Placenta xxx (2014) 1e6
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Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes* € m c, A.C. Petersen b, G.V. Eriksen a, N. Uldbjerg a M. Meinert a, *, A. Malmstro a b c
Department of Obstetrics and Gynaecology, Aarhus University Hospital, 8200 Aarhus N, Denmark Department of Pathology, Aalborg University Hospital, 9100 Aalborg, Denmark Department of Cell and Molecular Biology, Biomedical Center, C13, 221 84 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 6 May 2014
Objective: The proteoglycan decorin stabilizes collagen whereas biglycan and hyaluronan disrupt wellorganized collagen. The aim was to determine the concentrations of these constituents in fetal membranes in relation to gestational age, preterm labour, PPROM and chorioamnionitis. Study design: Preterm fetal membranes (24e34 weeks gestation) were obtained from elective caesarean deliveries (N ¼ 4), from PPROM (N ¼ 14), and from preterm labour (N ¼ 14). Term fetal membranes from elective caesarean deliveries (N ¼ 9) and spontaneous vaginal deliveries (N ¼ 11) were used for comparison. Chorioamnionitis was assessed histologically. The proteoglycans were analysed using alcian blue precipitation, SDS-PAGE and immunostaining. Hyaluronan was estimated by a radioimmunoassay. Results: Preterm amniotic membranes with chorioamnionitis displayed a 8-fold decrease in hyaluronan concentration as well as a pronounced (88%) degradation of decorin and biglycan (p < 0.05). The amnion from preterm elective caesarean sections had higher decorin (3.2 vs. 1.7 mg/mg, p < 0.05) and lower biglycan (0.4 vs. 1.0 mg/mg, p < 0.05) concentrations as compared to similar term amnion (p < 0.05), whereas the hyaluronan concentrations were not associated with gestational age. Also the chorio-decidua from preterm caesarean sections had higher decorin concentrations (1.8 vs. 1.0 mg/mg, p < 0.05) whereas the biglycan concentration was unchanged. Labour (term as well as preterm) was characterized by increased hyaluronan and biglycan concentrations in the amnion (not statistically significant). Conclusion: The biglycan/decorin balance increases during third trimester of pregnancy and during active labour. This relation might contribute to mechanical weakening of the membranes. Chorioamnionitis induces dramatic degradation of both proteoglycans and hyaluronan, which can explain the decreased biomechanical strength. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Decorin Biglycan Hyaluronan PPROM Amnion Chorioamnionitis
1. Introduction Forty percent of preterm deliveries are initiated by preterm prelabour rupture of membranes (PPROM). One pathway leading to PPROM is chorioamnionitis [1e3], which causes a destruction of the
* Supported by grants from The Danish Medical Research Council, The Institute of Clinical Experimental Research, The University of Aarhus, The Danish Medical Association Fond, Consultant,PhD Karl Moeller Bek and The Swedish Medical Research Council. * Corresponding author. Y Research Laboratory, Department of Obstetrics and Gynaecology, Aarhus University Hospital, 8200 Aarhus N, Denmark. Tel.: þ45 78453721, þ45 40104052; fax: þ45 78453725. E-mail address: [email protected] (M. Meinert).
connective tissue of the fetal membranes. Another possible pathway could be a biomechanical insufficiency of the connective tissue in the fetal membranes. The biomechanical strength of the fetal membranes is determined mainly by the collagen component, the concentration of which decreases by 20e30% during the third trimester [4]. PPROM, however, is not associated with decreased collagen concentration in the amnion or along the rupture line [4,5], and results concerning the type III/type I collagen ratio are inconsistent [6,7]. Therefore connective constituents other than collagen may be important for the pathophysiology of PPROM. Such constituents could be decorin and biglycan, which are small leucine-rich proteoglycans highly expressed in reproductive tissues [8e10]. Decorin binds to collagen fibres and stabilizes the
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Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
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M. Meinert et al. / Placenta xxx (2014) 1e6
architecture of the tissue [11,12], whereas biglycan destabilizes this decorin-collagen interaction [13,14]. Previously, we demonstrated that fetal membranes obtained after term labour have higher biglycan/decorin ratios than do fetal membranes from elective caesarean sections [15]. Furthermore, the connective tissue disease EhlerseDanlos syndrome (EDS) is associated with PPROM, a condition often caused by abnormal secretion of biglycan and decorin [14,16]. We therefore raise the hypothesis that altered concentrations of decorin and biglycan can lead to PPROM. Another constituent of the connective tissue in the fetal membranes is hyaluronan, a polysaccharide that binds large amounts of water and increases tissue compliance [17]. This is important for the visco-elastic properties of many tissues such as Wharton's jelly [8,17e19]. The hyaluronan concentration increases in the amnion during the third trimester of pregnancy [19], with a dramatic increase during labour [15]. Furthermore, it is found in huge concentrations in the gelatinous substance that appears between the amnion and chorio-decidua at labour [15]. The hyaluronan concentration is not changed in PPROM [19] but has not been studied in relation to chorioamnionitis. The aim of this study was therefore to describe the concentrations of biglycan, decorin and hyaluronan in fetal membranes in relation to gestation, preterm birth, PPROM and chorioamnionitis. 2. Methods 2.1. Materials 2.1.1. Patient details Fetal membranes were obtained from A. Four elective preterm caesarean deliveries before the onset of labour indicated by previous intrauterine fetal death (n ¼ 2), carcinoma in situ of the cervix (n ¼ 1) and fetal stress (n ¼ 1); median gestational age 34 weeks. B. Fourteen women with PPROM with a median gestational age of 33 weeks (range: 24þ4e33þ6 weeks gestation). In five patients caesarean delivery was performed, nine delivered vaginally. C. Fourteen women with spontaneous preterm labour with a median gestational age of 31 weeks (Range: 24þ2e33þ4 weeks gestation). In two patients caesarean deliveries were performed, 12 delivered vaginally. D. Nine term pregnant women who underwent elective caesarean delivery before start of labour and without histological signs of chorioamnionitis [15]. E. Eleven term pregnant women with spontaneous vaginal delivery and without histological signs of chorioamnionitis [15]. Fetal membranes from women with multiple pregnancies and those suffering from any systemic disease (including preeclampsia and diabetes) were excluded. Medication: All patients from group AeC were treated with betamethasone 24 mg for lung maturation and ampicillin. Tocolytica were used if relevant, including indomethacin. Informed consent was obtained from all patients. The ethics committee of Aarhus County, Denmark, approved the study (permission 1988/1349). 2.1.2. Tissue sampling The free fetal membranes were collected within 20 min after delivery. Biopsies for biochemical analysis of 1, 4, and 8 cm2 were punched out using a cutting instrument mounted with razor blades in parallel, separated in the amnion and the chorio-decidua, and finally frozen at 80 C [8]. Mid-zone biopsies were obtained from halfway between the cervical area and the placental edge as described previously [15]. The free fetal membranes for light microscopy including immunohistochemistry were rolled from the rupture site towards the placental edge and fixed in 3% glutaraldehyde with 5% sucrose for 24 h. 2.2. Analytical procedures 2.2.1. Collagen determination Biopsies (1 cm2) were analysed for hydroxyproline concentration as described previously [8]. 2.2.2. Proteoglycan determination The biopsies were cut into pieces and homogenized using a manual mortar and extracted for 48 h in 4 M guanidinium chloride, containing 0.05 M acetate and proteinase inhibitors. After centrifugation, the proteoglycans were precipitated by alcian blue (WiesLab AB, Lund, Sweden). The resulting pellet was resolved in guanidinium chloride-propanol, and the concentration of proteoglycans estimated by
the absorbance of alcian blue at 600 nm [20,21]. Electrophoresis, analysis of the gels and digestion with chondroitinase ABC lyase (5 unit/ml) were carried out according to our previous protocol [8,22]. The interassay coefficient of variation was 7.0% (n ¼ 6) [8]. 2.2.3. Hyaluronan determination The concentration of hyaluronan was determined in the same extracts by means of a radioimmunoassay (Amersham Pharmacia Biotech, Bucks, UK) [24]. The interassay coefficient of variation was 6.2% (n ¼ 5). 2.2.4. Light microscopy of HE-stained slides After fixation the membrane rolls were embedded in paraffin, cut into 3 mm sections and stained by HE. Chorioamnionitis was evaluated by light microscopy according to the criteria described by Redline [23]. Mild chorioamnionitis was defined as infiltration of granulocytes in the subchorionic fibrin and/or the trophoblastic layer of chorion (maternal inflammatory response stage 1: MIR1) and the fibrous chorion (maternal inflammatory response stage 2: MIR2) whereas severe chorioamnionitis was defined as diffuse infiltration through all layers of the fetal membranes with necrosis of the amniotic epithelium or a hypereosinophilic amniotic basal membrane (maternal inflammatory response stage 3: MIR3). 2.2.5. Immunohistochemical procedure (decorin and biglycan) After fixation the membrane rolls were embedded in paraffin and cut into 3 mm sections. The epitopes were demasked with Protease1® (Roche) for 4 min (decorin) and 12 min (biglycan) at 36 . The immunohistochemical staining for decorin (Antidecorin, Abcam/ab54728®; AH Diagnostics) and biglycan (Anti-biglycan, Abcam/ ab54855/GR105713-1®; AH Diagnostics) was performed on an Ultra Benchmark platform® (Roche). The tissue was incubated for 32 min at 36 with the antibody (decorin diluted 1:10,000; biglycan diluted 1:24,000) and counterstained for 8 min with haematoxylinII® (Roche). The evaluation was done by light microscopy. 2.3. Statistical analysis Wilcoxon signed rank tests were used in the comparisons between samples within the same patient groups. Comparisons between different patients groups were determined by ManneWhitney Rank Sum Tests. Data were expressed as median (25 and 75 percentiles). The level of significance used was p < 0.05.
3. Results 3.1. Histology In the non-inflamed membranes (Fig. 1B) we found a strong reaction for decorin in the extracellular substance in the fibrous, collagen-rich layers of amnion and chorio-decidua. A distinct cytoplasmic reaction for biglycan was seen in the amniotic epithelium, in the fibroblasts in the fibrous layers of amnion and chorion and in the trophoblastic and decidual cells (Fig 1C). In contrast, the two proteoglycans were almost completely degraded in the severely inflamed membranes (Fig. 2B,C). No reaction for decorin was seen in the vicinity of the neutrophils. A dissociated weak reaction for decorin was seen only in regions without neutrophils. The compact layer and the fibrous layer of amnion showed a decreased reaction (Fig. 2B). In most layers no reaction for biglycan was found. The amniotic epithelium, when preserved, exhibited only a weak staining for biglycan (Fig. 2C). 3.2. Gestational age All biopsies from preterm and term elective caesarean deliveries were examined histologically and showed no signs of chorioamnionitis. Compared to term amnion, the preterm amnion had high decorin, low biglycan and unchanged hyaluronan concentrations (Table 1). The preterm chorio-decidua also displayed a high decorin concentration. Both decorin and biglycan gave welldefined bands on SDS-PAGE, which indicates the presence of intact proteoglycans (Fig 3). 3.3. Preterm labour without chorioamnionitis Among the preterm deliveries initiated by contractions, 29% were without chorioamnionitis. These amniotic membranes had
Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
M. Meinert et al. / Placenta xxx (2014) 1e6
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Fig. 1. HE-staining and immunostaining for decorin and biglycan. Fetal membranes from a planned cesarean section in week 34 and 1 day due to carcinoma in situ of the cervix. A: HE-staining showing no inflammation. AE: amniotic epithelium. CL: compact layer. FA: fibrous layer of amnion. FC: fibrous layer of chorion. TC: trophoblastic layer of chorion. DL: decidual layer. B: Decorin: A strong diffuse reaction for decorin in the extracellular substance in the fibrous layers of amnion and chorion and a cytoplasmic reaction in the decidual cells. C: Biglycan: A distinct cytoplasmic reaction for biglycan was seen in the amniotic epithelium, in the fibroblasts in the fibrous layers of amnion and chorion and in the trophoblastic and decidual cells.
increased hyaluronan (p ¼ 0.17) and unchanged decorin and biglycan concentrations as compared to elective preterm caesarean deliveries (Table 1). The chorio-decidua had unchanged hyaluronan, decorin and biglycan concentrations. 3.4. PPROM without chorioamnionitis Among the preterm deliveries initiated by PPROM, 36% were without chorioamnionitis. These amniotic membranes and the chorio-decidua had unchanged hyaluronan, decorin and biglycan concentrations as compared to preterm elective caesarean sections (Table 1). 3.5. Chorioamnionitis Among the preterm deliveries (PPROM and spontaneous labour), 68% displayed chorioamnionitis. In the remaining 32% of membranes without chorioamnionitis, decorin and biglycan from both amnion and chorio-decidua
migrated as intact proteoglycans on SDS-PAGE (Fig. 3). In seven of the eight amnions with severe chorioamnionitis, the proteoglycans had migrated further towards the front of the gel (Fig. 4 and Table 2), indicating fragmentation of the molecules. Electrophoresis on 2% agarose gel revealed that these fragmented molecules consisted of free polysaccharide chains (data not shown). 3.5.1. Hyaluronan Severe chorioamnionitis was associated with a 8-fold decrease in the concentration of hyaluronan in the amnion as compared to no and mild chorioamnionitis (Table 2). A similar but less pronounced decrease was seen in chorio-decidua (1.2 vs. 0.49 mg/mg wet weight, p < 0.05). 3.5.2. Hydroxyproline The hydroxyproline concentration in the amnion from elective caesarean section in week 34 and week 39 were identical 43 (39e62) vs. 47 (39e61) mg/mg dry weight.
Fig. 2. HE- staining and immunostaining for decorin and biglycan. Fetal membranes from a spontaneous preterm vaginal delivery in week 25. Contractions for 14 h. Previously conebiopsy (since 2 years). HE: Chorioamnionitis with maternal inflammatory response stage 3. Infiltration of neutrophils in all layers and necrosis of the amniotic epithelium. Decorin: A dissociated weak reaction in most layers. Minimal reaction in the basal membrane and fibrous layer of amnion. Biglycan: Almost no reaction. Only a weak staining in the preserved amniotic epithelium.
Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
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M. Meinert et al. / Placenta xxx (2014) 1e6
Table 1 Proteoglycans and hyaluronan (mg/mg wet weight) in amnion and chorio-decidual mid-zone samples without chorioamnionitis. Data are expressed as median (25e75% range).
N Amnion Hyaluronan Biglycan Decorin Chorio-decidua Hyaluronan Biglycan Decorin
34.4 weeks
32.3 weeks
33.3 weeks
39.1 weeks
39.6 weeks
Elective caesarean delivery
PPROM
Spontaneous labour
Elective caesarean delivery
Spontaneous labour
4
5
4
9
11
2.2 (1.0e3.6) 0.4* (0.2e0.4) 3.2* (2.7e4.0)
1.7 (1.3e2.6) 0.2 (0.2e0.7) 3.2 (2.5e4.1)
3.1 (1.6e5.2) 0.8 (0.2e1.6) 2.5 (2.0e4.1)
1.0 (0.7e5.8) 1.0 (0.7e1.4) 1.7 (1.6e2.2)
4.9 (3.8e7.1) 0.8 (0.6e1.1) 1.9 (1.4e2.5)
0.9 (0.5e1.8) 1.1 (0.9e1.4) 1.8* (1.7e2.2)
1.1 (0.6e1.9) 0.7 (0.5e0.9) 2.3 (1.9e3.0)
1.3 (0.4e3.1) 1.2 (0.5e2.2) 2.0 (1.8e2.4)
0.7 (0e1.8) 1.3 (0.6e1.6) 1.0 (0.8e1.5)
2.3 (1.9e3.2) 1.8 (1.4e2.9) 0.8 (0.7e1.4)
*Preterm vs. term elective caesarean deliveries, p < 0.05.
On the other hand, the hydroxyproline concentration in the amnion was significantly reduced (39 vs. 52 mg/mg dry weight) in cases of severe chorioamnionitis (Table 2). 4. Discussion The main finding of this study was that the decorin concentration in the preterm amnion without chorioamnionitis is 2-times that of term amnions. This is visualised by the strong immunostaining for decorin in a week 34 elective C-section membrane (Fig 1B). In the cases of severe chorioamnionitis, a dramatic decomposition of both hyaluronan and proteoglycans occurred, thus leaving free glycosaminoglycans without a protein core. This finding is strongly supported by immunostaining which revealed a very weak staining for decorin and biglycan in severely inflamed membranes with no staining in the proximity of granulocyte infiltration. The
Fig. 3. SDS PAGE gel of three preterm delivered membranes from elective caesarean delivery, PPROM, and spontaneous preterm delivery in the absence of chorioamnionitis. Am: amnion and Cho-D: chorio-decidua. Lane 1, small proteoglycan from bovine sclera used as standard. Lanes 2 and 3, amnion and chorio-decidua mid-zone from preterm elective caesarean delivery (CS). Lanes 4 and 5, amnion mid-zone and rupture site in PPROM. Lanes 6 and 7, chorio-decidua mid-zone and rupture site in PPROM. Lanes 8 and 9, amniotic and chorio-decidual mid-zone samples from preterm labour. (# In the cases of PPROM the proteoglycans was almost exclusively decorin. This is exemplified by amnion mid-zone in this one patient (Lane 3), which contained 95% decorin and 5% biglycan (with median 91% decorin and 9% biglycan in the PPROM group) in contrast to the preterm labour group with median 74% decorin and 26% biglycan (NS) (Lane 7).)
staining for decorin is only present away from the neutrophils and is lying as thin curtains in the vicinity of collagen fibres (Fig 2B). One strength of this study is that preterm membranes with and without chorioamnionitis were described separately (as opposed to the study by Skinner [4]). Another important aspect is that we only studied membranes from very preterm deliveries (24 h rupture of membranes before delivery Degraded proteoglycans (% of patients in each group) Hyaluronan mg/mg wet weight Hydroxyproline mg/mg dry weight
No chorioamnionitis (N ¼ 9)
Mild inflammation (N ¼ 11)
Severe inflammation (N ¼ 8)
32.3
30.3
26.4**
18%
55%
37%
0%
27%
88%*
2.3 (1.3e3.5)
2.3 (1.8e2.7)
0.3* (0.0e1.7)
52 (45e62)
62 (52e69)
39* (24e55)
*Mild inflammation (MIR1 þ 2) versus severe inflammation (MIR3) (p < 0.05). **No inflammation versus severe inflammation (MIR3) (p < 0.05). MIR: maternal inflammatory response.
Firstly, the association between term and preterm labour and low decorin concentrations in the amnion in this study on humans supports a recent publication on a mouse model of spontaneous preterm birth [16]. The study of Calmus et al. was based on the genetic ablation of biglycan and decorin and found that the loss of two of the four possible wild-type alleles for biglycan and decorin in the pregnant mouse was not associated with preterm delivery, whereas the loss of three alleles, and even more dramatically, the loss of all four alleles, resulted in a significantly increased risk of preterm birth [16]. Furthermore, these pregnancies displayed uterine dysfunction [27] and abnormal architecture with large diameters and irregular contours of the collagen fibrils in the decidualized stroma of the endometrium [28]. In pregnancies characterized with fetal loss these alleles had higher risk of growth restriction, whereas the risk of preterm birth was not significantly increased [16]. The second pathophysiological aspect of our study is that chorioamnionitis causes partial degradation of collagen and almost complete decomposition of hyaluronan and the proteoglycans. This explains why chorioamnionitis very often leads to PPROM [29]. Hyaluronidase from bacteria such as group B streptococci is probably of major importance as well as matrix metalloproteinases from macrophages and polymorphnuclear leucocytes which degrades the proteoglycans [30,31]. This phenomenon may be associated with PROM at term of pregnancy as an increased leucocyte density, high levels of inflammatory markers, and a lost collagen integrity has been demonstrated at the rupture zone of the chorio-decidual membranes from such cases [32,33]. It may be of importance that decorin binds and neutralizes different cytokines including TGF-b [34]. This anti-inflammatory effect of decorin in the fetal membranes may interplay with the antibacterial potential of the cervical mucus plug that inhibits ascending infections [31,32]. From this study, we conclude that proteoglycans and hyaluronan are involved in the physiological maturation of fetal membranes as well as in the pathophysiology of preterm delivery.
Conflict of interest We confirm and have checked that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes* € m c, A.C. Petersen b, G.V. Eriksen a, N. Uldbjerg a M. Meinert a, *, A. Malmstro a b c
Department of Obstetrics and Gynaecology, Aarhus University Hospital, 8200 Aarhus N, Denmark Department of Pathology, Aalborg University Hospital, 9100 Aalborg, Denmark Department of Cell and Molecular Biology, Biomedical Center, C13, 221 84 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 6 May 2014
Objective: The proteoglycan decorin stabilizes collagen whereas biglycan and hyaluronan disrupt wellorganized collagen. The aim was to determine the concentrations of these constituents in fetal membranes in relation to gestational age, preterm labour, PPROM and chorioamnionitis. Study design: Preterm fetal membranes (24e34 weeks gestation) were obtained from elective caesarean deliveries (N ¼ 4), from PPROM (N ¼ 14), and from preterm labour (N ¼ 14). Term fetal membranes from elective caesarean deliveries (N ¼ 9) and spontaneous vaginal deliveries (N ¼ 11) were used for comparison. Chorioamnionitis was assessed histologically. The proteoglycans were analysed using alcian blue precipitation, SDS-PAGE and immunostaining. Hyaluronan was estimated by a radioimmunoassay. Results: Preterm amniotic membranes with chorioamnionitis displayed a 8-fold decrease in hyaluronan concentration as well as a pronounced (88%) degradation of decorin and biglycan (p < 0.05). The amnion from preterm elective caesarean sections had higher decorin (3.2 vs. 1.7 mg/mg, p < 0.05) and lower biglycan (0.4 vs. 1.0 mg/mg, p < 0.05) concentrations as compared to similar term amnion (p < 0.05), whereas the hyaluronan concentrations were not associated with gestational age. Also the chorio-decidua from preterm caesarean sections had higher decorin concentrations (1.8 vs. 1.0 mg/mg, p < 0.05) whereas the biglycan concentration was unchanged. Labour (term as well as preterm) was characterized by increased hyaluronan and biglycan concentrations in the amnion (not statistically significant). Conclusion: The biglycan/decorin balance increases during third trimester of pregnancy and during active labour. This relation might contribute to mechanical weakening of the membranes. Chorioamnionitis induces dramatic degradation of both proteoglycans and hyaluronan, which can explain the decreased biomechanical strength. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Decorin Biglycan Hyaluronan PPROM Amnion Chorioamnionitis
1. Introduction Forty percent of preterm deliveries are initiated by preterm prelabour rupture of membranes (PPROM). One pathway leading to PPROM is chorioamnionitis [1e3], which causes a destruction of the
* Supported by grants from The Danish Medical Research Council, The Institute of Clinical Experimental Research, The University of Aarhus, The Danish Medical Association Fond, Consultant,PhD Karl Moeller Bek and The Swedish Medical Research Council. * Corresponding author. Y Research Laboratory, Department of Obstetrics and Gynaecology, Aarhus University Hospital, 8200 Aarhus N, Denmark. Tel.: þ45 78453721, þ45 40104052; fax: þ45 78453725. E-mail address: [email protected] (M. Meinert).
connective tissue of the fetal membranes. Another possible pathway could be a biomechanical insufficiency of the connective tissue in the fetal membranes. The biomechanical strength of the fetal membranes is determined mainly by the collagen component, the concentration of which decreases by 20e30% during the third trimester [4]. PPROM, however, is not associated with decreased collagen concentration in the amnion or along the rupture line [4,5], and results concerning the type III/type I collagen ratio are inconsistent [6,7]. Therefore connective constituents other than collagen may be important for the pathophysiology of PPROM. Such constituents could be decorin and biglycan, which are small leucine-rich proteoglycans highly expressed in reproductive tissues [8e10]. Decorin binds to collagen fibres and stabilizes the
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Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
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architecture of the tissue [11,12], whereas biglycan destabilizes this decorin-collagen interaction [13,14]. Previously, we demonstrated that fetal membranes obtained after term labour have higher biglycan/decorin ratios than do fetal membranes from elective caesarean sections [15]. Furthermore, the connective tissue disease EhlerseDanlos syndrome (EDS) is associated with PPROM, a condition often caused by abnormal secretion of biglycan and decorin [14,16]. We therefore raise the hypothesis that altered concentrations of decorin and biglycan can lead to PPROM. Another constituent of the connective tissue in the fetal membranes is hyaluronan, a polysaccharide that binds large amounts of water and increases tissue compliance [17]. This is important for the visco-elastic properties of many tissues such as Wharton's jelly [8,17e19]. The hyaluronan concentration increases in the amnion during the third trimester of pregnancy [19], with a dramatic increase during labour [15]. Furthermore, it is found in huge concentrations in the gelatinous substance that appears between the amnion and chorio-decidua at labour [15]. The hyaluronan concentration is not changed in PPROM [19] but has not been studied in relation to chorioamnionitis. The aim of this study was therefore to describe the concentrations of biglycan, decorin and hyaluronan in fetal membranes in relation to gestation, preterm birth, PPROM and chorioamnionitis. 2. Methods 2.1. Materials 2.1.1. Patient details Fetal membranes were obtained from A. Four elective preterm caesarean deliveries before the onset of labour indicated by previous intrauterine fetal death (n ¼ 2), carcinoma in situ of the cervix (n ¼ 1) and fetal stress (n ¼ 1); median gestational age 34 weeks. B. Fourteen women with PPROM with a median gestational age of 33 weeks (range: 24þ4e33þ6 weeks gestation). In five patients caesarean delivery was performed, nine delivered vaginally. C. Fourteen women with spontaneous preterm labour with a median gestational age of 31 weeks (Range: 24þ2e33þ4 weeks gestation). In two patients caesarean deliveries were performed, 12 delivered vaginally. D. Nine term pregnant women who underwent elective caesarean delivery before start of labour and without histological signs of chorioamnionitis [15]. E. Eleven term pregnant women with spontaneous vaginal delivery and without histological signs of chorioamnionitis [15]. Fetal membranes from women with multiple pregnancies and those suffering from any systemic disease (including preeclampsia and diabetes) were excluded. Medication: All patients from group AeC were treated with betamethasone 24 mg for lung maturation and ampicillin. Tocolytica were used if relevant, including indomethacin. Informed consent was obtained from all patients. The ethics committee of Aarhus County, Denmark, approved the study (permission 1988/1349). 2.1.2. Tissue sampling The free fetal membranes were collected within 20 min after delivery. Biopsies for biochemical analysis of 1, 4, and 8 cm2 were punched out using a cutting instrument mounted with razor blades in parallel, separated in the amnion and the chorio-decidua, and finally frozen at 80 C [8]. Mid-zone biopsies were obtained from halfway between the cervical area and the placental edge as described previously [15]. The free fetal membranes for light microscopy including immunohistochemistry were rolled from the rupture site towards the placental edge and fixed in 3% glutaraldehyde with 5% sucrose for 24 h. 2.2. Analytical procedures 2.2.1. Collagen determination Biopsies (1 cm2) were analysed for hydroxyproline concentration as described previously [8]. 2.2.2. Proteoglycan determination The biopsies were cut into pieces and homogenized using a manual mortar and extracted for 48 h in 4 M guanidinium chloride, containing 0.05 M acetate and proteinase inhibitors. After centrifugation, the proteoglycans were precipitated by alcian blue (WiesLab AB, Lund, Sweden). The resulting pellet was resolved in guanidinium chloride-propanol, and the concentration of proteoglycans estimated by
the absorbance of alcian blue at 600 nm [20,21]. Electrophoresis, analysis of the gels and digestion with chondroitinase ABC lyase (5 unit/ml) were carried out according to our previous protocol [8,22]. The interassay coefficient of variation was 7.0% (n ¼ 6) [8]. 2.2.3. Hyaluronan determination The concentration of hyaluronan was determined in the same extracts by means of a radioimmunoassay (Amersham Pharmacia Biotech, Bucks, UK) [24]. The interassay coefficient of variation was 6.2% (n ¼ 5). 2.2.4. Light microscopy of HE-stained slides After fixation the membrane rolls were embedded in paraffin, cut into 3 mm sections and stained by HE. Chorioamnionitis was evaluated by light microscopy according to the criteria described by Redline [23]. Mild chorioamnionitis was defined as infiltration of granulocytes in the subchorionic fibrin and/or the trophoblastic layer of chorion (maternal inflammatory response stage 1: MIR1) and the fibrous chorion (maternal inflammatory response stage 2: MIR2) whereas severe chorioamnionitis was defined as diffuse infiltration through all layers of the fetal membranes with necrosis of the amniotic epithelium or a hypereosinophilic amniotic basal membrane (maternal inflammatory response stage 3: MIR3). 2.2.5. Immunohistochemical procedure (decorin and biglycan) After fixation the membrane rolls were embedded in paraffin and cut into 3 mm sections. The epitopes were demasked with Protease1® (Roche) for 4 min (decorin) and 12 min (biglycan) at 36 . The immunohistochemical staining for decorin (Antidecorin, Abcam/ab54728®; AH Diagnostics) and biglycan (Anti-biglycan, Abcam/ ab54855/GR105713-1®; AH Diagnostics) was performed on an Ultra Benchmark platform® (Roche). The tissue was incubated for 32 min at 36 with the antibody (decorin diluted 1:10,000; biglycan diluted 1:24,000) and counterstained for 8 min with haematoxylinII® (Roche). The evaluation was done by light microscopy. 2.3. Statistical analysis Wilcoxon signed rank tests were used in the comparisons between samples within the same patient groups. Comparisons between different patients groups were determined by ManneWhitney Rank Sum Tests. Data were expressed as median (25 and 75 percentiles). The level of significance used was p < 0.05.
3. Results 3.1. Histology In the non-inflamed membranes (Fig. 1B) we found a strong reaction for decorin in the extracellular substance in the fibrous, collagen-rich layers of amnion and chorio-decidua. A distinct cytoplasmic reaction for biglycan was seen in the amniotic epithelium, in the fibroblasts in the fibrous layers of amnion and chorion and in the trophoblastic and decidual cells (Fig 1C). In contrast, the two proteoglycans were almost completely degraded in the severely inflamed membranes (Fig. 2B,C). No reaction for decorin was seen in the vicinity of the neutrophils. A dissociated weak reaction for decorin was seen only in regions without neutrophils. The compact layer and the fibrous layer of amnion showed a decreased reaction (Fig. 2B). In most layers no reaction for biglycan was found. The amniotic epithelium, when preserved, exhibited only a weak staining for biglycan (Fig. 2C). 3.2. Gestational age All biopsies from preterm and term elective caesarean deliveries were examined histologically and showed no signs of chorioamnionitis. Compared to term amnion, the preterm amnion had high decorin, low biglycan and unchanged hyaluronan concentrations (Table 1). The preterm chorio-decidua also displayed a high decorin concentration. Both decorin and biglycan gave welldefined bands on SDS-PAGE, which indicates the presence of intact proteoglycans (Fig 3). 3.3. Preterm labour without chorioamnionitis Among the preterm deliveries initiated by contractions, 29% were without chorioamnionitis. These amniotic membranes had
Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
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Fig. 1. HE-staining and immunostaining for decorin and biglycan. Fetal membranes from a planned cesarean section in week 34 and 1 day due to carcinoma in situ of the cervix. A: HE-staining showing no inflammation. AE: amniotic epithelium. CL: compact layer. FA: fibrous layer of amnion. FC: fibrous layer of chorion. TC: trophoblastic layer of chorion. DL: decidual layer. B: Decorin: A strong diffuse reaction for decorin in the extracellular substance in the fibrous layers of amnion and chorion and a cytoplasmic reaction in the decidual cells. C: Biglycan: A distinct cytoplasmic reaction for biglycan was seen in the amniotic epithelium, in the fibroblasts in the fibrous layers of amnion and chorion and in the trophoblastic and decidual cells.
increased hyaluronan (p ¼ 0.17) and unchanged decorin and biglycan concentrations as compared to elective preterm caesarean deliveries (Table 1). The chorio-decidua had unchanged hyaluronan, decorin and biglycan concentrations. 3.4. PPROM without chorioamnionitis Among the preterm deliveries initiated by PPROM, 36% were without chorioamnionitis. These amniotic membranes and the chorio-decidua had unchanged hyaluronan, decorin and biglycan concentrations as compared to preterm elective caesarean sections (Table 1). 3.5. Chorioamnionitis Among the preterm deliveries (PPROM and spontaneous labour), 68% displayed chorioamnionitis. In the remaining 32% of membranes without chorioamnionitis, decorin and biglycan from both amnion and chorio-decidua
migrated as intact proteoglycans on SDS-PAGE (Fig. 3). In seven of the eight amnions with severe chorioamnionitis, the proteoglycans had migrated further towards the front of the gel (Fig. 4 and Table 2), indicating fragmentation of the molecules. Electrophoresis on 2% agarose gel revealed that these fragmented molecules consisted of free polysaccharide chains (data not shown). 3.5.1. Hyaluronan Severe chorioamnionitis was associated with a 8-fold decrease in the concentration of hyaluronan in the amnion as compared to no and mild chorioamnionitis (Table 2). A similar but less pronounced decrease was seen in chorio-decidua (1.2 vs. 0.49 mg/mg wet weight, p < 0.05). 3.5.2. Hydroxyproline The hydroxyproline concentration in the amnion from elective caesarean section in week 34 and week 39 were identical 43 (39e62) vs. 47 (39e61) mg/mg dry weight.
Fig. 2. HE- staining and immunostaining for decorin and biglycan. Fetal membranes from a spontaneous preterm vaginal delivery in week 25. Contractions for 14 h. Previously conebiopsy (since 2 years). HE: Chorioamnionitis with maternal inflammatory response stage 3. Infiltration of neutrophils in all layers and necrosis of the amniotic epithelium. Decorin: A dissociated weak reaction in most layers. Minimal reaction in the basal membrane and fibrous layer of amnion. Biglycan: Almost no reaction. Only a weak staining in the preserved amniotic epithelium.
Please cite this article in press as: Meinert M, et al., Chorioamniontis in preterm delivery is associated with degradation of decorin and biglycan and depletion of hyaluronan in fetal membranes, Placenta (2014), http://dx.doi.org/10.1016/j.placenta.2014.05.004
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M. Meinert et al. / Placenta xxx (2014) 1e6
Table 1 Proteoglycans and hyaluronan (mg/mg wet weight) in amnion and chorio-decidual mid-zone samples without chorioamnionitis. Data are expressed as median (25e75% range).
N Amnion Hyaluronan Biglycan Decorin Chorio-decidua Hyaluronan Biglycan Decorin
34.4 weeks
32.3 weeks
33.3 weeks
39.1 weeks
39.6 weeks
Elective caesarean delivery
PPROM
Spontaneous labour
Elective caesarean delivery
Spontaneous labour
4
5
4
9
11
2.2 (1.0e3.6) 0.4* (0.2e0.4) 3.2* (2.7e4.0)
1.7 (1.3e2.6) 0.2 (0.2e0.7) 3.2 (2.5e4.1)
3.1 (1.6e5.2) 0.8 (0.2e1.6) 2.5 (2.0e4.1)
1.0 (0.7e5.8) 1.0 (0.7e1.4) 1.7 (1.6e2.2)
4.9 (3.8e7.1) 0.8 (0.6e1.1) 1.9 (1.4e2.5)
0.9 (0.5e1.8) 1.1 (0.9e1.4) 1.8* (1.7e2.2)
1.1 (0.6e1.9) 0.7 (0.5e0.9) 2.3 (1.9e3.0)
1.3 (0.4e3.1) 1.2 (0.5e2.2) 2.0 (1.8e2.4)
0.7 (0e1.8) 1.3 (0.6e1.6) 1.0 (0.8e1.5)
2.3 (1.9e3.2) 1.8 (1.4e2.9) 0.8 (0.7e1.4)
*Preterm vs. term elective caesarean deliveries, p < 0.05.
On the other hand, the hydroxyproline concentration in the amnion was significantly reduced (39 vs. 52 mg/mg dry weight) in cases of severe chorioamnionitis (Table 2). 4. Discussion The main finding of this study was that the decorin concentration in the preterm amnion without chorioamnionitis is 2-times that of term amnions. This is visualised by the strong immunostaining for decorin in a week 34 elective C-section membrane (Fig 1B). In the cases of severe chorioamnionitis, a dramatic decomposition of both hyaluronan and proteoglycans occurred, thus leaving free glycosaminoglycans without a protein core. This finding is strongly supported by immunostaining which revealed a very weak staining for decorin and biglycan in severely inflamed membranes with no staining in the proximity of granulocyte infiltration. The
Fig. 3. SDS PAGE gel of three preterm delivered membranes from elective caesarean delivery, PPROM, and spontaneous preterm delivery in the absence of chorioamnionitis. Am: amnion and Cho-D: chorio-decidua. Lane 1, small proteoglycan from bovine sclera used as standard. Lanes 2 and 3, amnion and chorio-decidua mid-zone from preterm elective caesarean delivery (CS). Lanes 4 and 5, amnion mid-zone and rupture site in PPROM. Lanes 6 and 7, chorio-decidua mid-zone and rupture site in PPROM. Lanes 8 and 9, amniotic and chorio-decidual mid-zone samples from preterm labour. (# In the cases of PPROM the proteoglycans was almost exclusively decorin. This is exemplified by amnion mid-zone in this one patient (Lane 3), which contained 95% decorin and 5% biglycan (with median 91% decorin and 9% biglycan in the PPROM group) in contrast to the preterm labour group with median 74% decorin and 26% biglycan (NS) (Lane 7).)
staining for decorin is only present away from the neutrophils and is lying as thin curtains in the vicinity of collagen fibres (Fig 2B). One strength of this study is that preterm membranes with and without chorioamnionitis were described separately (as opposed to the study by Skinner [4]). Another important aspect is that we only studied membranes from very preterm deliveries (24 h rupture of membranes before delivery Degraded proteoglycans (% of patients in each group) Hyaluronan mg/mg wet weight Hydroxyproline mg/mg dry weight
No chorioamnionitis (N ¼ 9)
Mild inflammation (N ¼ 11)
Severe inflammation (N ¼ 8)
32.3
30.3
26.4**
18%
55%
37%
0%
27%
88%*
2.3 (1.3e3.5)
2.3 (1.8e2.7)
0.3* (0.0e1.7)
52 (45e62)
62 (52e69)
39* (24e55)
*Mild inflammation (MIR1 þ 2) versus severe inflammation (MIR3) (p < 0.05). **No inflammation versus severe inflammation (MIR3) (p < 0.05). MIR: maternal inflammatory response.
Firstly, the association between term and preterm labour and low decorin concentrations in the amnion in this study on humans supports a recent publication on a mouse model of spontaneous preterm birth [16]. The study of Calmus et al. was based on the genetic ablation of biglycan and decorin and found that the loss of two of the four possible wild-type alleles for biglycan and decorin in the pregnant mouse was not associated with preterm delivery, whereas the loss of three alleles, and even more dramatically, the loss of all four alleles, resulted in a significantly increased risk of preterm birth [16]. Furthermore, these pregnancies displayed uterine dysfunction [27] and abnormal architecture with large diameters and irregular contours of the collagen fibrils in the decidualized stroma of the endometrium [28]. In pregnancies characterized with fetal loss these alleles had higher risk of growth restriction, whereas the risk of preterm birth was not significantly increased [16]. The second pathophysiological aspect of our study is that chorioamnionitis causes partial degradation of collagen and almost complete decomposition of hyaluronan and the proteoglycans. This explains why chorioamnionitis very often leads to PPROM [29]. Hyaluronidase from bacteria such as group B streptococci is probably of major importance as well as matrix metalloproteinases from macrophages and polymorphnuclear leucocytes which degrades the proteoglycans [30,31]. This phenomenon may be associated with PROM at term of pregnancy as an increased leucocyte density, high levels of inflammatory markers, and a lost collagen integrity has been demonstrated at the rupture zone of the chorio-decidual membranes from such cases [32,33]. It may be of importance that decorin binds and neutralizes different cytokines including TGF-b [34]. This anti-inflammatory effect of decorin in the fetal membranes may interplay with the antibacterial potential of the cervical mucus plug that inhibits ascending infections [31,32]. From this study, we conclude that proteoglycans and hyaluronan are involved in the physiological maturation of fetal membranes as well as in the pathophysiology of preterm delivery.
Conflict of interest We confirm and have checked that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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