1 Introduction Preterm labor (PTL) is the delivery in

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					                                                                                     1


       EXPLORATION OF ACTIVITIES AND GENETIC PATTERN OF
     ELASTASE IN PATIENTS WITH THREATENED PRETERM LABOR

                                Jusuf Sulaeman Effendi

                      Department of Obstetrics and Gynecology
                      Medical Faculty of Padjadjaran University
                                      Bandung
                                      Indonesia


Introduction 1
                                                     Beside the failure of respiration
      Preterm labor (PTL) is the               system as the cause of preterm infant
delivery in gestational age between 20         death, the morbidity raise as well as
weeks and more than or same with 37            cerebral hemorrhage, nervous system
weeks, while normal labor between 37           disturbance, mental retarded, deafness
to 42 weeks of pregnancy.                      and growth disturbance.
      The incidence of PTL vary                      Primary prevention of PTL by
between 5 to 12 % from all delvery  i          eliminated risk factors were impossible
with the low incidence in the d       eve-     done because risk factors know for n
loped country between 5 – 7 %. In              PTL such as history of PTL, history of
Indonesia we still not have the national       recurrent    abortions and habitu      al
rate, but some researchers giv the e           factors. In this stage of prevention we
hospital rate.                                 have to perform close antenatal care,
      Joesoef reported the incidence of        and perform secondary prevention.
PTL in several hospital in Jak   arta in             Secondary prevention are include
1991 about 13.3 %, whil in oure                early detection of PTL. Clinically we
hospital, Hasan Sadikin Hospit l in a          can perform detection by evaluating of
Bandung in 2001 the incidence                  uterine contraction and the condition of
reported 9.9 %.                                the cervix. We can measure the
Most of PTL are spontaneously about            cervical length by ultrasound, bio-
72 %, while the remain occur because           chemical markers those are pH of
the pregnancy must be terminated pre-          vagina and fetal fibronectin rom     f
termly because of medical indications.         cervicovaginal secret. Unfortu    nately
      Neonatal death in PTL contri-            those efforts did not give the real
buted 70 % of perinatal mortalty ini           advantages to be applied as mode of
Indonesia, and neonatal death of               detection of PTL widely.
preterm babies in the first year of their            Morbidity and mortality rate of
live in Hasan Sadikin Hospital range           preterm infant will be higher if the
between 40–70%. The preterm infant             baby born too early or the baby weight
death in Hasan Sadikin Hospita           l     too small, so the effort to prolong the
Bandung was 56,6% of all pe       rinatal      gestational period was the important
death.                                         thing to be done in threatened PTL.



1
    Presented in Asia Oceania Congress of
    Obstetry and Gynecology 2009, Auckland,
    New Zealand
                                                                                         2


      The effort has been done through           matrix and resulting the rupture of the
medication, but the result unsatisfied,          membrane. The increase of elasase    t
because of advances of the labr           o      activity in pregnancy compartm         ent
process.                                                                          c
                                                 those include in canalis cervi alis in
There are many suggestion that the               the patient with threatened PTL, can be
pathophysiology of PTL is the inflam-            choose as a tool to predict the PTL.
mation process in the pregnanc             y
compartment.       This    process        will   Research method
activate cytokine network whic             h
resulting Interleukin-1 (Il-1), Inter-           This research conducted throug          h
leukin-6 (Il-6) and Tumor Necrosis               analytic comparative study with cross-
Faktor α (TNF α) because of activated            sectional design.
macrophag.                                       The research was done in 14 mo      nths
Both cytokine will stimulate he           t      period and the end in 2005. I thatn
process of Interleukin-8 (Il-8) forma-           period we recruit 32 patients with
tion by decidua, chorion and a    mnion          criteria threatened preterm labor, and
and then together with Colony                    in the same time we choose 34 women
Stimulating Factor-1 (CSF-1) recruit             with normal pregnancy as a control at
polymorphonuclear (PMN) cell nto        i        random. Both of two groups of patients
the pregnancy compartment those                  with      PTL and control group ar      e
include uterine cervix, and th theen             include in inclusion and excluion   s
PMN produce the elastase.                        criteria, and both of the subj       ects
      Elastase is the proteolytic enzy-          received the same investigation.
me which found in pancreas, sp        leen       Threatened PTL is the pregnancy with
and neutrophyl. In the neutropyl        h        gestational age 28 weeks or more and
elastase is found in neutrophy              l    less than 37 weeks with the signs such
lysozyme, and neutrophyl was found in            as uterine contraction with interval less
the pregnant uterine cervix as the               than 10 minutes and observe in 30
                                  t
consequence of leucocyte infil ration            minutes minimally using cardiotoco-
in that area.                                    graphy, the dilation of cervix ≤ 4 cm
      In the normal pregnancy, elastase          by internal examination and th          e
detected at 20 weeks of pregnancy in             membranes still intact. As the control
the endocervix and then raise gradually          group are the pregnant women in the
as the raise of gestational age, and the         same gestational age without any signs
concentration raise abruptly 4 – 5               of labor.
weeks before initial of labor.                   Both of groups received many
This enzyme cleavage the elastin, the            investigation such as :
protein of connective tissue w        hich       1. Taken out of 10 cc of blood from
resistant to many proteinase, and this               the cubiti vein for haemoglobi ,   n
connective tissue are the esse tial   n              leucocyte      count,      -Reactive
                                                                                C
component in the chorion, amnion and                 Protein and for DNA examination.
the cervix. The increases of activity of         2. Taken out the swab from canali       s
this enzyme in pregnancy compartment                 cervicalis for elastase activi     ty
will cleavage of extracellular matrix                examination.
and resulting changes in these tissue.
Uterine cervix become soften, the                Hb, leucocyte count and CRP
cervix will be dilated and shorten those         examination using standard examina-
are the early sign of labor. n the  I            tion in our hospital, elastase exami-
chorion and amnion membrane th            is     nation using Elisa Method and for
enzyme will cleavage the extracellular           DNA elastase examination from
                                                                                   3


isolated leucocyte from the blood and        table. Mann-Whitney test for compar-
amplified elastase gen by PCR                ing the differences of two medan    i
technique. This amplification was done       value from the data with no no    rmal
using the pair of primer those F and R       distribution, t-test for comparing the
elastase primer :                            differences of two average data with
                                             normal distribution, and Kruskal-Walis
                                             test for comparing the differences of 2
5’-GTATCACGGGGCCCTGGATAA-3’ (F)              median value with no              normal
5’-CGGCCCGCCCGTGCCTCCCCG-3’ (R)              distribution. Significancy is decided
                                             with p value <0.05.
This pair of primer amplified the
promoter area of elastase gen which          RESULT
range from nt 800 to nt 1, totally 800
pb.                                                                       tients
                                             In this research we have 32 pa
Test of significancy was done by Chi-                                       i
                                             with threatened PTL and 34 patents
square    test    for         ces
                       differen      of      normal pregnancy as a control     .
                               n
percentation for data in conti gency
.

Table 1. Characteristic
                                        Group
     Characteristic           Threatened       Normal            Significancy
                                 PTL          Pregnancy
                               (n = 32)        (n = 34)
1. Age (year)
          < 20                      4               1
         20-24                      7               8
         25-29                      6              10
         30-34                      9               9
          > 35                      5               6
                                                                   t = 0,93
         X (SB)                 27,5 (6,2)     28,9 (6,3)
                                                                   p = 0,355
2. Parity
             0                     16              18              X2 = 0,06
            1-3                    15              15              p = 0,972
            >4                      1               1
3. Gestational Age (w)
         28-30                     10              10
         31-33                      8              17
         34-36                     14               7
                                                                   t = 0,76
         X (SB)                 32,0 (2,6)     31,6 (2,1)
                                                                   p = 0,448
Note : t = t-test; X2 = chi-square test.

Table 1 shows the characteristic of the      Most of the women of the both groups
subject those are include age of the         with the age range between 20-35 year,
women, parity and gestational age.           the optimal reproductive age. The
                                             differences no significant statistically
                                                                                     4


(p>0.05). Most of the women of both                                               t
                                                 most of the patient with threaened
groups with the parity 0, more than 50           PTL with the gestational age of 34–36
% for both groiups. The differences no           weeks. The differences no significant
significant statistically (p>0.05).              statistically (p>0.05).
Fifty percent of control group with the          Based on this homogeneity both
gestational age of 31–33 weeks, while            groups are comparable.

Table 2      Elastase concentration in canalis cervicalis in both groups
                                            Group
Elastase concentration
                                   Case              Control         Significancy
        (ng/ml)
                                 (n = 32)            (n = 34)
          X (SB)             2,68 (3,99)            1,31 (1,94)      ZM-W = 3,06
          Median                1,72                   0,52           p = 0,002
          Distance           0,09-22,07             0,11-9,98
Note : ZM-W = Mann-Whitney test

Table 2 shows the significa  nce dif-            tase in patient with threatened PTL is
                               i
ference of elastase concentraton in              1.72 ng/ml more than 1 ng/ml, while in
canalis cervicalis between two groups                                             ,
                                                 normal pregnancy is 0.52 ng/ml less
(p=0,002). The concentration of elas-            than 1 ng/ml yaitu 0,52 ng/ml. ……..

Table 3 Cut off Point of elastase concentration in predicting threatened PTL
     Cut off Point              Group                   Sens.     Spec.   Accuracy
       Elastase            Case     Control             (%)        (%)      (%)
    concentration        (n = 32)    (n = 34)
       (ng/ml)
 1.          >4              5               2          15,6      94,1      56,1
             <4             27              32
 2.          >3              8               4           25       88,2      57,6
             <3             24              30
 3.         > 1,7           16               6           50       82,4      66,7
            < 1,7           16              28
 4.         > 1,0           21              12          65,6      64,7      65,2
            < 1,0           11              22
 5.        > 0,75           23              14          71,9      58,8      65,2
           < 0,75            9              20
 6.        > 0,55           27              16          84,4      52,9      68,2
           < 0,55            5              18
 7.        ≥ 0,52           28              16          87,5      52,9      69,7
           < 0,52            4              18
 8.         > 0,5           29              18          90,6      47,0      68,2
            < 0,5            3              16
                                                                                   5


Table 3 shows 8 elastase conc     entra-    accuracy of 69.7%. If we use the
tion in canalis cervicalis are designed     elastase concentration with hi       gher
as a cut off point and are analyzed in      sensitivity, the specificity w beill
two groups. In those concentrations are     lesser. If we use the elastase concen-
evaluated sensitivity, specificity and                                      y
                                            tration with higher sensitivit , the
accuracy. From eight elastase concen-                                        l
                                            specificity will be lesser whie the
trations which are evaluated as a cut off   accuracy remain not changes. W          e
point in predicting PTL, the elastase       arranged the eight elastase concen      -
concentration of 0.52 ng/ml is choosed      trations in the curve of Receiver
as a cut off point with sensitivity of      Operating Characteristic as follows :
87.5%, specificity of 52.9% an         d




                     Figure 1. ROC of Elastase Concentration


After the eight concentrations are          examined by PCR technique to amplify
arranged in the curve of ROC                elastase gene. This amplification was
(Receiver Operating Characteris    tic),    done by using the pair of F an R  d
                th
apparently the 7 elastase with concen-      primer of elastase :
                                 l
tration of 0.52 ng/ml, has a ongest
distance from diagonal line of the          5’-GTATCACGGGGCCCTGGATAA-3’ (F)
curve. The selected cut-off point was       5’-CGGCCCGCCCGTGCCTCCCCG-3’ (R)
elastase concentration of 0,52 ng/ml.
                                            The pair of primer amplifi the  ed
                                            promoter region elastase gene which
DNA Elastase Examination Analyzes
                                            lied between 800 till 1 nt, the amount
Analyzed DNA from isolated leuco-
                                            of 800 base pair.
cyte taken out from the blood are
                                                                                     6


                           c1 c2                c3 c4




             800 pb




                      Note : pb : Pair of bases ; c : cases of PTL


Figure 2 PCR amplification result of Elastase gene in Threatened Preterm Labor
         Patients


Figure 2 show 6 sample of DNA                   from 6 patients with threatene        d
patients with Threatened Pretem  r              preterm labor with the size 800 pair of
Labor after amplificate with he    t            bases.
primers. The DNA tape which isolated

                               n1         n2   n3




         800 pb
                                                                                  7


                                 n1       n2     n3     n4




         800 pb




                        Note : pb : Pair of bases ; n : normal cases


Figure 3 PCR amplification result of Elastase Gene in normal pregnancy

Figure 3 shows 7 sample of D      NA              DNA tape which isolated from 7
patients with normal pregnancy after              patients with normal pregnancy has
amplification with these primers. The             size of 800 pair of bases.

                                 c1       c2       c3




            800 pb


            220 pb
            184 pb
            92 pb
            50 pb
            20 pb
                                                                                     8


                                c4




             800 pb


             220 pb
             184 pb
             92 pb
             50 pb
             20 pb




                         Note : pb : Pair of bases ; c : PTL cases

Figure 4. Fragmen DNA fragment of Elastase Gene after cut by HpaII enzyme
          in Threatened PTL group

Figure 4 shows the DNA tape re     sult          are 50 pb, 184 pb, 184 pb, 92 pb, 220
after PCR which come from               4                                        ly
                                                 pb, 50 pb and 20 pb respective of
pregnant women with threatened PTL               those DNA samples.
cutting by HpaII restriction enzyme              HpaII enzyme has ability to cut at the
The segment of tape resulting after its          sequence CCGG, and from DNA tape
cut are measured the length of pair of           with length of 800 pb there ar 7e
base. The restriction by this enzyme             sequence CCGG so the cutting resulted
resulting of 7 segments of DNA those             7 slices DNA tape.

                                                 n1        n2




              800 pb




              220 pb
              184 pb
              92 pb
              50 pb
              20 pb
                                                                                      9


                                n3




            800 pb




            220 pb
            184 pb
            92 pb
            50 pb
            20 pb




                          Note : pb : Pair base ; n : normal cases


Figure 5.   DNA fragment of Elastase Gene after cut by HpaII enzyme in Normal
            Pregnancy


Figure 5 show the DNA tape after PCR              restriction enzyme were 7 same DNA
from three normal pregnant wom        en          slices of both groups which are 50 pb,
which cut by HpaII enzyme. The                    184 pb, 184 pb, 92 pb, 220 pb, 50 pb
segment cutting is measured of its                dan 20 pb consecutively.
length of base. The result of cutting by
                                                                                      10




                                      k1          n            k2




             800 pb



             220 pb
             184 pb
             92 pb
             50 pb

             20 pb




                 Note : k : Threat.PTL;    n : Normal cases;        pb : Pair base


Figure 6. DNA fragment of Elastase Gene after cut by HpaII Enzyme from the
           patients with threatened PTL and normal pregnancy


Figure 6 show the DNA tape after PCR              pb, 92 pb, 220 pb, 50 pb dan 2 pb 0
from two pregnant women with                      consecutively.
threatened PTL (c1 and c2) and one                The result of segments of DNA after
normal pregnant woman (n) which cut               cutting by this restriction enyme z
by HpaII enzyme. The segment cutting              shows the same result between normal
is measured of its length of base. The            pregnancy and threatened PTL, and we
result of cutting by restriction enzyme           conclude that there is no difference in
were 7 same DNA slices of both                    gene expression in both groups.
groups which are 50 pb, 184 pb, 184


Conclusion

1. We      found      the    significance         2. We did not find the difference of
   difference of elastase concentration              elastase gene expression between
   in canalis cervicalis between                     normal pregnancy and threatened
   normal pregnancy and threatened                   preterm labor.
   preterm labor.
                                                                                  11


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