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Arachidonate lipoxygenase metabolites in amniotic fluid of women with intra-amniotic infection and preterm labor
Arachidonate lipoxygenase metabolites in arnniotic fluid of wornen with intra-arnniotic infection and preterm labor Roberto Romero, M.D ., * Ruben Quintero, M.D., Mohamed Emamian, M.D., Macor Wan, M.D., Camille Grzyboski, C.L.T., John C. Hobbins, M.D., and Murray D. MitchelI, D.PhiI. NeU' H auen , Connecticut, and San Diego, California
This study was undertaken to examine Ihe effects of intrauterine infecl ion and prelerm labor on the amniotic fluid concen trations of arachidonate Iipoxygenase metabolites in women with premature rupture of memb ranes. Amniotic fluid was obtained trom four groups of women with prernature rupture of membran es: group 1, wome n without labor orinfection; group 2, women with labor but without infection; group 3, women wilh intra-amniotic infection but wilhout labor; and group 4, women wilh both inteclion and labor. 12-Hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, and leukol riene 8 _ were measu red by radioimmunoassays. Amniotic fluid concent rations of 12-hydroxyeicosatetraenoic acid were found not to differ significantly among Ihe four groups. Amniotic fluid concentrations of 15-hydroxye icosatetraenoic acid in group 4 were significanlly higher than in women in groups 1 and 3 (p < 0.05) . In addition , amniotic fluid concentrations in leukolriene 8_were significanlly higher in group 4 than in any of the other three groups (p < 0.05). Leukotriene 8. concentralions were higher in groups 2 and 3 than in group 1, suggesling that the presence of both labor and infecl ion increases the concentration of Ihis metabolite in amniotic fluid. Infection and labor had an additive effect in the elevation of amniotic fluid concenlrations of leukotriene 8•. These results suggest that the amniotic fluid concentrat ions of arachldonate Iipoxygenase metabolites are affected differently by the presence of infeclion and labor in women with premalure rupture of membranes . (AM J Ossrer G VNECOl 1987;157:1454-60.)
Key words: Arachidonat e lipoxygenase , intra -amn iotic infection, prern ature rupture of membranes
Intra-arn nio tic in fection is fre q ue n tly associa ted with th e o nse t of Iab or.' T he mechanisms resp onsible fo r parturition in these cases ha ve not bee n esta blishe d . Partu ritio n at term is assoc iated with mob ilizati on of a rachido nic acid fro m fe ta l mernbranes." Arachidonic acid ca n be metabol ized eithe r by way o f the cyclooxy genase 0 1' lipoxygenase pathways. The former pathway leads to the syn thesis of prostaglandins a nd thrornboxanes, wh ereas the lau er pa thwa y leads to the format ion o f lip oxygenase products suc h as hyd roxyeicosatetraen oic ac ids an d leuk ot ri en es, Arac hidonate lip oxygenase products are ge ne ra ted during th e co u rse of inFrom tlte Departments of Obstetrics and Gynecology, Yale Uni versity School ofMediane, and the Depa rtment of R eproductive M ediane, the Uni uersits of Cal iforni a, San Diego. Suppor ted by grants f rom the Walte r Scott Foundation [or M edical Research and National Institutes of Hea l üi grafts H D 2074 7 a nd HD 2 0 77 9. Present ed in part at the T hirtyl ourth Annual M eeting of the Society fo r Gynecologic Inuestigation, Atlanta, Georgia, March I8-2 l, 198 7. Reprint requests: R oberto Romero, M .D ., Department of Obstetrics and Gynecology, Yale Un iversity School of M edicine, P . O. Box 3333, Ne w Hauen, CT 06510. *Recipient of a Physician Scientist Award [rom the N ational I nstitu tes of H ealth.
1454
flarn rna to ry reacrion s"a nd can be syn thesize d by human intrauterine tissues .' T hese products hav e been found to be involved in the d evelopment of uterine contractions,' :" a nd they ma y participate in the no rma l p ro cess o f lab or 0 1' in the me ch an isrn o f lab or th at is assoc iated with 0 1' th e result of an inAammat ory rea ction (e.g., chorioamnionitis). The purpose of this study was to deterrnine the concentra tions of arach idonate lipoxygenase rnet ab olites in a m niotic fluid ofwomen with and without in tra- arnn iot ic in fect ion s a nd to e valuate th e rel ati ve in flu en ce of la bor a nd in fection o n the a m niotic fluid levels o f these co m po u nds .
Material and methods Study design. A cr oss-sectional stud y was construct ed accordin g to the resu lts of a m nio tic flu id cu ltures in women with premature ru ptu re of mernbranes. Fou l' groups wcre created: g rou p 1, women without labor 0 1' infection ; gro u p 2, wo rnen with labor bu t without in fecti on ; gro u p 3, wo rnen with a n intra-amniotic infection but witho u t labor ; a nd gr o u p 4 , wo me n with both infec tion an d lab or. An intra-am n iotic in feet ion was defined as the p res ence of a positive amnio tic fluid cu lture. None of th e patients in this st ud y developed
Volume 157 Number 6
signs and symptoms of infection. Labor was defined as the presence of regular uterine contractions that led to cervical dilatation and spontaneous vaginal delivery within 24 hours of the performance of the amniocentesis. Pelvic examination was not performed at the time of amniocentesis, since this could result in a higher incidence of infection.' Women were cIassified according to the results of amniotic fluid culture and their labor status at the time of amniocentesis. Retrieval of amniotic fluid. Amniotic fluid was obtained by transabdominal amniocentesis in women admitted to Yale--New Haven Hospital with the diagnosis of rupture of membranes at a gestational age of less than 37 weeks. Our success rate in obtaining fluid from these women is 95%. Amniotic fluid was processed for Gram stain, culture, lecithin/sphingomyelin ratio, and phosphatidylglycerol, and any remaining fluid was stored at - 20° C. Immediately before amniocentesis, the presence 01' absence of uterine contractions was recorded by external tocodynamometry. Culture technique, Amniotic fluid obtained by transabdominal amniocentesis was transported to the laboratory in a capped, plastic syringe immediately after collection of the specimen. These conditions precluded air contact with the specimen. Plating occurred within 30 minutes of collection in all cases. The sam pies were plated on blood agar, MacConkey agar, Columbia colistin-nalidixic acid agar, chocolate agar (36° C in 8% carbon dioxide) for aerobic culture and blood agar, bacteroides and bile-esculin agar/laked kanamycin and vancomycin agar, and Martin-Lewis agar (all prereduced) for anaerobic culture. Amniotic fluid was also inoculated in thioglycolate broth. Anaerobic cultures were grown in an anaerobic chamber in a Forma Seientific anaerobic system (Model 1024, Division of Mallinckrodt, Marietta, Ohio). The number of colonyforming units per milliliter was determined by plating the specimen with a 0.01 and 0.001 calibrated urine loop. Each colonial morphotype was identified to species by Food and Drug Administration-Centers for Disease Control-approved commercial systems supplemented by conventional tests when needed. An identification was accepted only when it yielded a normalized probability of 97% 01' greater. When more than three organisms were grown from an amniotic fluid sample, the infection was referred to as "mixed aerobic 01' anaerobic flora." Species identification was not conducted in these cases. This protocol evolved, since it was frequently difficult to identify all baeterial species involved, and there was no evidence that this effort altered patient management. There was also no evidence that amniotic fluid concentrations of lipoxygenase products would be preferentially affected by some organisms. Identification of anaerobes was conducted with the IDS RapID ANA system (Innovative Di-
Intra-amniotic infection and Iipoxygenase products
1455
agnostic System, Atlanta, Ga.). Agar was obtained from Baltimore Biological Laboratories, Cockeysville, Md. Mycoplasma and Ureaplasma cultures. Amniotic fluid was centrifuged at 2000 g for 20 minutes and the sediment was placed into 2 ml of holding media (pleuropneumonia-like organism broth, which was supplemented with fresh yeast extract and horse serum, penicillin, polymyxin B, and amphotericin). SampIes werestored at -70°C until culture (Mycotrim-GU, Berkeley, Calif.). Lipoxygenase metabolite determinations. Arachidonate lipoxygenase products were extracted from arnniotic fluid into acidified diethyl ether (five volumes). The solvent was evaporated under nitrogen, and the residue was dissolved in assay buffer and assayed using specific radioimmunoassays for 15-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B., (Seragen, Inc., Boston, Mass.). Extraction efficiency was determined by assessment of the recovery of each radiolabeled lipoxygenase product added to an aliquot of an amniotic fluid pool. The extraction efficiency was 76% for leukotriene B4 , 9 I % for 12-hydroxyeicosatetraenoic acid, and 87% for 15hydroxyeicosatetraenoic acid. All measurements of arnniotic fluid sam pies were conducted in the same assay. One hunclred microliters of amniotic fluid was used in the radioimmunoassay. Intra-assay coefficients of variation were 12% for leukotriene B4 , 11% for 15-hydroxyeicosatetraenoic acid, and 10% for 12hydroxyeicosatetraenoic acid. Sensitivities of the assays for 12-hydroxyeicosatetraenoic acid and 15hydroxyeicosatetraenoic acid were 8 pg, whereas it was 4 pg for leukotriene B 4 • Inhibition of the binding of the radioligancl to the antibody by 50% was achieved by 90 pg for leukotriene B1> 170 pg for 15-hydroxyeicosatetraenoic acid, and 260 pg for 12hydroxyeicosatetraenoic acid. Se rial sarnple clilutions were parallel to the standard curve for each assay (slope comparisons: p> 0.05). Solvent blanks for leukotriene B" 15-hydroxyeicosatetraenoic acid, and 12hydroxyeicosatetraenoic acid were 8 pg, 29 pg, and 98 pg, respectively. Solvent blanks were subtracted from measured values. Statistical analysis. The Kolmogorov-Smirnov test was used to test the hypothesis that lipoxygenase products were normally distributed. Logarithmic transforrnation was required for normality in 15hydroxyeicosatetraenoic acid. The amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid were normally distributed, but those of Ieukotriene Bi were not. One-way analysis of variance was used to compare the levels of lipoxygenase metabolites in the four different groups for I2-hydroxyeicosatetraenoic acid and log 15-hydroxyeicosatetraenoic acid. Duncan's multiple
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Romero et al.
Decem ber 198 7 Am J Obs tet Gynecol
16'0~
o
15.5 15.0 14.5 5.5
0
5.0 15HETE
ng/ml
4.5 4.0
0 0
3.5
'b
0
3.0 2.5
0
2.0
0
0
1.5
0
0
~
1.0 0.5 0.0
0
0 0 0 0
d'b
0
' - --
-----""""''----
I No Labor No Infection (n = 15)
--''''''-'''=----
---->;3iJ...---- - - ' - --
11 Labor No Infection (n = 15)
111
No Labor Infection (n = 14)
----'
IV Labor Infection (n = 12)
Fig. 1. Amniotic fluid co nce ntra tions of 15-h yd roxyeicosatetraen oic acid (I 5-HETE) in the following four groups of women: group 1, wom en with out labo r or infection ; group 2, women with labor but with out infection; group 3, women with an int ra-amn iotic in fectio n bu t with ou t labor; and gro up 4, women with bo th infection and labor. Log arith mic tra ns for mation was re q uired for normalit y to be achieved . 'O ne-way analys is of var ian ce was used 10 co m pare the am niotic fluid co ncentra tions of l ö-hvd roxyeicosa tetraenoic acid am on g th e four di fferent groups ( F = 4.02; p < 0.05). A sign ificant di ffer en ce was fou nd o nly between gro u ps 1 an d 4 and groups 3 and 4 (p< 0.05 , Dun can 's multiple range test ). The median co ncentra tion o f each gro up is represented by th e line in the scattergra m of each grou p.
range test was used for compari son between the diffe rent gro u ps. A p value < 0.05 was consid ered statistically sig nificant. Kruskal-WaIlis test was used to cornpare the amniotic fluid concen tratio ns of leukotriene . B, am ong different groups. A logarithm ic transforrnation was not used because many patients had nondetectable levels . Multiple co mpa riso ns amo ng the four groups were car ried out foIIowing the procedure described by Conover." Results
The co ncen trations of 15-hydroxyeicosatetraenoic acid, leukotriene B" and 12-h ydrox yeicosatetraenoic acid in amniotic fluid frorn women in the four differen t groups are iIIustrated in Figs. 1 through 3, respectively. The concentration of 12-hydroxyeicosatetraenoic acid in amniotic fluid did not vary significa ntl y among gr ou ps (ana lysis of varian ce, p > 0.05), whereas differences were detected in th e levels o f 15-h ydrox yeicosate tra eno ic acid and leukotriene B, (analysis of variance, p < 0.05). Amniotic fluid con centrations of 15h ydrox yeicosatetraenoic acid were gre ater in group 4 th an in grou p I (p < 0.05, Duncan's multiple ra nge test) and greater in grou p 4 th an in group 3 (p < 0.05, Duncan's multiple range test) . Amniotic fluid con centrations of leukotriene B, were sign ificantly different
amo ng aII gr oups excep t for the comparison between gr oups 2 a nd 3. T able I illustrates the descriptive statistics for ea ch group. T able s 1I and III show the re sults of quantitative microbiologyand amniotic fluid concentrations o f lipoxygenase products of women with intra-amniotic infections (gro u ps 3 a nd 4). No consistent relationship between arachid o nic lipoxygenase metabolite con centrations in amniotic fluid and specifi c organisms isolated from the fluid cou ld be d ernonstrated . No difference in the time in terval be tween arnniocentesis and delivery was found in the groups of women in labor at th e tim e of the amniocentesis (grou ps 2 and 4) (p > 0.5, WiIcoxon rank test) . Comment
The results of this stud y suggest th at the arnniotic fluid con centrations of I2-hydroxyeicosatetraenoic acid , l 5-h ydroxyeicosat etraenoic acid, and leukotriene B, are a ffected d ifferentlyby infection and labor in women with premature ruptu re of mernbranes. No differences were detected in the levels of I2 -h ydroxyeicosatetra enoic acid in a mnio tic fluid among the fou r groups; this was not the case with amniotic fluid concen trations of leukotriene B, a nd 15hydroxyeicosatetracnoic acid . T herefo re it appears that differen ces exist in the activity of th e enzyma tic path-:
Intra-arnniotic intection and lipoxygenase products
Volurne 157 Number 6
500 0
450 400 350
0
300 LTB4 pg/ml
0 0
250 200 0
150
0 0
100
0
50
0
0
I
No Labor No Intection (n = 15)
-e&
0
II
Labor No Intection (n = 15)
Il!
No Labor Intection (n = 14)
IV
Labor Infection (n = 12)
Fig. 2. Arnniotic fluid concentrations of leukotriene B, (LTB4) in the following four groups of wornen are displayed: group 1, women without labor or infection; group 2, women with labor but without infection; group 3, women with an intra-amniotic infection but without labor;and group 4, women with both infection and labor. A Kruskal-Wallis analysis of variance was perforrned to compare amniotic fluid concentrations among the different groups (p < 0.01). Amniotic fluid concentrations of leukotriene B, were significantly greater in group 4 than in any of the other groups. Amniotic fluid concentrations of leukotriene B, were significantly lower in group 1 than in groups 2 and 3. There were no differences between groups 2 and 3 (p > 0.05). The median concentration of each group is represented by the line in the scattergram of each group.
40 35 0
30 25 12HETE nglml
20
0 0 0 0
15 10
<9
~ 0 0
0
e
I
No Labor No Infection (n = 15)
-i% 0
s 0
11
Labor Ne lnfection (n = 15)
0
0
8
0
0
5
0
0 0
0
0
~
0
.....,.. ~ 0
0 0
111
No Labor Intectien {n = 14)
<9
IV
Labor Intectien {n = 12)
Fig. 3. Amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid (12-HETE) in the following four groups of women are displayed: group 1, women without labor or infection; group 2, women with labor but wirheut infection; group 3, women with an intra-amniotic infection but without labor; and group 4, women with both infection and labor. One-way analysis of variance was used to compare the arnniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid. There were no significant differences in amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid among the four different groups (F = 1.80, P > 0.05). The mean concentration of each group is represented by the line in the scattergram of each group.
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1458 Romero et aJ. Am
December 1987
J Obstet Gynecol
Table I. Descriptive statistics for concentrations (pg/ml) of amniotic fluid lipoxygenase metabolites in the four study groups
I 12 HETE No. observations Mean
Standard deviation Standard error of the mean Median Range 15 HETE No. observations Mean Standard deviation Standard error of the mean Median Range LTB4 No. observations Mean Standard deviation Standard error of the mean Median Range
GTOUP 1
Group 2
Group 3
Group 4
15 8546 5873 1516 5773 (292 I -2 I ,964)
15 11,496 6182 1597 10,315 (4728-24;972)
14 13,865 7792 2082 10,391 4296-31,198)
12 14,644 8432 2434 14.644 (2910-31,995)
14 908 899 240 677 (226-3498)
12 2192 1622 468 1817 (788-5744)
15 556 761 197 200 (237-2716)
15 2110 3994 1031 386 (382- I 5,965)
15 7.8 15.0 3.8 0 (0-50)
15 15.8 16.8 4.3 10.0 (0-46)
14 61.6 124.9 33.4 15.5 (0-465)
12 129.4 98 28.3 98.0 (8-331)
Table 11. Amniotic fluid concentrations (pg/ml) of arachidonate lipoxygenase products and microbiology of the amniotic fluid in women with intra-amniotic infections but without preterm labor (group 111) Patient identification No.
1I1 141 195 105 1071 36 1203A 1236A 1183A 1187A 1276 1294 36 37
M icroorganism Haemophilus inftuenzae Mycoplasma hominis M. hominis
Lactobacillus Group B streptococcus Fusobacterium species Bacteroides oralis M. hominis Fusobacterium species Group B streptococcus Fusobacterium species M. hominis Ureaplasma -urealsticum Staphylococcus aureus M. hominis Capnocytophaga Gardnerella oaginalis G. vaginalis U. urealyticum
Colony count*
12-Hydroxyeicosatetraenoic
15-Hydroxyeicosatetraenoic
Leukotriene B 4
30271 15717 3369
3204 1015 626
93 22 0
9131 8121
1440 285
33 17
875,) 11227
107 348
0 0
22713
728
14
23637 16484 9555 20073 7721 7337
1696 1508 69 0 141 1539
465 46 0 164 2 7
>10' >10" > 10" >10', >10',
>10" io >10"
> 10"
25 X 10" >10"
*Colony count is expressed in colony-forming units per milliliter. When quantitative microbiology was unavailable, it is indicated by-.
ways or the degradation of these compounds in intrauterine tissues. Infection and premature labor may not resuit in activation of the pathway responsible for the biosynthesis of 12-hydroxyeicosatetraenoic acid; however, these clinical conditions may result in effects on the pathways responsible for the biosynthesis 01' degradation of 15-hydroxyeicosatetraenoic acid and leukotriene B4 - Alternatively, these results may be explained by differences in the concentration of arachi-
donic acid. Depending on the Michaelis constant of the enzymes of the different lipoxygenase pathways for arachidonic acid and other substrates, the changes observed could be partially caused by alterations in the concentrations of arachidonic acid. We have found that premature labor alone in women with premature rupture of membranes does not alter the amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid 01' 15-hydroxyeicosatetraenoic acid but
Intra-amniotic infeetion and Iipoxygenase products
Volume 157 Number 6
1459
Table III. Amniotic fluid co nce n tratio ns (pg/m l) of arachidonate lip ox ygenase products and microbiolog y of the a m nio tic fluid in women with intra -a rnn iotic infections and preterm labor (grou p IV) Patient identijication N o.
150 39 70 11 32 33 1239A 1206A 1297 1255 1093 171A 1000
M icroorganism Gardnerella vaginalis
Fusobacterium speci es
Mycoplasma homiuis Mixed anaerobic flo ra Fusobacterium species G. vag ina lis Mixed an ae robic flora Gaffkya anaerobica G. vaginalis Fusobacterium nucleatum Mixed anae ro bic flor a Listerio monocytogenes Haemophilus influenza/' Gardnerella vaginalis
Colony count*
12-Hydroxyeicosatetraenoic
l i-Hvdroxyeicosatetraenoic
Leukotriene B.
3+ > 10' 1+
14789 1983 10095
5450 494 612
83 246 134
17369 24053
11 23 2749
32 72
> 10; > 10' 25 x 10" > 10' > 10" > 10' > 10'
15402 14499 2300 5255 13477 15632 31068
1075 1084 549 3060 2510 3425 4 177
258 102 8 94
> lW > 10;
72
121 331
*Co lony COU Ill is expressed in colony-form ing uni ts per m illiliter. When quant itati ve microbi ology was un available, it is indi cat ed
by - .
only that o f leukotriene B,. This findin g is interesting because it a ppea rs to be at variance with th e results of our earlier study in which women in term labor had sig nificantly eleva ted co nce n tra tio ns of aIl th ese compounds." There are severaI reasons wh y such diffe rences ma y exist. For ins ta nce, in our earlier stud y, aIl wornen we re in acrive lab or with a ce rvical dilatation o f 6 cm or grea te r, where as in the cu rre nt study, th e wom en a re likel y [0 have been in mu ch ea rlie r labo r. Data o n ce r vical d ilat ati on are not avai lable because pel vic examinations wer e not performed in women with prernature ruptu re of mem branes, sin ce thi s procedu re may in crease the incidence of infection ." It can also be a rgued that a maturational eye nt may acco u nt for the d ifferential re lease of lipox ygenase products in to arn niotic fluid . Women in preterrn labor ha ve lower concentration s of cycloox ygenase metabolites (i.e., p rostaglandins) in amnioti c fluid and plasm a tha n laboring wome n at terrn. " :" A similar phenomen on ma y occu r with lip ox ygenase rnet abolites. Evidence su p po r ting this concept is that man y women with preterm premature r u pt ure of membranes had nondetectable co ncen tr ati on s of leukotriene B, in the present study a rid that aJl nonlaboring wom en at te rm with in tact membranes had detectable concentrations of leu kotriene B•.9 H owe ver, these two gro u ps of women were not id entical with respect to membran e status. Intraamniotic infections alo ne seemed to affe et only the amnio tic fluid co ncentrations of leukot riene B I (corn pariso n bet ween gro ups 1 a nd 3). It remain s to be establi sh ed wh ether the in cr eased a mniotic fluid co ncentration of th is compound is the re sult of bacterial or host signals on the me ta bolism 01' ava ilability of arachidonic ac id . When both lab o r and in fectio n are present, the po-
tential rel ati ve effects of these two clini cal conditions on th e a m niotic fluid conc en tratio n ofli po xyge nase rnetabolites are not easy to interpret. Amniotic fluid co ncentrations of leuko trie ne BI were significantly ele vated in women infected and in labor (group 4) compared with all o the r gro u ps. This see ms to imply that labor a nd infection ha ve an ad d itive effect o n a mnio tic fluid concentrations of leukotriene B.,. .Since am niotic fluid concentrarions of p ro staglandin s a re increased with progressive ce r vical d ilatati on ," it may be hypothesized that the d ifference o bse rved in a m n iotic fluid co nce nt ra tions of leukotriene B., between groups 2 and 4 cou ld be attribu ted to differing ce rvical dilatat ion at the time o f am niocentesis between the grou ps. However, thi s is not Iikely to be the case because the time interval betwee n arnniocentesis and d elivery was not different between these two groups . Significant d ifferences in a m ni otic flu id co nce nt ra tions of 15-hydroxyeicosatetraenoic acid were o nl y detected when labor a nd in fec tio n we re present (gro u p 4) co m pa red with gro u ps 1 a nd 3 but not co rnpared with group 2. Further studies will be required to est abl ish the relative contribution of preterm labor and infection to amniotic fluid co ncen tra tio ns of 15-h yd roxye icosat e trae noic acid as this study is not conc1usive. In co nclu sio n, our data indicate that a m n iotic fluid conce nt rations of arach id o na te lipoxygenase merabolites are affect ed by th e prese nce of lab or a nd infeetion in women wit h p rete r m premature rupture of m em bran es. Pre te rm lab or is associa te d with increased amniotic fluid co nce n tratio n of leukotriene B•. Intra-amnio tic in fectio n without labor is also associat ed with incr eased amniotic fluid concen tra tions of Ieukotriene B,. The prese nce of preterm la bor and intra-
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Romero et al.
amniotic infection had an additive effect on the increase of amniotic fluid concentrations of leukotriene B 1 • Neither preterm labor nor infection was associated with significant changes in amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid. These observations are consistent with the possibility of an involvernent of the lipoxygenase pathway of arachidonic acid metabolism in the mechanism of human parturition associated with preterm labor and intra-amniotic infection. REFERENCES
1. Garite TJ, Freeman RK. Chorioamnionitis in the preterm gestation. Obstet Gynecol 1982;59:539. 2. Okita JR, MacDonald PC, Johnston jM. Mobilization of arachidonic acid from specific glycerophospholipids of human fetal membranes during early labor. j Biol Chem 1982;257: 14029. 3. Hansson G, Malmsten C, Radmark O. The leukotrienes and other lipoxygenase products. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. Vol 5. Amsterdam: Elsevier, 1983: 127·69. 4. Saeed SA, Mitchell MD. Formation of arachidonate lipoxygenase metabolites by human fetal mernbranes, uterine decidua vera and placenta. Prostaglandins Leukotrienes Med 1982;8:635.
December 1987 Am J Obstet Gynecol
5. Carraher R, Hahn DW, Ritchie DM, et aI. Involvement of lipoxygenase products in myometrial contractions. Prostaglandins 1983;26:23. 6. Ritchie DM, Hahn DW, McGuire JL. Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid. Life Sei 1984;34:509. 7. Schutte MF, Treffers PE, Kloosterman Gj, et aI. Management of premature rupture of membranes: the risk of vaginal examination to the infant. AM j OBSTET Gy" NECOL 1983;146:395. 8. Conover WJ. Practical nonparametric statistics. New York: john Wiley, 1981:229. 9. Romero R, Emamian M, Wan M, Grsyboski C, Hobbins J. Mitchell M. Increased concentrations of arachidonic acid lipoxygenase metabolites in amniotic fluid during parturition. Obstet Gynecol 1987;70:849. 10. TambyRaja RL, Salmon JA, Karim SMM, et aI. F Prostaglandin levels in amniotic fluid in premature labor. Prostaglandins 1977;13:339. 1I. Sellers S, Mitchell MD, Bibby jG, et aI. A comparison of plasma prostagiandin levels in term and preterm labour. Br J Obstet Gynaecol 1981;88:362. 12. Keirse MjNC. Endogenous prostaglandins in human parturition. In: Kierse MjNC, Anderson ABM, Bennebroek Gravenhorstj, eds. Human parturition. The Hague: Martinus Nijhoff, 1979:101-42.
Correction
In the article by Key, Giuffrida, and Moore, entitled "Predictive value of early pregnancy glycohemoglobin in the insulin-treated diabetic patient" (AM J ÜBSTET GVNECOL 1987;156:1096-100), the abstract is incorrect, The correct abstract appears below. The influence of early pregnancy glycemic control as measured by hemoglobin A te concentration al1d the incidence of congenital anomalies and spontaneous abortions were evaluated in women presenting for prenatal care with insulin-treated diabetes in a population whose glycemic control was poor. Thirty-one abnormaloutcomes were seen in 83 pregnancies (37%). There were 22 spontaneous abortions and nine major congenital anomalies. No woman with an early pregnancy hemoglobin A'e value <9.5% had an infant with a congenital anomaly and a single woman experienced a spontaneous abortion (4%). Converseiy, in women with an early pregnancy hemoglobin A'e value ~9.5%, congenital anomalies occurred in 24% and spontaneous abortion in 35%. Outcomes of pregnancies in type I and type II diabetic women were comparable. A strong statistical relationship between hemoglobin A'e and adverse pregnancy outcomes was demonstrated. These results strongly suggest that poor glycemic control during early pregnancy adversely influences pregnancy outcomes; the greater the degree of poor control, the greater the impact on pregnancy outcome. The data further justify the need for preconceptional control in diabetic woman and for careful evaluation of the fetus during pregnancy in the woman with insulin-treated diabetes.
This study was undertaken to examine Ihe effects of intrauterine infecl ion and prelerm labor on the amniotic fluid concen trations of arachidonate Iipoxygenase metabolites in women with premature rupture of memb ranes. Amniotic fluid was obtained trom four groups of women with prernature rupture of membran es: group 1, wome n without labor orinfection; group 2, women with labor but without infection; group 3, women wilh intra-amniotic infection but wilhout labor; and group 4, women wilh both inteclion and labor. 12-Hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, and leukol riene 8 _ were measu red by radioimmunoassays. Amniotic fluid concent rations of 12-hydroxyeicosatetraenoic acid were found not to differ significantly among Ihe four groups. Amniotic fluid concentrations of 15-hydroxye icosatetraenoic acid in group 4 were significanlly higher than in women in groups 1 and 3 (p < 0.05) . In addition , amniotic fluid concentrations in leukolriene 8_were significanlly higher in group 4 than in any of the other three groups (p < 0.05). Leukotriene 8. concentralions were higher in groups 2 and 3 than in group 1, suggesling that the presence of both labor and infecl ion increases the concentration of Ihis metabolite in amniotic fluid. Infection and labor had an additive effect in the elevation of amniotic fluid concenlrations of leukotriene 8•. These results suggest that the amniotic fluid concentrat ions of arachldonate Iipoxygenase metabolites are affected differently by the presence of infeclion and labor in women with premalure rupture of membranes . (AM J Ossrer G VNECOl 1987;157:1454-60.)
Key words: Arachidonat e lipoxygenase , intra -amn iotic infection, prern ature rupture of membranes
Intra-arn nio tic in fection is fre q ue n tly associa ted with th e o nse t of Iab or.' T he mechanisms resp onsible fo r parturition in these cases ha ve not bee n esta blishe d . Partu ritio n at term is assoc iated with mob ilizati on of a rachido nic acid fro m fe ta l mernbranes." Arachidonic acid ca n be metabol ized eithe r by way o f the cyclooxy genase 0 1' lipoxygenase pathways. The former pathway leads to the syn thesis of prostaglandins a nd thrornboxanes, wh ereas the lau er pa thwa y leads to the format ion o f lip oxygenase products suc h as hyd roxyeicosatetraen oic ac ids an d leuk ot ri en es, Arac hidonate lip oxygenase products are ge ne ra ted during th e co u rse of inFrom tlte Departments of Obstetrics and Gynecology, Yale Uni versity School ofMediane, and the Depa rtment of R eproductive M ediane, the Uni uersits of Cal iforni a, San Diego. Suppor ted by grants f rom the Walte r Scott Foundation [or M edical Research and National Institutes of Hea l üi grafts H D 2074 7 a nd HD 2 0 77 9. Present ed in part at the T hirtyl ourth Annual M eeting of the Society fo r Gynecologic Inuestigation, Atlanta, Georgia, March I8-2 l, 198 7. Reprint requests: R oberto Romero, M .D ., Department of Obstetrics and Gynecology, Yale Un iversity School of M edicine, P . O. Box 3333, Ne w Hauen, CT 06510. *Recipient of a Physician Scientist Award [rom the N ational I nstitu tes of H ealth.
1454
flarn rna to ry reacrion s"a nd can be syn thesize d by human intrauterine tissues .' T hese products hav e been found to be involved in the d evelopment of uterine contractions,' :" a nd they ma y participate in the no rma l p ro cess o f lab or 0 1' in the me ch an isrn o f lab or th at is assoc iated with 0 1' th e result of an inAammat ory rea ction (e.g., chorioamnionitis). The purpose of this study was to deterrnine the concentra tions of arach idonate lipoxygenase rnet ab olites in a m niotic fluid ofwomen with and without in tra- arnn iot ic in fect ion s a nd to e valuate th e rel ati ve in flu en ce of la bor a nd in fection o n the a m niotic fluid levels o f these co m po u nds .
Material and methods Study design. A cr oss-sectional stud y was construct ed accordin g to the resu lts of a m nio tic flu id cu ltures in women with premature ru ptu re of mernbranes. Fou l' groups wcre created: g rou p 1, women without labor 0 1' infection ; gro u p 2, wo rnen with labor bu t without in fecti on ; gro u p 3, wo rnen with a n intra-amniotic infection but witho u t labor ; a nd gr o u p 4 , wo me n with both infec tion an d lab or. An intra-am n iotic in feet ion was defined as the p res ence of a positive amnio tic fluid cu lture. None of th e patients in this st ud y developed
Volume 157 Number 6
signs and symptoms of infection. Labor was defined as the presence of regular uterine contractions that led to cervical dilatation and spontaneous vaginal delivery within 24 hours of the performance of the amniocentesis. Pelvic examination was not performed at the time of amniocentesis, since this could result in a higher incidence of infection.' Women were cIassified according to the results of amniotic fluid culture and their labor status at the time of amniocentesis. Retrieval of amniotic fluid. Amniotic fluid was obtained by transabdominal amniocentesis in women admitted to Yale--New Haven Hospital with the diagnosis of rupture of membranes at a gestational age of less than 37 weeks. Our success rate in obtaining fluid from these women is 95%. Amniotic fluid was processed for Gram stain, culture, lecithin/sphingomyelin ratio, and phosphatidylglycerol, and any remaining fluid was stored at - 20° C. Immediately before amniocentesis, the presence 01' absence of uterine contractions was recorded by external tocodynamometry. Culture technique, Amniotic fluid obtained by transabdominal amniocentesis was transported to the laboratory in a capped, plastic syringe immediately after collection of the specimen. These conditions precluded air contact with the specimen. Plating occurred within 30 minutes of collection in all cases. The sam pies were plated on blood agar, MacConkey agar, Columbia colistin-nalidixic acid agar, chocolate agar (36° C in 8% carbon dioxide) for aerobic culture and blood agar, bacteroides and bile-esculin agar/laked kanamycin and vancomycin agar, and Martin-Lewis agar (all prereduced) for anaerobic culture. Amniotic fluid was also inoculated in thioglycolate broth. Anaerobic cultures were grown in an anaerobic chamber in a Forma Seientific anaerobic system (Model 1024, Division of Mallinckrodt, Marietta, Ohio). The number of colonyforming units per milliliter was determined by plating the specimen with a 0.01 and 0.001 calibrated urine loop. Each colonial morphotype was identified to species by Food and Drug Administration-Centers for Disease Control-approved commercial systems supplemented by conventional tests when needed. An identification was accepted only when it yielded a normalized probability of 97% 01' greater. When more than three organisms were grown from an amniotic fluid sample, the infection was referred to as "mixed aerobic 01' anaerobic flora." Species identification was not conducted in these cases. This protocol evolved, since it was frequently difficult to identify all baeterial species involved, and there was no evidence that this effort altered patient management. There was also no evidence that amniotic fluid concentrations of lipoxygenase products would be preferentially affected by some organisms. Identification of anaerobes was conducted with the IDS RapID ANA system (Innovative Di-
Intra-amniotic infection and Iipoxygenase products
1455
agnostic System, Atlanta, Ga.). Agar was obtained from Baltimore Biological Laboratories, Cockeysville, Md. Mycoplasma and Ureaplasma cultures. Amniotic fluid was centrifuged at 2000 g for 20 minutes and the sediment was placed into 2 ml of holding media (pleuropneumonia-like organism broth, which was supplemented with fresh yeast extract and horse serum, penicillin, polymyxin B, and amphotericin). SampIes werestored at -70°C until culture (Mycotrim-GU, Berkeley, Calif.). Lipoxygenase metabolite determinations. Arachidonate lipoxygenase products were extracted from arnniotic fluid into acidified diethyl ether (five volumes). The solvent was evaporated under nitrogen, and the residue was dissolved in assay buffer and assayed using specific radioimmunoassays for 15-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B., (Seragen, Inc., Boston, Mass.). Extraction efficiency was determined by assessment of the recovery of each radiolabeled lipoxygenase product added to an aliquot of an amniotic fluid pool. The extraction efficiency was 76% for leukotriene B4 , 9 I % for 12-hydroxyeicosatetraenoic acid, and 87% for 15hydroxyeicosatetraenoic acid. All measurements of arnniotic fluid sam pies were conducted in the same assay. One hunclred microliters of amniotic fluid was used in the radioimmunoassay. Intra-assay coefficients of variation were 12% for leukotriene B4 , 11% for 15-hydroxyeicosatetraenoic acid, and 10% for 12hydroxyeicosatetraenoic acid. Sensitivities of the assays for 12-hydroxyeicosatetraenoic acid and 15hydroxyeicosatetraenoic acid were 8 pg, whereas it was 4 pg for leukotriene B 4 • Inhibition of the binding of the radioligancl to the antibody by 50% was achieved by 90 pg for leukotriene B1> 170 pg for 15-hydroxyeicosatetraenoic acid, and 260 pg for 12hydroxyeicosatetraenoic acid. Se rial sarnple clilutions were parallel to the standard curve for each assay (slope comparisons: p> 0.05). Solvent blanks for leukotriene B" 15-hydroxyeicosatetraenoic acid, and 12hydroxyeicosatetraenoic acid were 8 pg, 29 pg, and 98 pg, respectively. Solvent blanks were subtracted from measured values. Statistical analysis. The Kolmogorov-Smirnov test was used to test the hypothesis that lipoxygenase products were normally distributed. Logarithmic transforrnation was required for normality in 15hydroxyeicosatetraenoic acid. The amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid were normally distributed, but those of Ieukotriene Bi were not. One-way analysis of variance was used to compare the levels of lipoxygenase metabolites in the four different groups for I2-hydroxyeicosatetraenoic acid and log 15-hydroxyeicosatetraenoic acid. Duncan's multiple
1456
Romero et al.
Decem ber 198 7 Am J Obs tet Gynecol
16'0~
o
15.5 15.0 14.5 5.5
0
5.0 15HETE
ng/ml
4.5 4.0
0 0
3.5
'b
0
3.0 2.5
0
2.0
0
0
1.5
0
0
~
1.0 0.5 0.0
0
0 0 0 0
d'b
0
' - --
-----""""''----
I No Labor No Infection (n = 15)
--''''''-'''=----
---->;3iJ...---- - - ' - --
11 Labor No Infection (n = 15)
111
No Labor Infection (n = 14)
----'
IV Labor Infection (n = 12)
Fig. 1. Amniotic fluid co nce ntra tions of 15-h yd roxyeicosatetraen oic acid (I 5-HETE) in the following four groups of women: group 1, wom en with out labo r or infection ; group 2, women with labor but with out infection; group 3, women with an int ra-amn iotic in fectio n bu t with ou t labor; and gro up 4, women with bo th infection and labor. Log arith mic tra ns for mation was re q uired for normalit y to be achieved . 'O ne-way analys is of var ian ce was used 10 co m pare the am niotic fluid co ncentra tions of l ö-hvd roxyeicosa tetraenoic acid am on g th e four di fferent groups ( F = 4.02; p < 0.05). A sign ificant di ffer en ce was fou nd o nly between gro u ps 1 an d 4 and groups 3 and 4 (p< 0.05 , Dun can 's multiple range test ). The median co ncentra tion o f each gro up is represented by th e line in the scattergra m of each grou p.
range test was used for compari son between the diffe rent gro u ps. A p value < 0.05 was consid ered statistically sig nificant. Kruskal-WaIlis test was used to cornpare the amniotic fluid concen tratio ns of leukotriene . B, am ong different groups. A logarithm ic transforrnation was not used because many patients had nondetectable levels . Multiple co mpa riso ns amo ng the four groups were car ried out foIIowing the procedure described by Conover." Results
The co ncen trations of 15-hydroxyeicosatetraenoic acid, leukotriene B" and 12-h ydrox yeicosatetraenoic acid in amniotic fluid frorn women in the four differen t groups are iIIustrated in Figs. 1 through 3, respectively. The concentration of 12-hydroxyeicosatetraenoic acid in amniotic fluid did not vary significa ntl y among gr ou ps (ana lysis of varian ce, p > 0.05), whereas differences were detected in th e levels o f 15-h ydrox yeicosate tra eno ic acid and leukotriene B, (analysis of variance, p < 0.05). Amniotic fluid con centrations of 15h ydrox yeicosatetraenoic acid were gre ater in group 4 th an in grou p I (p < 0.05, Duncan's multiple ra nge test) and greater in grou p 4 th an in group 3 (p < 0.05, Duncan's multiple range test) . Amniotic fluid con centrations of leukotriene B, were sign ificantly different
amo ng aII gr oups excep t for the comparison between gr oups 2 a nd 3. T able I illustrates the descriptive statistics for ea ch group. T able s 1I and III show the re sults of quantitative microbiologyand amniotic fluid concentrations o f lipoxygenase products of women with intra-amniotic infections (gro u ps 3 a nd 4). No consistent relationship between arachid o nic lipoxygenase metabolite con centrations in amniotic fluid and specifi c organisms isolated from the fluid cou ld be d ernonstrated . No difference in the time in terval be tween arnniocentesis and delivery was found in the groups of women in labor at th e tim e of the amniocentesis (grou ps 2 and 4) (p > 0.5, WiIcoxon rank test) . Comment
The results of this stud y suggest th at the arnniotic fluid con centrations of I2-hydroxyeicosatetraenoic acid , l 5-h ydroxyeicosat etraenoic acid, and leukotriene B, are a ffected d ifferentlyby infection and labor in women with premature ruptu re of mernbranes. No differences were detected in the levels of I2 -h ydroxyeicosatetra enoic acid in a mnio tic fluid among the fou r groups; this was not the case with amniotic fluid concen trations of leukotriene B, a nd 15hydroxyeicosatetracnoic acid . T herefo re it appears that differen ces exist in the activity of th e enzyma tic path-:
Intra-arnniotic intection and lipoxygenase products
Volurne 157 Number 6
500 0
450 400 350
0
300 LTB4 pg/ml
0 0
250 200 0
150
0 0
100
0
50
0
0
I
No Labor No Intection (n = 15)
-e&
0
II
Labor No Intection (n = 15)
Il!
No Labor Intection (n = 14)
IV
Labor Infection (n = 12)
Fig. 2. Arnniotic fluid concentrations of leukotriene B, (LTB4) in the following four groups of wornen are displayed: group 1, women without labor or infection; group 2, women with labor but without infection; group 3, women with an intra-amniotic infection but without labor;and group 4, women with both infection and labor. A Kruskal-Wallis analysis of variance was perforrned to compare amniotic fluid concentrations among the different groups (p < 0.01). Amniotic fluid concentrations of leukotriene B, were significantly greater in group 4 than in any of the other groups. Amniotic fluid concentrations of leukotriene B, were significantly lower in group 1 than in groups 2 and 3. There were no differences between groups 2 and 3 (p > 0.05). The median concentration of each group is represented by the line in the scattergram of each group.
40 35 0
30 25 12HETE nglml
20
0 0 0 0
15 10
<9
~ 0 0
0
e
I
No Labor No Infection (n = 15)
-i% 0
s 0
11
Labor Ne lnfection (n = 15)
0
0
8
0
0
5
0
0 0
0
0
~
0
.....,.. ~ 0
0 0
111
No Labor Intectien {n = 14)
<9
IV
Labor Intectien {n = 12)
Fig. 3. Amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid (12-HETE) in the following four groups of women are displayed: group 1, women without labor or infection; group 2, women with labor but wirheut infection; group 3, women with an intra-amniotic infection but without labor; and group 4, women with both infection and labor. One-way analysis of variance was used to compare the arnniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid. There were no significant differences in amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid among the four different groups (F = 1.80, P > 0.05). The mean concentration of each group is represented by the line in the scattergram of each group.
1457
1458 Romero et aJ. Am
December 1987
J Obstet Gynecol
Table I. Descriptive statistics for concentrations (pg/ml) of amniotic fluid lipoxygenase metabolites in the four study groups
I 12 HETE No. observations Mean
Standard deviation Standard error of the mean Median Range 15 HETE No. observations Mean Standard deviation Standard error of the mean Median Range LTB4 No. observations Mean Standard deviation Standard error of the mean Median Range
GTOUP 1
Group 2
Group 3
Group 4
15 8546 5873 1516 5773 (292 I -2 I ,964)
15 11,496 6182 1597 10,315 (4728-24;972)
14 13,865 7792 2082 10,391 4296-31,198)
12 14,644 8432 2434 14.644 (2910-31,995)
14 908 899 240 677 (226-3498)
12 2192 1622 468 1817 (788-5744)
15 556 761 197 200 (237-2716)
15 2110 3994 1031 386 (382- I 5,965)
15 7.8 15.0 3.8 0 (0-50)
15 15.8 16.8 4.3 10.0 (0-46)
14 61.6 124.9 33.4 15.5 (0-465)
12 129.4 98 28.3 98.0 (8-331)
Table 11. Amniotic fluid concentrations (pg/ml) of arachidonate lipoxygenase products and microbiology of the amniotic fluid in women with intra-amniotic infections but without preterm labor (group 111) Patient identification No.
1I1 141 195 105 1071 36 1203A 1236A 1183A 1187A 1276 1294 36 37
M icroorganism Haemophilus inftuenzae Mycoplasma hominis M. hominis
Lactobacillus Group B streptococcus Fusobacterium species Bacteroides oralis M. hominis Fusobacterium species Group B streptococcus Fusobacterium species M. hominis Ureaplasma -urealsticum Staphylococcus aureus M. hominis Capnocytophaga Gardnerella oaginalis G. vaginalis U. urealyticum
Colony count*
12-Hydroxyeicosatetraenoic
15-Hydroxyeicosatetraenoic
Leukotriene B 4
30271 15717 3369
3204 1015 626
93 22 0
9131 8121
1440 285
33 17
875,) 11227
107 348
0 0
22713
728
14
23637 16484 9555 20073 7721 7337
1696 1508 69 0 141 1539
465 46 0 164 2 7
>10' >10" > 10" >10', >10',
>10" io >10"
> 10"
25 X 10" >10"
*Colony count is expressed in colony-forming units per milliliter. When quantitative microbiology was unavailable, it is indicated by-.
ways or the degradation of these compounds in intrauterine tissues. Infection and premature labor may not resuit in activation of the pathway responsible for the biosynthesis of 12-hydroxyeicosatetraenoic acid; however, these clinical conditions may result in effects on the pathways responsible for the biosynthesis 01' degradation of 15-hydroxyeicosatetraenoic acid and leukotriene B4 - Alternatively, these results may be explained by differences in the concentration of arachi-
donic acid. Depending on the Michaelis constant of the enzymes of the different lipoxygenase pathways for arachidonic acid and other substrates, the changes observed could be partially caused by alterations in the concentrations of arachidonic acid. We have found that premature labor alone in women with premature rupture of membranes does not alter the amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid 01' 15-hydroxyeicosatetraenoic acid but
Intra-amniotic infeetion and Iipoxygenase products
Volume 157 Number 6
1459
Table III. Amniotic fluid co nce n tratio ns (pg/m l) of arachidonate lip ox ygenase products and microbiolog y of the a m nio tic fluid in women with intra -a rnn iotic infections and preterm labor (grou p IV) Patient identijication N o.
150 39 70 11 32 33 1239A 1206A 1297 1255 1093 171A 1000
M icroorganism Gardnerella vaginalis
Fusobacterium speci es
Mycoplasma homiuis Mixed anaerobic flo ra Fusobacterium species G. vag ina lis Mixed an ae robic flora Gaffkya anaerobica G. vaginalis Fusobacterium nucleatum Mixed anae ro bic flor a Listerio monocytogenes Haemophilus influenza/' Gardnerella vaginalis
Colony count*
12-Hydroxyeicosatetraenoic
l i-Hvdroxyeicosatetraenoic
Leukotriene B.
3+ > 10' 1+
14789 1983 10095
5450 494 612
83 246 134
17369 24053
11 23 2749
32 72
> 10; > 10' 25 x 10" > 10' > 10" > 10' > 10'
15402 14499 2300 5255 13477 15632 31068
1075 1084 549 3060 2510 3425 4 177
258 102 8 94
> lW > 10;
72
121 331
*Co lony COU Ill is expressed in colony-form ing uni ts per m illiliter. When quant itati ve microbi ology was un available, it is indi cat ed
by - .
only that o f leukotriene B,. This findin g is interesting because it a ppea rs to be at variance with th e results of our earlier study in which women in term labor had sig nificantly eleva ted co nce n tra tio ns of aIl th ese compounds." There are severaI reasons wh y such diffe rences ma y exist. For ins ta nce, in our earlier stud y, aIl wornen we re in acrive lab or with a ce rvical dilatation o f 6 cm or grea te r, where as in the cu rre nt study, th e wom en a re likel y [0 have been in mu ch ea rlie r labo r. Data o n ce r vical d ilat ati on are not avai lable because pel vic examinations wer e not performed in women with prernature ruptu re of mem branes, sin ce thi s procedu re may in crease the incidence of infection ." It can also be a rgued that a maturational eye nt may acco u nt for the d ifferential re lease of lipox ygenase products in to arn niotic fluid . Women in preterrn labor ha ve lower concentration s of cycloox ygenase metabolites (i.e., p rostaglandins) in amnioti c fluid and plasm a tha n laboring wome n at terrn. " :" A similar phenomen on ma y occu r with lip ox ygenase rnet abolites. Evidence su p po r ting this concept is that man y women with preterm premature r u pt ure of membranes had nondetectable co ncen tr ati on s of leukotriene B, in the present study a rid that aJl nonlaboring wom en at te rm with in tact membranes had detectable concentrations of leu kotriene B•.9 H owe ver, these two gro u ps of women were not id entical with respect to membran e status. Intraamniotic infections alo ne seemed to affe et only the amnio tic fluid co ncentrations of leukot riene B I (corn pariso n bet ween gro ups 1 a nd 3). It remain s to be establi sh ed wh ether the in cr eased a mniotic fluid co ncentration of th is compound is the re sult of bacterial or host signals on the me ta bolism 01' ava ilability of arachidonic ac id . When both lab o r and in fectio n are present, the po-
tential rel ati ve effects of these two clini cal conditions on th e a m niotic fluid conc en tratio n ofli po xyge nase rnetabolites are not easy to interpret. Amniotic fluid co ncentrations of leuko trie ne BI were significantly ele vated in women infected and in labor (group 4) compared with all o the r gro u ps. This see ms to imply that labor a nd infection ha ve an ad d itive effect o n a mnio tic fluid concentrations of leukotriene B.,. .Since am niotic fluid concentrarions of p ro staglandin s a re increased with progressive ce r vical d ilatati on ," it may be hypothesized that the d ifference o bse rved in a m n iotic fluid co nce nt ra tions of leukotriene B., between groups 2 and 4 cou ld be attribu ted to differing ce rvical dilatat ion at the time o f am niocentesis between the grou ps. However, thi s is not Iikely to be the case because the time interval betwee n arnniocentesis and d elivery was not different between these two groups . Significant d ifferences in a m ni otic flu id co nce nt ra tions of 15-hydroxyeicosatetraenoic acid were o nl y detected when labor a nd in fec tio n we re present (gro u p 4) co m pa red with gro u ps 1 a nd 3 but not co rnpared with group 2. Further studies will be required to est abl ish the relative contribution of preterm labor and infection to amniotic fluid co ncen tra tio ns of 15-h yd roxye icosat e trae noic acid as this study is not conc1usive. In co nclu sio n, our data indicate that a m n iotic fluid conce nt rations of arach id o na te lipoxygenase merabolites are affect ed by th e prese nce of lab or a nd infeetion in women wit h p rete r m premature rupture of m em bran es. Pre te rm lab or is associa te d with increased amniotic fluid co nce n tratio n of leukotriene B•. Intra-amnio tic in fectio n without labor is also associat ed with incr eased amniotic fluid concen tra tions of Ieukotriene B,. The prese nce of preterm la bor and intra-
1460
Romero et al.
amniotic infection had an additive effect on the increase of amniotic fluid concentrations of leukotriene B 1 • Neither preterm labor nor infection was associated with significant changes in amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid. These observations are consistent with the possibility of an involvernent of the lipoxygenase pathway of arachidonic acid metabolism in the mechanism of human parturition associated with preterm labor and intra-amniotic infection. REFERENCES
1. Garite TJ, Freeman RK. Chorioamnionitis in the preterm gestation. Obstet Gynecol 1982;59:539. 2. Okita JR, MacDonald PC, Johnston jM. Mobilization of arachidonic acid from specific glycerophospholipids of human fetal membranes during early labor. j Biol Chem 1982;257: 14029. 3. Hansson G, Malmsten C, Radmark O. The leukotrienes and other lipoxygenase products. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. Vol 5. Amsterdam: Elsevier, 1983: 127·69. 4. Saeed SA, Mitchell MD. Formation of arachidonate lipoxygenase metabolites by human fetal mernbranes, uterine decidua vera and placenta. Prostaglandins Leukotrienes Med 1982;8:635.
December 1987 Am J Obstet Gynecol
5. Carraher R, Hahn DW, Ritchie DM, et aI. Involvement of lipoxygenase products in myometrial contractions. Prostaglandins 1983;26:23. 6. Ritchie DM, Hahn DW, McGuire JL. Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid. Life Sei 1984;34:509. 7. Schutte MF, Treffers PE, Kloosterman Gj, et aI. Management of premature rupture of membranes: the risk of vaginal examination to the infant. AM j OBSTET Gy" NECOL 1983;146:395. 8. Conover WJ. Practical nonparametric statistics. New York: john Wiley, 1981:229. 9. Romero R, Emamian M, Wan M, Grsyboski C, Hobbins J. Mitchell M. Increased concentrations of arachidonic acid lipoxygenase metabolites in amniotic fluid during parturition. Obstet Gynecol 1987;70:849. 10. TambyRaja RL, Salmon JA, Karim SMM, et aI. F Prostaglandin levels in amniotic fluid in premature labor. Prostaglandins 1977;13:339. 1I. Sellers S, Mitchell MD, Bibby jG, et aI. A comparison of plasma prostagiandin levels in term and preterm labour. Br J Obstet Gynaecol 1981;88:362. 12. Keirse MjNC. Endogenous prostaglandins in human parturition. In: Kierse MjNC, Anderson ABM, Bennebroek Gravenhorstj, eds. Human parturition. The Hague: Martinus Nijhoff, 1979:101-42.
Correction
In the article by Key, Giuffrida, and Moore, entitled "Predictive value of early pregnancy glycohemoglobin in the insulin-treated diabetic patient" (AM J ÜBSTET GVNECOL 1987;156:1096-100), the abstract is incorrect, The correct abstract appears below. The influence of early pregnancy glycemic control as measured by hemoglobin A te concentration al1d the incidence of congenital anomalies and spontaneous abortions were evaluated in women presenting for prenatal care with insulin-treated diabetes in a population whose glycemic control was poor. Thirty-one abnormaloutcomes were seen in 83 pregnancies (37%). There were 22 spontaneous abortions and nine major congenital anomalies. No woman with an early pregnancy hemoglobin A'e value <9.5% had an infant with a congenital anomaly and a single woman experienced a spontaneous abortion (4%). Converseiy, in women with an early pregnancy hemoglobin A'e value ~9.5%, congenital anomalies occurred in 24% and spontaneous abortion in 35%. Outcomes of pregnancies in type I and type II diabetic women were comparable. A strong statistical relationship between hemoglobin A'e and adverse pregnancy outcomes was demonstrated. These results strongly suggest that poor glycemic control during early pregnancy adversely influences pregnancy outcomes; the greater the degree of poor control, the greater the impact on pregnancy outcome. The data further justify the need for preconceptional control in diabetic woman and for careful evaluation of the fetus during pregnancy in the woman with insulin-treated diabetes.