-----

No Infection No Delivery n=21

No Infection Delivery n=19

Infection n = 17

1 fmol = 2.5 pg Fig. 2. Amniotic fluid concentrations of endothelin-I ,2 in women in preterm labor according to response to tocolysis and amniotic fluid culture results. Subgroup 4a, women with negative amniotic fluid cultures who responded to tocolysis: median 55 fmol/ml, range 17 to 83 fmol/ml; subgroup 4b, women with negative amniotic fluid cultures who did not respond to tocolysis and were delivered of preterm neonates: median 62 fmol/ml, range 31 to 110 fmol/ml; subgroup 4c, women with positive amniotic fluid cultures who were delivered of preterm neonates: median 150 fmol/ml, range 41 to 640 fmol/m!. No difference was found between subgroups 4a and 4b; concentration in subgroup 4c was greater than that in subgroups 4a and 4b (P < 0.05).

analysis of variance. The Dunn test was used for post hoc multiple comparisons among groups (True Epistat, Epistat Services, Richardson, Tex.). Results

Immunoreactive endothelin-I,2 was detected in all amniotic fluid samples from patients in the second and third trimesters of pregnancy (groups 1 and 2, Fig. I). No changes were observed with gestational age in the amniotic fluid concentrations of endothelin-I,2. Spontaneous labor at term was not associated with an increased concentration of immunoreactive endothelin1,2 in amniotic fluid (p > 0.05) (Fig. I). Patients in preterm labor with positive amniotic fluid cultures for microorganisms (group 4c) had significantly greater concentrations of amniotic fluid endothelin-I,2 than did women without intraamniotic infection (subgroups 4a and 4b, Fig. 2) (p < 0.05). Failure to respond to tocolysis in women with preterm labor

was not associated with higher amniotic fluid concentrations of endothelin-I,2 when compared with those of women in preterm labor who were responsive to tocolysis. Table I displays the clinical and microbiologic information for patients with preterm labor and microbial invasion of the amniotic cavity. Comment

This study documents the presence of immunoreactive endothelin-I,2 in normal amniotic fluid from women in the second and third trimesters of pregnancy. It is interesting to note that amniotic fluid concentrations of endothelin-l,2 were approximately tenfold higher than those observed in peripheral blood or in the umbilical circulation. 13. H No change in the amniotic fluid concentration of endothelin-I,2 was observed with gestational age. The role of these endothelins is uncertain. Since endothelin can induce smooth muscle contraction, we pos-

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January 1992 Am J Obstet Gynecol

Table I. Clinical and microbiologic data of patients with preterm labor and microbial invasion of amniotic cavity Pa!zen! identification No.

GestatIOnal age (wk)

26 2

25

3 4 5

22 33 24

6

28

7 8 9

29 28 24

10 11 12 13

24 28 24 33

14 15 16 17

25 27 25 33

Colonv co un! (cf~/ml)

OrganISm ClostridIUm sp. Capnocytophaga sp.

Mixed aerobic grampositive flora

Gardnerella vaginahs Fusobacterium sp Gardnerella vagznalis Peptostreptococcus sp. Mycoplasma hommls Fusobacterium sp. Gardnerella vagmalis Ureaplasma urealY/lcum Ureaplasma urealytlcum Streptococcus agalac/iae Ureaplasma urealyticum Streptococcus vindans Bacteroides sp. mixed Candida albicans Staphylococcus aureus Streptococcus agalactiae

Mixed anaerobes

Ureaplasma urealytlcum Streptococcus vmdans

tulated that it might playa role in the control of human parturition. Indeed, myometrium is extremely sensitive to the action of endothelin with a contractile effect demonstrated at very low concentrations of the peptide. l5 This action is mediated by an increase in intracellular calcium concentration and phosphorylation of the myosin light chain. l5 Additionally, endothelin-l can stimulate uterotonic prostaglandin output by myometrium!6 and thus induce contractions indirectly. Our data, however, do not indicate a change in amniotic fluid concentrations of endothelin-l,2 with spontaneous parturition at term. In contrast, women with preterm labor and microbial invasion of the amniotic cavity have higher amniotic fluid concentrations of endothelin-l.2 than do those with negative amniotic fluid cultures for microorganisms. These results are consistent with a role for endothelin-l,2 in the mechanism of preterm labor associated with intraamniotic infection. Alternatively, the increased availability of endothelin-I,2 in amniotic fluid could be an epiphenomenon associated with parturition in the setting of infection. Further investigation of the intrauterine sources and local regulation of the production of endothelin is required. It is unknown whether bacteria could be a source of endothelin. It has been documented, however, that human amnion produces endothelin 17 and that cytokines such as interleukin-ll3 can increase endothelin production by

10' 10'

2000 >10' 100

Ammotic flUid endothelzn-l ifmoUrnl)

115 220 125 150 440

>10' >10'

160 100 180

>10'

63 280 63 120

>10' >10' 10' >10' >10' 10'

41 170 220 150 640

amnion.!8 Interleukin-ll3 is found in the amniotic fluid of women with pre term labor and intraamniotic infection !9. 20 and is produced by human decidua in response to bacterial products!! Therefore cytokines produced during the host response to intraamniotic infection may be responsible for the increased availability of endothelin-l,2. Endothelins may playa role in the regulation of myometrial contractility in infection-associated preterm labor. Further studies are required to determine whether amniotic fluid endothelin can cross intact chorioamniotic membranes and reach the myometrium to exert this biologic action.

I.

2.

3.

4.

5.

REFERENCES Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-45. Itoh Y, Yanagisawa M, Ohkubo S, et al. Cloning and sequence analysis of eDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBS Lett 1988;231 :440-4. Yanagisawa M, Inoue A, Ishikawa T, et al. Primary structure, synthesis, and biological activity of rat endothelin, and endothelin-derived vasoconstrictor peptide. Proc Nat! Acad Sci USA 1988;85:6964-7. Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Nat! Acad Sci USA 1989;86:2863-7. Kozuka M, Ito T, Hirose S, Takahashi K, Hagiwara H. Endothelin induces two types of contractions of rat

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uterus : phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels. Biochem Biophys Res Commun 1989;159:317-23. Resink TJ. Scott-Burden T. Buhler FR. Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158:279-86. Resink TJ. Scott-Burden T. Buhler FR. Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988;157: 1360-8. Sugiura M. Inagami T, Hare GMT,JohnsJA. Endothelin action : inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun 1989; 158: 170-6. Kosaka T, Suzuki N, Matsumoto H, et al. Synthesis of the vasoconstrictor peptide endothelin in kidney cells. FEBS Lett 1989;249:42-6. Sunnergren KP, Word RA, SambrookJF, MacDonald PC, Casey ML. Expression and regulation of endothelin precursor mRNA in avascular human amnion. Mol Cell Endocrmol 1990;68 :R7-R14 . Mitchell MD, Lundin-Schiller S, Edwin S. Endothelin production by cells derived from gestational tissues [Abstract 149). In : Proceedings of the thirty-seventh annual meetmg of the Society for Gynecologic Investigation. St. Louis, Missouri. March 21-24, 1990. Sl. Louis: Society for Gynecologic Investigation, 1990. Romero R, Quintero R. Emamian M, et al. Arachidonate lipoxygenase metabolites in amniotic fluid of women with intraamniotic infection and preterm labor. AM J OBSTET GYNECOL 1987;157:1454-60. Haegerstrand A, Hemsen A, GIllis C. et al. Endothehn:

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presence in human umbilical vessels, high levels in fetal blood and potent constrictor effect. Acta Physiol Scand 1989;137:541-2. Nakamura T, Kasai K. Konuma S, et al. Immunoreactive endothelin concentrations in maternal and fetal blood. Life Sci 1990;46: 1045-50. Word RA, Kamm KE, Stull JT, Casey ML. Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium . AM J OBSTET GYNECOL 1990; 162 :1103-8. Romero R, Avila C, Mitchell MD. Endothelin-I in human parturition [Abstract 510]. In : Proceedings of the thirtyseventh annual meeting of the Society for Gynecologic Investigation, Sl. Louis, Missouri, March 21-24, 1990. St. Louis: Society for Gynecologic Investigation, 1990. Mitchell MD, Edwin S, Lepera R, Rittenhouse L, Romero R. Actions of endothelin-I on prostaglandin production by gestatIonal tissues [Abstract 150]. In : Proceedings of the thirty-seventh annual meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. St. Louis: Society for Gynecologic Investigation. 1990. Mitchell MD. Endothelins in perinatal biology. Semin Perinatol 1991; 15:79-85. Romero R, Brody DT, Oyarzun E, et al. Infection and labor. III. Interleukin-l: a signal for the onset of parturition. AM J OBSTET GYNECOL 1989; 160: 1117-23. Romero R, Mazor M, Brandt F, Sepulveda W, Avila C, Dinarello CA. Interleukin-1n and interleukin-113 in human preterm and term parturition. Am J Reprod Immunol [In press]. Romero R, Wu YK, Brody DT, Oyarzun E, Duff GW, Durum SK. Human decidua : a source of interleukin-l. AM J OBSTET GYNECOL 1989;73:31-4.