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
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.
Presented in Asia Oceania Congress of
Obstetry and Gynecology 2009, Auckland,
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
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
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
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
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
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 .
percentation for data in conti gency
Table 1. Characteristic
Characteristic Threatened Normal Significancy
(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
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
(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
Case Control Significancy
(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
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)
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
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
apparently the 7 elastase with concen- primer of elastase :
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
c1 c2 c3 c4
Note : pb : Pair of bases ; c : cases of PTL
Figure 2 PCR amplification result of Elastase gene in Threatened Preterm Labor
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
n1 n2 n3 n4
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
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.
Note : pb : Pair base ; n : normal cases
Figure 5. DNA fragment of Elastase Gene after cut by HpaII enzyme in Normal
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
k1 n k2
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
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.
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