Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation

All women gave written informed consent according to the requirements of the North Somerset and South Bristol Research Ethics Committee. Placenta and gestational membranes were collected immediately post-partum from the following groups of women: preterm (25–36 weeks gestation) not-in-labour (PNIL), delivery by caesarean section for maternal or fetal complications; spontaneous preterm labour (SPL), with vaginal delivery; term (≥ 37 weeks gestation) not-in-labour (TNIL), delivery by elective caesarean section indicated by previous section and/or breech presentation; spontaneous term labour (STL), with vaginal delivery; term following induction of labour (IOL) with intravaginal PGE₂ pessary and/or intravenous oxytocin infusion, with delivery vaginally or by emergency caesarean section (failure to progress). The women were of mixed parity and all delivered live singletons. None of the women in preterm labour received steroid treatment. Tissues were also collected from a group of women (INF) with evidence of inflammation, as suggested by clinical features of the women (pyrexia or uterine tenderness) and gross pathology of the delivered placentas, and confirmed histologically by the presence of leucocyte infiltration in the fetal membranes (chorioamnionitis), decidua (deciduitis) or placenta (intervillositis), with or without maternal pyrexia or uterine tenderness [4]. Clinical information for the women providing uterine tissues for this study is given in Table 1. Tissues from 36 women were used in this study; tissues from 31 of these women were previously among those used to study overall levels of prostaglandin pathway gene expression in placenta and gestational membranes [13]. Myometrial tissues used in the previous study were taken from a separate group of women. Gestational membranes were dissected from between 1 cm and 4 cm from the placental border. Placental tissue was dissected from > 5 mm beneath the maternal surface of the placenta. Tissue samples were dissected immediately after delivery (amnion and choriodecidua were separated by blunt dissection), washed in sterile phosphate-buffered saline (PBS), snap-frozen and stored in liquid nitrogen. Tissues were also fixed and paraffin-embedded following standard procedures for immunohistochemistry.

Total RNA was extracted from 100 mg tissue samples by the guanidine isothiocyanate/phenol method using 1 ml TRIzol (Invitrogen, Carlsbad, CA, US), giving yields of 10–150 μg. RNA was quantified using a GeneQuant II spectrophotometer (GE Healthcare, Little Chalfont, UK). 2 μg total RNA was used as a template for cDNA synthesis primed by random primers using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, US). cDNA was diluted fourfold and 2 μl used as template for qPCR with the Power SYBR Green PCR Master Mix (Applied Biosystems), with reaction volume of 20 μl, forward and reverse primer concentrations of 75 nM, and 45 cycles of 95 C for 15 s and 60 C for 60 s, followed by a dissociation stage, using a 7500 Real-Time PCR System (Applied Biosystems). Two genes with least Ct variability, POLR2A (polymerase (RNA) II (DNA directed) polypeptide A, 220 kDa) and ARHGDIA (Rho GDP dissociation inhibitor (GDI) alpha), were chosen from five candidates for use as endogenous controls. PCR reaction efficiencies for all primer pairs were tested by serial template dilution, and were between 90% and 110%. The ‘sample maximization’ method was used, with reactions for each gene run on the minimum number of plates. A standard set of inter-run calibrators was included on each plate. Analysis was as previously described [13]. Sequences for all primers used in this study are given in Table 2.

Slide-mounted, paraffin-embedded tissue sections were dewaxed in Histoclear (National Diagnostics, Atlanta, GA), hydrated in a graded ethanol series (absolute, 90%, 70% ethanol) and incubated in 1% (w/w) aqueous hydrogen peroxide solution for 15 min to block endogenous peroxidase activity. Antigen retrieval was achieved by incubation in citrate buffer (10 mM sodium citrate, pH6.0, 0.05% (v/v) Tween-20) at 95°C for 20 min. Slides were incubated for 20 min with 2% (v/v) blocking serum, washed with PBS and incubated overnight with primary antibody at the following dilutions: PTGS1 (prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)) 1:60 (sc-1752, Santa Cruz Biotechnology, Santa Cruz, CA); PTGS2 1:60 (sc-1745); AKR1B1 (aldo-keto reductase family 1, member B1 (aldose reductase)) 1:200 (in house, Fortier MA); AKR1C3 (aldo-keto reductase family 1, member C3) 1:200 (ab27491, Abcam, Cambridge, UK); CBR1 1:300 (ab4148); PTGES (prostaglandin E synthase) 1:200 (160140, Cayman Europe, Tallinn, Estonia); HPGD 1:300 (in house, Fortier MA); SLCO2A1 (solute carrier organic anion transporter family, member 2A1) 1:3500 (in house, Fortier MA); VIM (vimentin) 1:200/1:1000 (V4630, Sigma, Gillingham, UK/M7020, DAKO, Ely, UK) in PBS + 3% (w/v) BSA. Slides were washed three times in PBS then incubated for 2 h with biotinylated secondary antibody (Vectastain ABC signal enhancement kit, Vector Labs, Burlingame, CA) diluted 1:200 in PBS + 3% (w/v) BSA. Slides were washed with PBS and incubated for 30 min with Vectastain ABC streptavidin-HRP (horseradish peroxidase) conjugate, washed again and incubated with 3,3’-Diaminobenzidine (DAB, Sigma) for antibody-specific colour development, which was stopped by washing in PBS, before counterstaining nuclei with Mayer’s Haemalum, dehydrating in a graded ethanol series followed by Histoclear and finally coverslip mounting using DPX mountant.

Associations between levels of gene expression and continuous clinical variables (maternal and gestational age, duration of labour) were determined by measuring the probability of significance associated with the Pearson correlation coefficient (using the TDIST function in Excel). Comparisons of expression levels in distinct subgroups of subjects were made in Excel with Student’s t-tests (two-way, not assuming equal variances or equal sample size).

We investigated the possibility of relationships between clinical features of the subjects and prostaglandin gene expression levels in uterine tissues.

Significant correlation between gestational age at delivery and prostaglandin gene expression occurred with gene and tissue specificity, as shown in Figure 2. In women who were not in labour at delivery, there was a negative correlation (decreasing gene expression with increasing gestational age) for PTGES in amnion (p = 0.045), and positive correlation for HPGDS (hematopoietic prostaglandin D synthase) in amnion (p = 0.039), HPGDS, AKR1C3 and ABCC4 in placenta (p = 0.020, 0.024, 0.046). In women delivering following spontaneous labour, there was negative correlation for AKR1B1 and PTGIS (prostaglandin I2 (prostacyclin) synthase) in amnion (p = 0.049, < 0.001), and positive correlation for PTGS2 in amnion (p = 0.007) and AKR1C3 and PTGIS in choriodecidua (p = 0.026, 0.022). In these women, as expected, gestational age showed a strong positive correlation with birth weight (p < 0.001).

Gene expression was compared between groups of women matched for gestational age who delivered with or without spontaneous labour. With preterm deliveries, expression was higher with labour for AKR1B1 in choriodecidua and PTGIS in placenta (p = 0.032, 0.028). With term deliveries, expression was higher with labour for PTGES in amnion and AKR1C3 in choriodecidua (p = 0.045, 0.033), while levels of PTGIS, ABCC4 and HPGD in amnion were higher in deliveries without labour (p = 0.043, 0.049, 0.038).

Duration of labour in spontaneous and induced labour deliveries ranged from 33 minutes to 17 hours. Pearson correlation coefficients were calculated to determine the association between duration of labour and gene expression. Negative correlation, indicating decreasing expression with increasing duration, was seen with expression of CBR1 in amnion (p = 0.006), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), PTGES3 (prostaglandin E synthase 3 (cytosolic)), AKR1C3 and CBR1 in choriodecidua (p = 0.049, 0.011, 0.013, <0.001) and AKR1C3 in placenta (p = 0.031). Positive correlation was seen for PTGES2 (prostaglandin E synthase 2) in amnion (p = 0.022) and SLCO2A1 in choriodecidua (p = 0.010).

Placenta and gestational membranes were collected from women with uterine inflammation, and PG gene expression in this group was compared by t-test with expression in a subgroup of women with no inflammation that was matched for gestational age and mode of delivery (Figure 2). Effects of inflammation were limited to upregulation of PTGS2 in amnion and choriodecidua (p = 0.022, 0.038), and downregulation of CBR1 and HPGD in choriodecidua (p = 0.018, 0.011).

Women were assigned to the inflammation group on the basis of established histological criteria [4], and we further characterised the inflammatory status of all tissue samples by measurement of the expression of three genes known to be involved in inflammatory responses: IL8, S100A8 and TLR2 (Figure 3). All three genes were significantly upregulated in both amnion (p = 0.021, < 0.001, 0.012) and choriodecidua (p = 0.002, <0.001, 0.002) from women assigned to the inflammation (INF) group. In placenta, the only change was an increase in S100A8 (p = 0.037) with inflammation. Both S100A8 and TLR2 were expressed at significantly higher levels in choriodecidua from women in the STL compared to the TNIL group (p = 0.014, 0.010) confirming a degree of inflammatory activity in term labour. Levels of both genes also appeared to be higher in SPL rather than PNIL choriodecidua, but these differences were of borderline significance (p = 0.061, 0.057).

Low magnification images of H&E-stained placental sections in Figure 4A show (i) the fetal trophoblastic villi and intervillous space, which make up the great majority of the placenta, and (ii) the basal plate, which lies adjacent to the uterine wall. Figure 4B-I show placental immunolocalisation of eight of the PG pathway proteins, while Figure 4J shows the localisation of vimentin in villous fibroblasts, vascular cells, macrophages and decidual cells, but not trophoblasts.

In the chorionic plate (the surface of the placenta adjacent to the amniotic cavity), the amnion epithelium showed staining for PTGS2 and PTGES (not shown). Extravillous cytotrophoblasts, which form an incomplete layer at the inner border of the chorionic plate, showed staining for HPGD, PTGES, SLCO2A1, AKR1B1, AKR1C3 and CBR1.In the placental villi (Figure 4A-K(i)), syncytiotrophoblasts displayed staining for AKR1B1, HPGD PTGS2, SLCO2A1, CBR1, AKR1C3, and PTGES. Villous fibroblasts showed PTGS2 and SLCO2A1 staining and heterogeneous AKR1B1 staining. Villous macrophages were positive for PTGS1 and PTGES.

The basal plate of the placenta (Figure 4A-K(ii)) consists of maternal decidual cells and fetal extravillous cytotrophoblasts, in some areas arranged in distinct layers and in others partially or thoroughly interspersed. Both decidual cells and extravillous cytotrophoblasts showed staining for AKR1B1, PTGS2, HPGD, PTGES, SLCO2A1, AKR1C3, and CBR1. Staining in the two cell types varied from patient to patient and even in different regions of the same placental tissue section, notably with PTGES and HPGD in extravillous cytotrophoblasts. Extravillous cytotrophoblasts clustered in cell islands in the villous placenta had similar staining patterns (not shown). There was no noticeable staining for any of these proteins in fibrinoids of the basal plate (not shown). Protein distribution in the placental cell populations is summarised in Table 3, along with references to previous descriptions of these proteins.

Figure 5A-G shows the immunolocalisation of seven of the PG pathway proteins in amnion and choriodecidua (PTGS1 is not included as we observed no staining in these tissues); Figure 5H shows vimentin localisation in decidual cells, amnion epithelium and fibroblasts of the amnion and chorion, but not in chorionic trophoblasts. In each panel a lower magnification image (i) gives a view through a full section of the membranes, while higher magnification images show (ii) decidual cells, (iii) chorionic trophoblasts and chorionic fibroblasts, (iv) amniotic epithelium.

The decidual cells showed staining for AKR1B1, HPGD, AKR1C3, PTGS2, SLCO2A1 and CBR1. Chorionic trophoblasts had staining for HPGD, AKR1B1, CBR1, PTGS2, PTGES, AKR1C3 and SLCO2A1. AKR1B1, PTGS2, AKR1C3, HPGD and CBR1 were seen in amniotic and chorionic fibroblasts. PTGS2 and PTGES had immunological reactions in amniotic epithelium. This protein distribution is summarised in Table 3.

Inflammation results in disruption of the fetal membranes, with highly variable leukocytic infiltration and loss of integrity of the chorionic trophoblast layer. Within a tissue section it is common to see regions of massive infiltration with minimal remaining chorionic trophoblasts, alongside sections of membrane that appear relatively normal. Figure 6 shows immunolocalisation of prostaglandin proteins in membranes with a moderate inflammatory reaction, with considerable leukocytic infiltration but a relatively undiminished chorion. Prostaglandin pathway protein immunolocalisation in amniotic epithelium, amniotic and chorionic fibroblasts, and decidual cells was not noticeably altered by inflammation. In chorionic trophoblasts, heterogeneous expression of PTGS2, PTGES, CBR1 and HPGD was seen (Figure 6A, B, E & G). In inflammatory leukocytes there was expression of PTGS2, AKR1C3, CBR1 and PTGES (Table 3 and Figure 6A, B, D & E).

As we have examined multiple members of the prostaglandin pathway in three uterine tissues, there is inevitably a degree of overlap with previous studies of prostaglandin pathway components. For descriptions of the immunolocalisation of prostaglandin pathway proteins, this overlap has been summarised in Table 3, from which it can be seen that we are now presenting novel evidence of uterine immunolocalisation for seven of the eight prostaglandin pathway proteins studied.

Previous descriptions of prostaglandin pathway gene expression have focused largely on the cyclooxygenase/prostaglandin H2 synthase genes PTGS1 and PTGS2 (formerly Cox1 and Cox2). Not all previous observations can be reconciled with each other.

In the placenta, there is evidence suggesting no change in PTGS1 expression with gestational age [15], and contrasting evidence of decreasing expression with increasing gestational age at labour [25]. In gestational membranes, increasing gestational age has been associated with increased [26, 27], unchanged [27, 28], and decreased [29]PTGS1 expression. Likewise, the incidence of labour has been associated with increased [26, 27] and unchanged [30‐36]PTGS1 expression.

In the placenta, the existing evidence suggests that there is no change in expression of PTGS2 with gestational age or clinical chorioamnionitis [25]. In the gestational membranes, several studies have shown higher PTGS2 expression with increasing gestational age [26‐29]. There is evidence supporting both increased PTGS2 expression following labour [26‐28, 31‐35] and no change with labour [20, 36, 37].

Information relating to intrauterine expression of other prostaglandin pathway genes is limited. Our previous work demonstrated expression of the 15 prostaglandin pathway genes in placenta, amnion and choriodecidua [13]. In addition, PLA2G4A (phospholipase A2, group IVA (cytosolic, calcium-dependent)) expression has been identified in human placenta and gestational membranes [38], as has expression of PTGDS and HPGDS[39]. In placenta and membranes, PTGES expression has shown no change with labour [21]. Expression of AKR1B1, AKR1C3, HPGD and SLCO2A1 has been demonstrated in amnion and choriodecidua [19]. Evidence has been presented in support of unchanged placental expression of HPGD in response to gestational age, labour and intrauterine infection [25, 40], but also in support of increased expression with gestational age [41]. In choriodecidua, there is evidence for lower levels of HPGD mRNA in labour than not-in-labour [24, 37, 40, 42], with further reductions occurring in the presence of intrauterine infection [40].