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Stretching of fetal membranes increases the concentration of interleukin-8 and collagenase activity

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Stretching of fetal membranes increases the concentration of interleukin-8 and collagenase activity
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  Stretching of fetal membranes increases interleukin-8 and collagenase activity the concentration of Emad E1 Maradny, PhD, Naohiro Kanayama, PhD, Abdul Halim, MB, BS, Kayoko Maehara, MD, and Toshihiko Terao, PhD Hamamatsu, Japan OBJECTIVE The aim of this study was to determine whether stretcNng of fetal membranes can increase interleukin-8 concentration and collagenase activ]ty. STLIDY DESIöN: Strips of whole fetal membranes, amnion, or muscles of the Iower uterine segment were stretched for 2 or 4 hours. Interleukin-8 and collagenase activity were measured in homogenized control and stretched samples. Immunohistochemical staining for interleukin-8 was carried out. RESULTS: The interleukin-8 concentration increased significantly after the whole fetal membranes were stretched for 2 and 4 hours (p < 0.0007 and 0.001, respectively). Also, stretcNng of amnion and muscles of the Iower uterine segment for 2 and 4 hours increased the concentrat]on of interleukin-8 significantly (p < 0.0007 after 2 and 4 hours, respectively). Collagenase activity was significantly increased after stretching of amnion, amniochorion, and muscles of the Iower uterine segment for 4 hours (p < 0.0007, 0.006, and 0.0007, respectively). After stretching, samples were darkly stained for interleukin-8 compared with control nonstretched samples. CONCLLI81ON: Stretching of amnion, amniochorion, and muscles of the Iower uterine segment increased interleukin-8 production and collagenase activity. (Am J Obstet Gynecol 1996;174:843-9.) Key words: Amnion, amniochorion, interleukin-8, collagenase activity, stretching, muscles of lower uterine segment During pregnancy both uterus and fetal membranes grow and stretch to accommodate the rapid growth of the fetus. Fetal membranes grow slower than the myome- trium does? After the second trimester the growth offetal membranes almost ceases. 2 Therefore tt?.ey have to stretch markedly to cope with the rapid growth rate of the uterus. 3 Fetal membranes may play a eentral role in the initia- tion of parturition. 40verstretching of the uterus and fetal membranes, as in cases of multiple pregnancies and hydramnios, iS usually accompanied by premature cer~~i- cal ripening and delivery. ~' 6 Mechanieal stretching of cuhured amnion cells and isolated myometrinm was found to increase synthesis and release of prostaglandin E2,: which has a great effect on cervical maturation and uterine contraction, s The ability of the fetal membranes and the uterine cervix to stretch and resist the intraamniotic pressure during pregnancy depends on their collagen-rich con- From the Department of Obstetrics and Gynecology, Hamamatsu Univet~ sity School of Medicine. Received for publication April 18, 1995; revised August 1, 1995; ac- cepted August 11, 1995. Reprint requests: Emad El Maradny, Department of Obstetrics and Gynecology, Hamamatsu Univ«si~y School of Medicine, 3600 Handa- cho, 431-31 Hamamatsu, Japan. Copyright © 1996 by Moslo~-Year Book, Inc. 0002-9378/96 5.00+ 0 6/1/68535 nective fissue. 1 A defect in the collagen content of fetal membranes may predispose to premature rupture of membranes. ~ A marked decrease in the collagen content of the cervix is known to occur during ripening. 1o Also, it was observed that the collagen content of the fetal mem- branes decreases significantly during the last 8 weeks of normal pregnancy/~ The mechanisms underlying col- lagen changes in fetal membranes are not clear, but they could be similar to the process that eauses cervical matu- ration. The decrease in the collagen content in the cervix was attributed to the effect of proteases enzymes, espe- cially collagenolytic enzymes. ~ These enzymes were found to be prodnced by cervical fibroblasts and neutro- phils, which invade the cervix during ripening. ~3 Interleukin-8 (IL-8) is a newly discovered chemotactic and acUvating factor for neutrophils. 14 It may play an essential role in cervical ripening and initiation of labor. ~5 IL-8 concentration in amniotic fluid was found to be increased gradnally in the third trimester of pregnancy? 6 Cultured amnion, chorion, and decidual cells produced IL-8 eonstimtively and in response to other cytokines.17' J~ Human uterine cervix was also found to be able to pro- duce large amounts of IL-8 near term. ~9 IL-8 conld ac- count for the neutrophil accumulation seen during cer- vital dilatation. Exogenous application of IL-8 was found to induce cervical ripening through increasing col- lagenolytic enzymes released by cervical fibroblasts and aceumulated neutrophils? ~ 843  844 El Maradny et al. March 1996 AmJ Obstet Gynecol Fig. 1. Machine used to stretch amnion, amniochorion, and uterine muscles. It has one fixed jaw, whereas other jaw is mobile and connected to spring balance. 70 65 60 55 50 45 4O 35 3O 100 Amnion Whole fetal membranes I Myometrium I I I I I 120 140 160 180 200 increase in length Fig. 2. Percentage of increase in length of amnion, arnniochorion, and uterine muscles in relation to traction in grams. It is unknown which factor(s) potentiates the produc- tion and release of IL-8. We hypothesized that stretching may be one of the factors that control IL-8 and collage- nase activity. Thus the aim of this research was to study the stretching effect of fetal membranes and muscles of the lower uterine segment on IL-8 concentration and colla- genase activity. Material and methods This research was approved by the research committee of Hamamatsu University. Written consent was obtained from all patients involved in this study. A muscle biopsy specimen was collected from the lower uterine segment during elective cesarean sections and before any clinical signs of labor (n = 15). Cesarean sec- tions were done because of previous cesarean sections (n = 10) or primigravid women with breech presentations (n = 5). All cases were between 38 and 40 weeks' of gesta- tion. Multiple specimens from the fetal membranes of the same patients were also collected immediately after deliv- ery of the placenta. Samples were washed thoroughly in warm saline solu- tion to remove blood clots and were transferred to the laboratory. Membranes were divided into equal 3 x 1 cm strips, whereas muscle biopsy specimens were divided into 2 x I × 0.5 cm strips. In membrane biopsy specimens we used either whole fetal membranes (amniochorion and attached remnant maternal decidua) or amnion alone after careful separation from chorion. Muscle, am- nion, or whole fetal membrane strips were clamped be- tween two jaws of a stretching machine (Fig. 1). One jaw is fixed, whereas the other jaw is mobile. The mobile jaw is attached to a spring balance to measure the stretching power in grams. Samples were immersed in phosphate- buffered saline solution at 37 ° C. Gradual stretching of the strips was carried out until maximal stretching was  Volume 174, Number 3 El Maradny et al. 845 AmJ Obstet Gynecol 1.8 1.6 1.4 ~o 1.2 o ~ .4 .2 p=O OOl i i * Am. : Amnion * Am-Oh : Amniochorion * myomet. : myometTium p=O.O007 all =O.r o07 p=O O007 Fig. 3. Concentration of IL-8 in amnion, anmiochorion, and muscles of lower uterine segment mea- sured after 2 and 4 hours of stretching. q { p=G0007 Cont. : Control | [~1 Stret. : Stretched .~ 2.5 O • N ~ c 2 .~ .~. ~, f p~o.oo6 .5 (cont.) (Stret. 4h) (cont.) (Stret. 4h) (cont.) (Stret. 4h) t j t I u j Ammon Amniochorion Muscle of lower uterine segment Fig, 4. Collagenase activity measured in amnion, amniochorion, and uterine muscles before and after stretching for 4 hours, achieved. The increases in length and stretching power in grams were calculated for each sample. We stretched the strips continuously for 2 or 4 hours. Then 100 mg (wet weight) of tissue was collected and homogenized with 1 ml of phosphate-buffered saline solution. After cen- trifugation of the homogenized strips, the supernatants were collected and kept at -80 ° C until used. The IL-8 concentration and collagenase activity were measured in nonstretched control samples and in stretched samples. IL-8 was measured with the commercially available kit Biotra IL-8 enzyme-linked immunosorbent assay System (Amersham, International pig Little Chatfont, Buck- inghamshire, United Kingdom). Steps of measurement were done according to the manufacturer's instructions. Coltagenase activity was measured with highly specific kits (coltagenase type 1 [matrix metalloproteinase -t] activity measurement, Cosmo Bio, Yagai, Tokyo). Control and su'etched specimens were fixed in parafor- maldehyde and embedded in paraffin wax. Paraffin sec- tions were cut at a thickness of 3 to 4 gin. After deparaf- finization, rehydrated tissue sections were stained by he- matoxylin and eosin to study the histopathologic changes after stretching. Also, immunohistochemical staining for IL-8 was carried out for control and stretched sections by  846 El Maradny et al. March 1996 AmJ Obstet Gynecol Fig. 5. A, Normal amnion stained few IL-8. h appears as single layer of smooth cuhoidal ceils. (Original magnification x40.) B, After stretching for 4 hours, amnion cells appeared as long columnar cells with surface projections. Also, the staining of the compact layer below the epithelium was increased. (Original magnification ×40.) use of a streptavidin-biotin complex-peroxidase kit (Dako, Calif.). First antibody (antihuman IL-8 antibody) was purchased from Oncogen Science, New York. Con- trol sections were subjected to the same methods, except that primary antibodies were replaced with Tris-buffered saline solution. Data are shown as mean +__SD, and the Wilcoxon signed-ranks test was used for statistical analysis. Results Amnion, whole fetal membranes, and muscle strips from the lower uterine segment increased in length in response to stretching for 4 hours. Fig. 2 shows the per- cent increase in length of stretched biopsy specimens in response to traction in grams. Amnion has a remarkable ability for stretching, compared with whole fetal mem- branes. High concentrations of IL-8 were found in control whole fetal membranes, anmion, and muscle biopsy Spec- imens (0.299 + 0.136, 0,205 _+ 0.097, and 0.111 + 0.044 ng per 100 mg wet.homogenized tissue, respectively). The concentration of IL-8 in control whole fetal membranes was higher but not significantly different from that in amnion biopsy specimens. Stretching of amnion cells led to a marked time-dependent increase in the IL-8 concen- tration. After 2 and 4 hours of stretching the homog- enized amnion cells showed a significant increase in 11~8 concentration (p = 0.0007). Also, stretching of the whole fetal membranes induced a significant increase in the IL-8 concentration after 2 and 4 hours (p= 0.0007 and  Volume i74, Nmnber 3 El Maradny et ak 847 AmJ Obstet (;ynecol Fig. 6. A, Normal amniochorion staiJ~cd for IL-8. (Original magnificado~~ ×20.) B, 32"ter stretchi~lg ~br 4 hours, staining of reticular layer of choHot~ and decidual cells was markedly increased. (Original mag~lification x20.) 0.001, respectively) (Fig. 3). The increased level of 1I.-8 concentration in whole fetal membranes was significa/ltly higher than in amnion cells after 2 and 4 hours of stretch- ing (p = 0.0001 and 0.01, respecdvely). The II.-8 concentration in stretched muscle strips from the lower uterine segment was increased time depen- dently. A significant increase in the 1L-8 concentration was found after 2 and 4 hours of stretching (p = 0.0007) Fig. 3). Collagenase activity was significantly increased after stretching ofamnion (p = 0.0007). Also, stretching of the whole fetaI membranes induced a significant increase of collagenase activity compared with the control group (p = 0.006) (Fig. 4). No significant change in collagenase activity of amnion or whole fetal membranes was found after 4 hours compared with 2 hours of suetching. Stretching of muscle strips from the lower uterhm seg- ment led to a significant increase in co/lagenase activity compared with nonstretched muscles (p= 0.0007) (Fig. 4). Also, we could E~ot observe significant cha~~ges in collagenase activity between 2 and 4 hours of stretching, In histologic sections stained by hematoxyiin and eosin, normal contro] arrmion cells appeared as a smooth layer of adherem cuboidal or short cohlmnar cel s. After stretching, most of the cells appeared as long columnar cel]s with surface projections. No other obvious changes could be observed after stretching of whole fetal mem- brane or muscle biopsy specimens, hnmunohistochemi- cal stainhlg for II:8 in amnion cells and whole fetal mem- branes showed a great difference before and after stretch- ing (Table I). After stretching, the immtmostaining for IL-8 was increased in the cytoplasm of amnion cells, indi-
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