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Urinary trypsin inhibitor levels in amniotic fluid of normalhuman pregnancy: Decreased levels observed at parturition

Hiroshi Kobayashi, MD, PhD,a Kazunao Suzuki, MD,a Dan Sugino, PhD,b and Toshihiko Terao,MD, PhDa

Shizuoka and Shiga, Japan

OBJECTIVE: Urinary trypsin inhibitor, a light chain of inter-α-trypsin inhibitor, is reported to inhibit uterinecontractility during pregnancy. The objective was to measure amniotic fluid urinary trypsin inhibitor levels inwomen throughout pregnancy.STUDY DESIGN: We determined levels of urinary trypsin inhibitor in human amniotic fluid (n = 40) by meansof Western blot analysis. For quantification, computerized scanning and densitometry were used. Resultswere analyzed for statistical significance with the Mann-Whitney U test.RESULTS: During pregnancy, mean amniotic fluid urinary trypsin inhibitor levels (±SD) were 1.5 ± 0.22, 1.2± 0.18, and 0.58 ± 0.24 μmol/L at 15 to 22 (n = 10), 30 to 35 (n = 5), and 37 to 40 (n = 10) weeks’ gestation,respectively. There was a significant negative correlation of urinary trypsin inhibitor level with gestational age.A significant difference was observed in amniotic fluid urinary trypsin inhibitor levels before and after (0.10 ±0.03 μmol/L; n = 15) the onset of labor.CONCLUSION: Amniotic fluid urinary trypsin inhibitor levels decrease significantly at term. These resultssupport the hypothesis that parturition may occur through the down-regulation of urinary trypsin inhibitor,which may decrease uterine contractility and maintain the uterus in a state of quiescence during pregnancy.(Am J Obstet Gynecol 1999;180:141-7.)

Key words: Amniotic fluid, onset of labor, parturition, urinary trypsin inhibitor

The mechanism by which uterine smooth muscleswitches from a quiescent state during pregnancy to con-traction at labor has been the subject of investigation foryears. The roles of innervation, hormones, nitric oxide,nitric oxide synthase,1 corticotropin-releasing factor, andcorticotropin-releasing factor binding protein2, 3 in medi-ating this switch have been extensively explored. Thesesubstances may keep the physically contracting uterinesmooth muscle from delivering the fetus before the opti-mal term gestation. It is our hypothesis that parturitionoccurs through the upward regulation of a group of pro-labor genes, such as those for oxytocin, oxytocin recep-tors,4, 5 gap junction proteins,6 and corticotropin-releas-ing factor2, 3 or through the down-regulation of a groupof genes for nitric oxide, nitric oxide synthase,1 corti-cotropin-releasing factor binding protein,2, 3 and urinarytrypsin inhibitor,7-12 which may decrease uterine contrac-tility and maintain the uterus in a state of quiescence dur-ing pregnancy.

Human inter-α-trypsin inhibitor (IαI) is composed of3 genetically different polypeptides, 2 heavy chains and 1light chain. The light chain, also referred to as urinarytrypsin inhibitor, appears to regulate protease activity.13

IαI occurs at a concentration near 0.5 mg/mL in plasmabut accounts for <5% of the total trypsin inhibitory activ-ity.14 In addition to their protease inhibitory activity, pro-teins of the IαI family are considered to have importantbiologic functions. Bovine proteins of the IαI family arethought to be involved in the stabilization of the extra-cellular matrix of cells that surround the developingovum.15 Another intriguing function of urinary trypsininhibitor is inhibition of uterine contractility duringpregnancy.6 The fetal liver13 and kidney16 and the amni-otic membrane17 are considered to be the sources of am-niotic fluid–derived urinary trypsin inhibitor.

In this study, which took advantage of the availability ofpurified urinary trypsin inhibitor, we developed a mono-clonal antibody raised against urinary trypsin inhibitor.We established a Western blot analysis and densitometricassay to measure human urinary trypsin inhibitor directlyin amniotic fluid, and we report here on amniotic fluidurinary trypsin inhibitor levels in women throughoutpregnancy. The amniotic fluid levels of urinary trypsin in-hibitor found in healthy pregnant women fell at 37 to 40weeks’ gestation. There was a significant decrease in theavailability of bioactive urinary trypsin inhibitor at term.Our studies of the control of parturition suggest that am-

From the Department of Obstetrics and Gynecology, HamamatsuUniversity School of Medicine,a and the Nissin Central ResearchInstitute, Nissin Food Products Company, Ltd.bReceived for publication April 7, 1998; revised and accepted July 28,1998.Reprint requests: Hiroshi Kobayashi, MD, Department of Obstetrics andGynecology, Hamamatsu University School of Medicine, Handacho3600, Hamamatsu, Shizuoka, 431-31, Japan.Copyright © 1999 by Mosby, Inc.0002-9378/99 $8.00 + 0 6/1/93482

142 Kobayashi et al January 1999Am J Obstet Gynecol

niotic fluid–derived urinary trypsin inhibitor may preventuterine contractility during pregnancy.

Material and methodsSubjects. Amniotic fluid samples (n = 40) were ob-

tained at amniocentesis for studies of karyotype and fetalpulmonary maturity and during delivery. Amniotic fluidsamples were obtained at 15 to 40 weeks’ gestation in nor-mal pregnancies (n = 25) and during delivery (n = 15). Atweeks 37 to 40, amniotic fluid samples were collected bycesarean delivery in 10 patients before the onset of labor.The indication for cesarean delivery was a previous ce-sarean delivery or breech presentation in an otherwiseuncomplicated pregnancy. Amniotic fluid was also col-lected from 15 patients with spontaneous labor and deliv-ery at term in uncomplicated pregnancies. Transvaginalamniocentesis was used to obtain amniotic fluid from la-boring patients. Samples obtained from patients whosepregnancies were complicated by multiple gestations andfetal anomalies were excluded. Patients who receivedprostaglandins or oxytocin for induction or augmenta-tion of labor were also excluded from this study.

Treatment decisions were made by the patients’ physi-cians in all cases and were not affected by participation inthis study. Informed consent was obtained from all sub-jects. This study was approved by our institutional reviewboard. All women gave their informed consent to partici-pate in this study, which was approved by the HamamatsuUniversity Hospital ethical committee. All amniotic fluidsamples were collected on ice, centrifuged (2000g) at4°C for 15 minutes, and stored at –20°C until assay.Urinary trypsin inhibitor in amniotic fluid was not re-moved during this process.

Purification of urinary trypsin inhibitor. In human be-ings, urinary trypsin inhibitor is found in plasma, urine,and amniotic fluid. Urinary trypsin inhibitor was purifiedto homogeneity from human urine according to themanufacturer’s suggested procedures, as described previ-ously.18 Elution of purified urinary trypsin inhibitor on aSephacryl S-200 sizing column (Pharmacia, Uppsala,Sweden) indicated that the molecule migrated as an un-complexed protein by molecular weight 50 to 60 kd (datanot shown). Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis under nonreducing conditions showedthat urinary trypsin inhibitor migrated as a polydispersedband of molecular weight 40 kd.18 By electroblotting di-rectly from sodium dodecyl sulfate–polyacrylamide gelelectrophoresis, we identified the 1 component on thebasis of the amino acid sequences. N-terminal sequenc-ing of the 40-kd urinary trypsin inhibitor preparationyielded the sequence 1Ala-Val-Lys-Pro-Gln-Glu-Glu-Glu-Gly9, which corresponds to that reported for urinarytrypsin inhibitor.13 Slight differences in reported molec-ular weight may be due to interlaboratory variations or tothe extent of the glycosylation of the molecules.

Production of monoclonal antibodies raised against uri-nary trypsin inhibitor. Male Balb/c mice were immunizedat 14-day intervals by intraperitoneal injection of 20 μgpurified urinary trypsin inhibitor. Three days after the lastbooster, spleen cells (1 × 108) were fused with the mousemyeloma cell line NS-1 and seeded according to standardprocedures.19 After 14 days supernatants were screenedby an enzyme-linked immunosorbent assay with purifiedurinary trypsin inhibitor–coated microtiter plates. Coat-ing was carried out by incubation of urinary trypsin in-hibitor (100 ng/well) at 4°C overnight in bicarbonatebuffer, pH 9.5. The wells were blocked by a 2-hour incu-bation with 2% bovine serum albumin in saline solutionbuffered with tris(hydroxymethyl)aminomethane and thenincubated for 2 hours with hybridoma supernatants. Anti-bodies bound to the urinary trypsin inhibitor–coatedwells were detected by peroxidase-conjugated rabbit anti-mouse immunoglobulin G (Dako, Copenhagen, Den-mark). After cloning from positive wells twice by limitingdilution, 6 clones were cultured in large scale for antibodyproduction. Antibodies were purified by protein A-Sepharose affinity chromatography. A monoclonal anti-body 4G12 was selected and used for further studies.

Sodium dodecyl sulfate–polyacrylamide gel electro-phoresis and Western blot analysis. Amniotic fluid wasmixed with an equal volume of sodium dodecyl sulfatesample buffer (10% sodium dodecyl sulfate, 10% glyc-erol, 0.01% bromophenol blue, and 0.625 mol/L tris(hy-droxymethyl)aminomethane hydrochloride, pH 6.8).The samples were electrophoresed on 1.5-mm thick, 8-cmlong, 15% sodium dodecyl sulfate–polyacrylamide slabgels after stacking on a 1.5-cm sodium dodecyl sulfate(3%)–polyacrylamide gel. Electrophoresis was done atconstant current (15 mA stacking, 25 mA separating). Af-ter polyacrylamide gel electrophoresis, the proteins werevisualized by staining with Coomassie blue or were trans-ferred to polyvinylidine difluoride paper with a semidryelectroblotting apparatus (Marysol, Tokyo, Japan) at 40mA/gel (90 minutes, 23°C). The polyvinylidine difluo-ride sheets were incubated in saline solution bufferedwith tris(hydroxymethyl)aminomethane and containing2% bovine serum albumin, and then incubated for 2hours at 23°C in saline solution buffered with tris(hy-droxymethyl)aminomethane containing 2% bovineserum albumin and a monoclonal antibody raised againsturinary trypsin inhibitor (4G12; 1:50 dilution). After in-cubation with 4G12, the paper was washed and incubatedfor 1 hour at 23°C in the secondary antibody, goat anti-mouse immunoglobulin G conjugated with biotin (1:500dilution; Dako). This paper was washed again and incu-bated for 1 hour at 23°C in avidin peroxidase solution(1:500 dilution; Dako) and then exposed to saline solu-tion buffered with tris(hydroxymethyl)aminomethanecontaining 4-chloro-1-naphthol (0.6 mg/mL) and hydro-gen peroxide (0.02 %), washed in distilled water, and air-

dried. For quantification, computerized scanning anddensitometry (Power Macintosh 7600/200-assisted FAS-IIand Electronic U.V. transilluminator; Toyobo Co Ltd,Tokyo, Japan) were used.

The specificity of the anti–urinary trypsin inhibitor an-tibody 4G12 was controlled by (1) omission of the pri-mary antibody, (2) incubation of the polyvinylidine diflu-oride membranes with normal mouse immunoglobulinG instead of the primary antibody, and (3) incubation ofthe polyvinylidine difluoride membranes with the pri-mary antibody previously absorbed with the urinarytrypsin inhibitor specifically produced to raise the 4G12.

Statistics. All statistical analysis was performed withStatView for Macintosh (Abacus Concepts, Inc, Berkeley,Calif). The Mann-Whitney U test was used for the com-parisons between different groups. One-way analysis ofvariance was performed with post hoc analysis by usingthe Scheffé F procedure for statistical interpretation. P <.05 was considered significant. Data are presented asmean ± SD. Comparison of amniotic fluid urinary trypsininhibitor levels with gestational week was performed bysimple regression analysis. The relationship between theamount of urinary trypsin inhibitor and the intensity ofbands was calculated by graphic analysis with StatView forMacintosh.

ResultsSodium dodecyl sulfate–polyacrylamide gel electro-

phoresis and Western blot analysis of human amniotic fluidurinary trypsin inhibitor. Proteins of the IαI family aremultichain proteins found in human plasma that arecomposed of urinary trypsin inhibitor and 1 or 2 heavychains. Human plasma urinary trypsin inhibitor is cova-lently bound to 3 homologous heavy chains (HC1, HC2,and HC3).20 Purified urinary trypsin inhibitor and humanurine, serum, and amniotic fluid were subjected tosodium dodecyl sulfate–15% polyacrylamide gel elec-trophoresis, and the urinary trypsin inhibitor proteinswere visualized by immunoblot analysis with monoclonalantibody 4G12 specific to the urinary trypsin inhibitormolecule (Fig 1). Incubation of polyvinylidine difluoridemembranes with the normal mouse immunoglobulin G orpreviously absorbed 4G12 abolished the specific im-munoreactive bands. This allowed us to discern and iden-tify the specific polypeptide chains. Western blot analysisof urinary trypsin inhibitor purified from human urineand amniotic fluid revealed a polydisperse band of ap-proximately 40 kd. Experiments were performed 4 timesand showed similar results. Small variations of molecularweight probably correspond to urinary trypsin inhibitorsof differing degrees of glycosylation. As shown by the West-ern blot analysis with 4G12, free urinary trypsin inhibitorand combinations of urinary trypsin inhibitor and heavychains have been identified in human plasma, for exam-ple IαI (urinary trypsin inhibitor plus HC1 plus HC2),

Volume 180, Number 1, Part 1 Kobayashi et al 143Am J Obstet Gynecol

pre-α-inhibitor (urinary trypsin inhibitor plus HC3), andurinary trypsin inhibitor/HC2 (urinary trypsin inhibitorplus HC2). After sodium dodecyl sulfate–polyacrylamidegel electrophoresis of human amniotic fluid, a polydis-perse single 40-kd band was seen on the correspondingWestern blot. Apparently amniotic fluid contains a glyco-sylated free urinary trypsin inhibitor. The intensity of im-munoreactive urinary trypsin inhibitor in amniotic fluidvaries considerably. The urinary trypsin inhibitor’s intensitymark-edly decreased at 40 weeks’ gestation.

Amniotic fluid content of immunoreactive urinarytrypsin inhibitor. Fig 2, A, shows a representative Westernblot in which varying amounts of purified urinary trypsininhibitor (50-6400 ng/lane) were applied to the gel. Be-cause the urinary trypsin inhibitor isolated from humanurine contained 35% to 50% carbohydrate, the antibody

Fig 1. Sodium dodecyl sulfate–polyacrylamide gel electrophore-sis and Western blot analysis of human amniotic fluid urinarytrypsin inhibitor. Analyses of immunoreactive urinary trypsin in-hibitor derived from human amniotic fluid, urine, and plasma.These proteins were analyzed by sodium dodecyl sulfate–poly-acrylamide gel electrophoresis under nonreducing conditionson 15% gels followed by Western blot analysis that used a mono-clonal antibody 4G12. The position of molecular mass standardsis shown in the left (in kilodaltons, lane 1); lane 2 shows purifiedurinary trypsin inhibitor (1 μg/lane), lane 3 shows human urine(1 μL/lane), lane 4 shows human plasma (1 μL/lane), lane 5shows amniotic fluid at 15 weeks’ gestation (1 μL/lane), andlane 6 shows amniotic fluid at 40 weeks’ gestation (1 μL/lane).4G12 reacts with IαI (arrowhead), pre-α-inhibitor and HC2/uri-nary trypsin inhibitor (single arrow), and urinary trypsin inhibitor(double arrow). Presented here is a representative Western blotfrom 4 independently performed experiments in which compa-rable results were obtained. Purified urinary trypsin inhibitor(lane 2) was used as a positive control preparation. Furthermore,incubation of the polyvinylidine difluoride membranes with thenormal mouse immunoglobulin G or 4G12 absorbed in advancewith urinary trypsin inhibitor abolished the specific immunore-active bands (data not shown).

stained a polydisperse band with an approximate molecu-lar mass of 40 kd. After Western blot analysis, 4G12 alsostained a glycoprotein with a molecular mass of 40 kd inamniotic fluid. Adding more amniotic fluid to each lanedid not improve the sensitivity of the assay.

The relationship between the amount of urinarytrypsin inhibitor and the intensity of bands was calcu-lated by graphic analysis with StatView for Macintosh.Densitometric scanning of the blots described a para-bolic relationship between the amount of purified uri-nary trypsin inhibitor added and the intensity of thebands. The analyses from the data obtained from repre-sentative experiments are illustrated in Fig 2, B.

Duplicate amniotic fluids were compared with the uri-nary trypsin inhibitor standards to determine the im-munoreactive urinary trypsin inhibitor content, whichwas expressed as millimoles of immunoreactive urinarytrypsin inhibitor per liter of amniotic fluid. The 40-kdband demonstrated specificity for urinary trypsin in-hibitor; its appearance was inhibited by the addition ofpurified urinary trypsin inhibitor to the primary antibody(data not shown). The 40-kd urinary trypsin inhibitorband appeared throughout the pregnancy (Fig 3, A).The data shown in Fig 3, A, exclude those women whosubsequently had premature labor and delivery. Urinary

144 Kobayashi et al January 1999Am J Obstet Gynecol

trypsin inhibitor was present in the amniotic fluid after15 weeks of pregnancy. There was significant negativecorrelation of urinary trypsin inhibitor level with gesta-tional age (r = –0.54; P < .05). The mean level of urinarytrypsin inhibitor in amniotic fluid in uncomplicatedpregnancies was significantly higher at 15 to 22 and 30 to35 weeks’ gestational age than at 37 to 40 weeks’ gesta-tional age (Fig 3, B). There was significant difference inamniotic fluid urinary trypsin inhibitor levels betweenmeasurements before and after the onset of labor. Theurinary trypsin inhibitor levels in amniotic fluid de-creased markedly after the onset of labor.

CommentThis study is the first to describe the measurement of

urinary trypsin inhibitor in human amniotic fluid by West-ern blot analysis with a specific monoclonal antibody.There are varying degrees of glycosylation of urinarytrypsin inhibitor, as evidenced on sodium dodecyl sul-fate–polyacrylamide gel electrophoresis; however, thisdoes not affect the specificity of the immunoblot becausea monoclonal antibody against urinary trypsin inhibitorrecognizes both the nonglycosylated and glycosylatedforms (Kobayashi H, et al. Unpublished data.). In this studyno patients had medical disorders that might have influ-

Fig 2. Western blot analysis of urinary trypsin inhibitor (UTI) standard and amniotic fluid. A, Western blot using a mon-oclonal antibody against urinary trypsin inhibitor (4G12). Purified urinary trypsin inhibitor was used as a standard.Lane 1 shows 6400 ng urinary trypsin inhibitor, lane 2 shows 3200 ng urinary trypsin inhibitor, lane 3 shows 1600 ng uri-nary trypsin inhibitor, lane 4 shows 800 ng urinary trypsin inhibitor, lane 5 shows 400 ng urinary trypsin inhibitor, lane6 shows 200 ng urinary trypsin inhibitor, lane 7 shows 100 ng urinary trypsin inhibitor, and lane 8 shows 50 ng urinarytrypsin inhibitor. B, An arbitrary unit measured by densitometric scanning was plotted against the urinary trypsin in-hibitor concentration, and a standard curve was constructed.

A

B

enced the results. No patients had evidence of intra-am-niotic infections or other infections, although urinarytrypsin inhibitor level in serum was reported to be dra-matically increased when infection was present.21 Urinarytrypsin inhibitor level is increased in patients with chronicrenal failure, who were excluded from this study.

The apparent molecular weight of the amniotic fluidurinary trypsin inhibitor was approximately 40 kd, whichis similar to that in normal human plasma and in urine.Consequently, the results reported here are absoluteamounts of urinary trypsin inhibitor in amniotic fluid.Amniotic fluid urinary trypsin inhibitor concentrationsduring the third trimester were significantly lower thanthe second-trimester levels, implying that the level of uri-

Volume 180, Number 1, Part 1 Kobayashi et al 145Am J Obstet Gynecol

nary trypsin inhibitor is not influenced by the increasingestrogen and progesterone levels found in pregnancy.Interestingly, reduced amniotic fluid urinary trypsin in-hibitor levels were found in the last few weeks of preg-nancy. Decreasing urinary trypsin inhibitor levels occurclose to term, at the time when uterine muscle contractil-ity has increased dramatically.7, 9-12 The demonstration ofa sixfold decrease in amniotic fluid urinary trypsin in-hibitor levels after labor and delivery strengthens our hy-pothesis that amniotic fluid urinary trypsin inhibitorcould prevent uterine contractions in a paracrine fashionduring pregnancy. This study suggests that the decreasedamniotic fluid levels of urinary trypsin inhibitor inwomen who had term labor were due to mechanisms that

Fig 3. Urinary trypsin inhibitor (UTI) levels in amniotic fluid at various stages of pregnancy. A, Western blot with 4G12.Lane 1 shows amniotic fluid urinary trypsin inhibitor at 15 weeks’ gestation, lane 2 shows amniotic fluid urinary trypsininhibitor at 22 weeks’ gestation, lane 3 shows amniotic fluid urinary trypsin inhibitor at 30 weeks’ gestation, lane 4 showsamniotic fluid urinary trypsin inhibitor at 35 weeks’ gestation, lane 5 shows amniotic fluid urinary trypsin inhibitor at37 weeks’ gestation, lane 6 shows amniotic fluid urinary trypsin inhibitor at 40 weeks’ gestation before the onset oflabor, and lane 7 shows amniotic fluid urinary trypsin inhibitor at 40 weeks’ gestation after the onset of labor (L). B,Urinary trypsin inhibitor levels in amniotic fluid were assayed as described in the Material and Methods section.Columns, Means; vertical lines, SDs. Asterisk, P < .05.

A

B

began before the onset of labor and may have been dueto the process of labor itself. It is impossible that the de-creased concentration of urinary trypsin inhibitorthrough gestation could be due to a dilutional effect re-lated to increasing amniotic fluid volume. In a parallelstudy, we attempted to determine the albumin concen-tration in the amniotic fluid and demonstrated that thealbumin concentration was not influenced by the labor.

Our recent study also demonstrates that urinarytrypsin inhibitor is produced not only in the fetal liverand kidney but also in the fetal membrane (Kobayashi etal18) (Kobayashi H, et al. Unpublished data.), althoughmuch of amniotic fluid urinary trypsin inhibitor may beproduced by the fetus. Fetal and amniotic fluid mem-brane urinary trypsin inhibitor seems to be producedafter ≥15 weeks of fetal life. We previously reported thatamniotic fluid urinary trypsin inhibitor functions as a po-tent mediator of smooth muscle relaxation.7 Earlier workdone in our laboratory investigated the role of urinarytrypsin inhibitor in relaxation of the actively laboringgravid uterus by means of an in vitro and an in vivo assaysystem.8-13 Oxytocin- or prostaglandin F2α–induced con-tractions in isolated pregnant uterine muscle strips werereversibly blocked by advance treatment with urinarytrypsin inhibitor.7 Furthermore, exogenous urinarytrypsin inhibitor, given in the form of a urinary trypsin in-hibitor suppository, arrested laboring uterine contrac-tions in human beings.8, 10-12

Reduction in amniotic fluid urinary trypsin inhibitoractivity at 37 to 40 weeks’ gestation suggests that urinarytrypsin inhibitor may contribute to the maintenance ofuterine contractility quiescence during gestation. Suppres-sion of uterine contractions at the myometrial level byamniotic fluid urinary trypsin inhibitor appeared to be-come insufficient at term, demonstrating that decreasedamniotic fluid urinary trypsin inhibitor levels may play arole in the initiation of parturition. We cannot concludewhether this decrease is sufficient to cause labor orwhether other factors are necessary for labor tooccur.22-24 We did not use premature delivery as the endpoint for this study because the success of treatment forpreterm labor depends on many factors unrelated to themechanisms of labor. Further studies are needed to de-termine whether the ability to suppress uterine contrac-tion was related to the amniotic fluid urinary trypsin in-hibitor concentrations. Nonetheless, these data dodemonstrate that amniotic fluid urinary trypsin inhibitorplays a role in suppressing a cascade of events leading tothe onset of premature labor.

This study demonstrated by immunologic assays thepresence of urinary trypsin inhibitor in human amnioticfluid and a significant decline in level at term. The re-duced capacity for endogenous urinary trypsin inhibitorproduction therefore occurs in a manner consistent witha causal role in the increased uterine contractions seen at

146 Kobayashi et al January 1999Am J Obstet Gynecol

term. Taken together with our previous observations thatexogenous urinary trypsin inhibitor relaxes uterinesmooth muscle and abates preterm labor in human be-ings, these studies suggest that amniotic fluid urinarytrypsin inhibitor could play a key role in the mainte-nance of uterine quiescence during pregnancy and thatreduced capacity for its production could promote theinitiation of labor at term. Additional studies are neededto determine whether amniotic fluid urinary trypsin in-hibitor serves a critical role in any of these processes.Furthermore, we must perform a series of experiments toexamine the biosynthesis of the urinary trypsin inhibitorprotein found in amniotic fluid.

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