Ann Hematol DOI 10.1007/s00277-016-2780-1

ORIGINAL ARTICLE

Abnormal expression of inflammatory genes in placentas of women with sickle cell anemia and sickle hemoglobin C disease Letícia C. Baptista 1 & Maria Laura Costa 2 & Regiane Ferreira 3 & Dulcinéia M. Albuquerque 3 & Carolina Lanaro 3 & Kleber Y. Fertrin 3 & Fernanda G. Surita 2 & Mary A. Parpinelli 2 & Fernando F. Costa 3 & Mônica Barbosa de Melo 1,4

Received: 27 June 2016 / Accepted: 1 August 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Sickle cell disease (SCD) is a complex disease that is characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane biology, endothelial activation, hemolysis, a procoagulant state, acute and chronic inflammation, and vaso-occlusion. Among the physiological changes that occur during pregnancy, oxygen is consumed by fetal growth, and pregnant women with SCD are more frequently exposed to low oxygen levels. This might lead to red blood cells sickling, and, consequently, to vaso-occlusion. The mechanisms by which SCD affects placental physiology are largely unknown, and chronic inflammation might be involved in this process. This study aimed to evaluate the gene expression profile of inflammatory response mediators in the placentas of pregnant women with sickle cell cell anemia (HbSS) and hemoglobinopathy SC (HbSC). Our results show differences in a number of these genes. For the HbSS group, when compared to the control group, the following genes

showed differential expression: IL1RAP (2.76-fold), BCL6 (4.49-fold), CXCL10 (−2.12-fold), CXCR1 (−3.66-fold), and C3 (−2.0-fold). On the other hand, the HbSC group presented differential expressions of the following genes, when compared to the control group: IL1RAP (4.33-fold), CXCL1 (3.05-fold), BCL6 (4.13-fold), CXCL10 (−3.32-fold), C3 (−2.0-fold), and TLR3 (2.38-fold). Taken together, these data strongly suggest a differential expression of several inflammatory genes in both SCD (HbSS and HbSC), indicating that the placenta might become an environment with hypoxia, and increased inflammation, which could lead to improper placental development. Keywords Sickle cell disease . Placenta . Pregnancy . Inflammation . Gene expression

Introduction The expression of some genes related to the inflammatory response is altered in the placentas of women with sickle cell disease. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2780-1) contains supplementary material, which is available to authorized users. * Mônica Barbosa de Melo [email protected] 1

Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas - UNICAMP, Campinas, SP, Brazil

2

Department of Obstetrics and Gynecology, University of Campinas UNICAMP, School of Medicine, Campinas, São Paulo, Brazil

3

Hematology and Hemotherapy Center, University of Campinas UNICAMP, Campinas, São Paulo, Brazil

4

Laboratory of Human Genetics, Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas UNICAMP, P.O. Box: 6010, Campinas, SP 13083-875, Brazil

Sickle cell disease (SCD) results from the homozygote state for hemoglobin (Hb) S, the combination of the HbS alelle with another abnormal Hb allele, beta thalassemia, or hereditary persistence of fetal Hb. It is a complex disease characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane biology, endothelial activation, hemolysis, a procoagulant state, acute and chronic inflammation, and vaso-occlusion [1]. Adhesive interactions between red blood cells (RBCs), platelets, leukocytes, and vascular endothelium result in the production of inflammatory mediators and elevated levels of cytokines that are associated with vaso-occlusive episodes. These complex chains of events are responsible for a number of different clinical manifestations [2, 3]. Pregnancy has always been a great concern for women with SCD because it is associated with very high rates of maternal and fetal mortality [4]. Currently, mortality rates

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have significantly decreased due to improved antenatal care, including vaccinations, the availability and safety of transfusions, and early diagnosis of complications by multidisciplinary teams responsible for medical care [5]. However, despite all of these advances, pregnancy in SCD patients is still considered to be a high-risk condition with increased possibility of adverse maternal and perinatal outcomes [6, 7]. Among the physiological changes during pregnancy, oxygen is increasingly consumed by fetal growth. Pregnant women with SCD are probably more frequently exposed to low oxygen levels. This may lead to more frequent RBC sickling and, consequently, to vaso-occlusion [8]. Vaso-occlusion in the placental circulation can contribute to the formation of areas of fibrosis, villous necrosis, and infarction [9]. Furthermore, increased blood viscosity and a certain degree of sickling and vaso-occlusion in the placenta [10] can result in decreased umbilical flow later in pregnancy [11]. Endothelial and inflammatory adaptations during pregnancy can contribute to the exacerbation of vaso-occlusive episodes, which may increase the occurrence and severity of obstetrical complications [6] such as anemia, increased risk of infection, and a procoagulant profile [12]. Maternal and fetal complications include prepartum and postpartum pain crises, thrombotic complications, preeclampsia (PE), premature delivery, and intrauterine growth restriction (IUGR) [13]. These complications are more frequent in patients homozygous for HbS. HbSC disease, in general, is considered a more benign condition; however, there is evidence to indicate that this disorder may cause serious problems, similar to those observed in HbSS during pregnancy [14]. Due to the increased production of inflammatory cytokines identified in the plasma and leukocytes of SCD patients [15], and the almost lack of information about the mechanisms by which this disease affects placental physiology, specifically in relation to the inflammatory pathway, this study aimed to evaluate the gene expression profile of inflammatory response mediators in the placentas of pregnant women with HbSS, HbSC, and uncomplicated controls.

Materials and methods Study group This study was approved by the local Ethics Board (number 201/2013). Pregnant women were recruited from the high-risk outpatient clinic and maternity of the State University of Campinas (UNICAMP), after providing written informed consent. This case-control study included 14 women with SCD (HbSS, n = 7 and HbSC, n = 7) and 8 women with non-complicated, control pregnancies (n = 8). Hemoglobinopathy diagnosis (HbSS or HbSC) was performed including clinical and laboratorial data, Hb electrophoresis, family analysis, and DNA

sequencing when necessary at the institution’s clinical laboratory [16]. Data on maternal demographic characteristics were collected (including age and previous pregnancy), as well as maternal complications, both antenatally and postnatally, and fetal outcome (including gender and weight and size at birth). For the SCD cases, specific data on the amount of time since the diagnosis, previous complications, and treatment were also recorded. The control group was constituted by healthy women at the same hospital. Documented cases of maternal hypertension, proteinuria, diabetes, fetal anomalies, and infections during prenatal care were exclusion criteria for the control group. Placental tissue collection and RNA extraction Placental villous tissue samples were randomly selected from the central areas close to the umbilical cord, as previously described [17]. The time between the delivery and the sampling was recorded, and the placenta was refrigerated after delivery. Before collection, the placentas were weighed and photographed. The samples were collected (≈200 mg) in three different regions avoiding macroscopically abnormal areas (without gross pathology); they were then separated into three equal size pieces and washed with phosphate saline buffer to remove residual maternal blood. Immediately after collection, the tissues were frozen in liquid nitrogen and stored at –80 °C until the RNA isolation procedure, with each sample having a pool from the four selected areas of the villous tissue. Total RNA was extracted using TRIzol Reagent (Ambion), followed by the RNeasy mini kit (Qiagen). The quantity and purity of the extracted RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific), and RNA integrity was determined using the Bioanalyzer 2100 system (Agilent Technologies). PCR array for human inflammatory response and autoimmunity Five hundred nanograms of RNAwere converted to complementary DNA (cDNA) using the RT 2 First Strand Kit (SABiosciences). Subsequently, complementary DNA was combined with the RT2 qPCR Master Mix, according to the manufacturer’s instructions, and added to the pathway-specific RT2 Profiler PCR Array (PAHS-077Z, SABiosciences). This array includes a total of 84 genes associated with inflammatory response and 12 controls (housekeeping genes, positive PCR controls, and reverse transcription controls). Quantitative real-time PCR (qPCR) was performed using ABI StepOne Plus equipment (Applied Biosystems) with the following cycling conditions: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 40 cycles of 60 °C for 1 min. The resulting threshold cycle values for all the genes were uploaded to the SABiosciences website for data analysis using

RT2 PCR Array Data Analysis Software version 3.5 (http://www.sabiosciences.com/pcr/arrayanalysis.

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php). All the gene expression levels were normalized to the housekeeping genes, ACTB, B2M, and GAPDH, which did not vary significantly between the study groups. The difference in gene expression was evaluated by calculating the fold change in the expression levels based on the criteria of at least a 2-fold up or down change in comparison to the gene expression levels of the control group.

Biosystems) with the following cycling conditions: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 40 cycles of 60 °C for 1 min. Amplification specificity was verified using a dissociation curve, and messenger RNA (mRNA) expression was calculated relative to the expression of ACTB, B2M, and GAPDH using the standard equation (2^(-ΔCt)), as previously described by Schmittgen [18].

Quantitative real-time PCR

Placental histology

To validate the results from the PCR array, qPCR was performed for selected genes that showed differential expression between the groups. The RNA samples were treated with DNAse I (Fermentas) and cDNA synthesized from 1 μg of RNA, using the RevertAid First Strand cDNA Synthesis Kit (Fermentas), according to the manufacturer’s protocol. Table 1 provides a summary of the primer sequences and the size of the fragments that were obtained; the annealing temperature for all of the primer sets was 60 °C. The PCR amplification was carried out in a 12 μl reaction volume containing 150 or 300 nM specific primers (3 μl), 10 ng cDNA (3 μl), and 6 μl SYBR Green PCR Master Mix (Applied Biosystems). All of the qPCR reactions were performed in duplicate using an ABI StepOne Plus equipment (Applied

The placental samples were fixed in formalin for 24 h, and they w e r e p a r a f f i n - e m b e d d e d f o r l i g h t m i c r o s c o p y. Five-micrometer-thick histological sections were obtained and stained with hematoxylin and eosin (H&E) for morphology examination. The tissue sections were deparaffinized in xylene, rehydrated in a series of ethanol, and washed in phosphate-buffered saline (PBS). Images were obtained using a Nikon E800 microscope with a color Olympus DT digital camera.

Table 1 Primers used for real time PCR analyses

Statistical analysis Statistical analysis was performed using GraphPad Prism version 5.00 software (GraphPad Software, CA, USA).

Gene

Primer sequences

Fragment size (bp)

BCL6

F-TAACATCGTTAACAGGTCCATGAC R-CTCTGCATGCTGTGGGGACT F-CTGTGCTGAGGAGAATTGCTTC R-GGTCTTGTACACATAGTCCACTCCT

121

C3 CXCL1 CXCL10 CXCL2 CXCR1 FASLG IL1RAP PTGS2 TLR3 ACTB B2M GAPDH

bp base pairs

F-CGAAGTCATAGCCACACTCAAGA R-GTCACTGTTCAGCATCTTTTCGA F-CCACGTGTTGAGATCATTGCTAC R-GAGATCTTTTAGACCTTTCCTTGCT F-CGAAGTCATAGCCACACTCAAGA R-GCCATTTTTCAGCATCTTTTCG F-CCGGTGCTTCAGTTAGATCAAA R-CTGTAATCTTCATCTGCAGGTGG F-CATTTAACAGGCAAGTCCAACTC R-AGGCCACCCTTCTTATACTTCAC F-AGGTAGTAGGCTCTCCAAAAAATG R-TGAGTAGCTCCTCTCCTGGTTCT F-CATGGGGTGGCATTAAATCATATT R-TCTGCCTGAGTATCTTTGACTGTG F-CATTATGCAAAAGATTCAAGGTACA R-CAGAGTGCATGGTTCAGTTTATAAT F-TGACCCAGATCATGTTTGAGACC R-CAGAGGCGTACAGGGATAGCA F-GTATGCCTGCCGTGTGAAC R-AAAGCAAGCAAGCAGAATTTGG F-AAGATCATCAGCAATGCCTCCT R-GGTCATGAGTCCTTCCACGATAC

106 97 121 97 128 101 96 140 121 81 139 96

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The Mann-Whitney U test was used in this analysis; a p value <0.05 was considered to be statistically significant.

Results Clinical complications The study evaluated seven women with HbSS, seven women with HbSC, and eight control healthy women without SCD. The HbSS and HbSC genotypes for SCD were both compared with the control group. No statistically significant differences were found among the study groups in terms of birth height and placental weight; however, the women with the HbSS genotype presented significantly lower gestational age at delivery and birth weight (35.0 ± 3.9 weeks, p = 0.02 and 2265 ± 872.8 g, p = 0.04) than the women in the control group (38.9 ± 1.7 weeks and 2946 ± 585.4 g). Sickle-related complications during pregnancy were more frequent in the HbSS group than in the HbSC group: vaso-occlusive crises (57.1 Table 2 Comparison of maternal-fetal characteristics between the examined groups

vs. 42.9 %), IUGR (28.6 vs. 0 %), perinatal mortality (14.2 vs. 0 %), hospital admission during pregnancy (71.4 vs. 28.6 %), and intensive care unit admission during pregnancy (14.2 vs. 0 %). The prevalence of acute chest syndrome did not differ between SCD groups. The data for each group are shown in Table 2. A total of 11 (78.6 %) pregnant women with SCD received prophylactic blood transfusions (six patients in the HbSS group and five patients in the HbSC group). In our institution, regular blood transfusion has been used as a prophylaxis to decrease adverse maternal and fetal outcomes [14]. Three of the patients (one HbSS and two HbSC) did not receive prophylactic transfusions for different reasons. In one case, preterm birth occurred at 28 weeks prior to the scheduled first transfusion, and neonatal death occurred shortly after that birth. The other two patients had difficulty adhering to treatment, due to personal issues. There was a trend toward lower placental and newborn weight in the HbSS group; however, the number of patients in our study was most likely not high enough to reach a clear conclusion. It is also important to

Characteristic

Control group n (%)

HbSS group n (%)

HbSC group n (%)

Total pregnancies Maternal age (years)—mean ± SD Parity Nulliparous Primiparous

8 26.7 ± 5.6

7 26.4 ± 5.8

7 27.9 ± 8.7

3 (37.5) 5 (62.5)

4 (57.1) 3 (42.9)

3 (42.9) 3 (42.9)

0

0

1 (14.2)

38.9 ± 1.7 1 (12.5)

35.0 ± 3.9* 3 (42.9)

37.6 ± 1.4 2 (28.6)

3 (37.5) 5 (62.5)

3 (42.9) 4 (57.1)

2 (28.6) 5 (71.4)

Multiparous Gestacional age (weeks)—mean ± SD Preterm labor Route of delivery Vaginal Cesarean section Birth weight (grams)—mean ± SD

2946 ± 585.4

Birth height (cm)—mean ± SD 47.4 ± 3.0 Placental weight (grams)—mean ± SD 546.0 ± 88.3 (n = 5) Baby’s sex Female 5 (62.5) Male 3 (37.5) Smoking 0 Sickle-related complications during pregnancy Vaso-occlusive crises – Acute chest syndrome – IUGR – Perinatal mortality – Programmed blood transfusion – Hospital admission during pregnancy ICU admission during pregnancy

– –

2265 ± 872.8*

3003 ± 442.6

44.2 ± 7.3 (n = 6) 431.4 ± 162.3

47.4 ± 1.3 540.0 ± 72.1 (n = 6)

2 (28.6) 5 (71.4) 0

2 (28.6) 5 (71.4) 1 (14.2)

4 (57.1) 2 (28.6) 2 (28.6) 1 (14.2) 6 (85.7)

3 (42.9) 2 (28.6) 0 0 5 (71.4)

5 (71.4) 1 (14.2)

2 (28.6) 0

IUGR intrauterine growth restriction, ICU intensive care unit, SD standard deviations *p < 0.05, Mann–Whitney U test

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consider that cases complicated by PE were also excluded, due to the possible influence in inflammatory response, and is well known that PE is one of the major obstetric complications in SCD pregnancies and that it is associated with IUGR. Placental histology As an example of the histological abnormality that may be found in the placenta, one HbSS patient was analyzed. Placental villous tissue samples were stained with H&E for morphological assessment. One HbSS patient (gestacional age 35 weeks) and a woman from the control group (gestacional age 36 weeks) were selected. The HbSS patient was admitted to the hospital several times during her pregnancy due to complications, such as pneumonia and a vaso-occlusive crisis. Histological examination revealed increased syncytial knots, dilation of villous capillaries, and maternal sickled red blood cells in the intervillous space (Fig. 1). The results of the histological analysis showed the presence of capillary dilation which can be considered as a compensatory adaptation change aimed at maintenance of placental circulation [19]. Expression profiles of the placental tissue The inflammatory response and autoimmunity PCR arrays was used for a comparison of the gene expression profiles in the villous tissue of five placentas from the HbSS group, five placentas from the HbSC group, and eight controls. In the HbSS group, 15 out of 84 inflammation-related genes showed significant differential expression. Among these genes, six were upregulated (BCL6, CCL24, CCR3, CXCL1, CXCL2, and IL5) and nine were downregulated (C3, CCL21, CXCL6, CXCL10, CXCR1, FASLG, KNG1, PTGS2, and

Fig. 1 Placental histopathology assessed by H&E staining. a Placental villous tissue from a control, uncomplicated 36 weeks pregnancy. b Placental villous tissue from a 35 weeks SCD pregnancy. Abnormal findings are pointed out in pannel b. Black arrow indicates syncytial

SELE) in comparison with the control group. In the HbSC group, 12 genes were differentially expressed: nine genes were upregulated (BCL6, CEBPB, CXCL1, CXCL2, IL1RAP, IL5, RIPK2, TLR3, and TLR9) and three genes were downregulated (CCL23, CRP, and CXCL6) in comparison to the control group. All of these genes are presented in Table 3. Four cytokines (CXCL1, CXCL2, CXCL6, and IL5) were equally differentially expressed in both the HbSS and HbSC groups. Gene array validation studies From the PCR arrays, 10 candidate genes were chosen among those differentially expressed for more detailed analysis: B cell lymphoma 6 (BCL6), complement component 3 (C3), chemokine ligand 1 (CXCL1), chemokine ligand 10 (CXCL10), chemokine ligand 2 (CXCL2), chemokine receptor 1 (CXCR1), fas ligand (FASLG), interleukin-1 receptor accessory protein (IL1RAP), prostaglandin-endoperoxide synthase 2 (PTGS2), and toll-like receptor 3 (TLR3), based on fold change (more than twofold difference) in the HbSS or HbSC groups as compared to the control group. For this study, samples from four additional women were included, two patients with HbSS and two patients with HbSC. In order to validate the PCR array findings for each candidate gene, qPCR was performed using individual RNA samples from the three study groups HbSS, HbSC, and control group (n = 7/group). ACTB, B2M, and GAPDH were used as housekeeping genes; their expressions did not differ among the three groups. The results derived from the array analysis were confirmed by significant fold changes in the genes BCL6 (4.49-fold, p = 0.01), C3 (−2.0-fold, p = 0.20), CXCL10 (−2.12-fold, p = 0.12), CXCR1 (−3.66-fold, p = 0.16), and IL1RAP (2.76-fold, p = 0.31) in the HbSS group (Fig. 2). For the HbSC group, the

knot, red arrow indicates dilation of villous capillaries, and circles indicate maternal sickled red blood cells in the intervillous space. Original magnification: x40 for a and b

Ann Hematol Table 3 mRNA expression profile of the inflammatory response in placentas of SCD groups Gene symbol HbSS vs. CON fold change HbSC vs. CON fold change BCL6

2.0

2.21

C3

−2.39

1.11

CCL11 CCL23

−2.1 1.14

−1.81 −3.04

CCL24

2.3

1.18

CCR3 CEBPB

2.65 1.72

1.29 2.2

CRP CXCL1

−1.35 4.41

−2.6 2.54

CXCL10

−2.11

−1.86

CXCL2

2.27

4.02

CXCL6 CXCR1 FASLG

−2.39 −2.52 −4.07

−2.57 1.0 −1.2

IL1RAP

1.8

2.18

IL5 KNG1 PTGS2

2.84 −2.2 −2.23

2.89 1.14 −1.89

RIPK2 SELE TLR3 TLR9

1.48 −2.2 1.87 1.05

2.0 −1.62 2.37 2.77

The values in italics showed significant differential expression (2-fold up or down change) in the SCD group when compared with the control group.CON control group

genes BCL6 (4.13-fold, p = 0.01), C3 (−2.0-fold, p = 0.31), CXCL1 (3.05-fold, p = 0.53), CXCL10 (−3.32-fold, p = 0.02), IL1RAP (4.33-fold, p = 0.02), and TLR3 (2.38-fold, p = 0.07) were validated (Fig. 3). The results of three genes (CXCL2, FASLG, and PTGS2) did not reach significant values for validation. All the results together are enough to validate the array data [20].

Discussion Prenatal care, clinical and laboratory follow-up and, probably, transfusions have had a major impact on reducing maternal and neonatal mortality in SCD. However, despite medical advances, pregnancy in women with SCD is still associated with many clinical and obstetric complications [21]. The mechanisms by which the disease can affect the development of the placenta are largely unknown. Inflammatory mediators not only play a role in innate immune response related to host-defense mechanisms, but they are also considered to be key components in reproductive processes, including the establishment and maintenance of pregnancy [22]. Inadequate

regulation of the inflammatory networks can lead to adverse pregnancy outcomes, such as IUGR, PE, miscarriage, and preterm labor [23, 24]. Vaso-occlusion is a characteristic event of SCD, which can lead to hypoxia as well as endothelial dysfunction, inflammation, and oxidative stress. We hypothesized that the occurrence of these events in the placental circulation can trigger an inflammatory response and lead to placental dysfunction. The results of this study demonstrated that the expression profile of a number of inflammatory response mediators in the placentas of pregnant women with sickle cell anemia (HbSS) showed significant differences in gene expression when compared to the controls. These genes were BCL6, CCL24, CCR3, CXCL1, C3, CCL21, CXCL6, CXCL10, CXCR1, ILIRAP, IL5, KNG1, and SELE. To the best of our knowledge, this is the first report concerning inflammatory gene expression in placental tissue of sickle cell anemia patients. These data suggest that there is increased inflammatory activity in the placenta of HbSS patients, since the upregulation of some of these genes may contribute to induce inflammatory mediator production or may indicate that an uncontrolled inflammatory response is occurring in this region. In fact, BCL6 is a transcriptional repressor associated with various cellular functions, including differentiation, survival, cell-cycle regulation, and DNA damage response [25]. Louwen et al. showed, for the first time, that preeclamptic placentas demonstrated a significant increase in the BCL6 protein that is mainly found in the nucleus of villous cytotrophoblasts [26]. However, the role of BCL6 in placentas still remains unknown. In macrophages, the BCL6 protein can bind to nuclear receptors and to their corepressors to reduce inflammation through cistromic antagonism of the toll-like receptor (TLR)–nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) subnetwork. BCL6-deficient mice are more prone to developing atherosclerosis, lethal neonatal pulmonary vasculitis, and myocarditis [27, 28]. In the present study, the increased gene expression of BCL6 in the placentas of the groups with SCD might be an indication that an uncontrolled inflammatory response is occurring. Thus, we speculate that BCL6 may act as a repressor of NFKB1 in placentas since NFKB1 is not differentially expressed (Supplementary Table 1). Although HbSC is a less severe condition, data in the literature indicate that, during pregnancy, patients with this condition may be subject to serious complications. The results of this study showed a probably very important inflammatory activity in the placenta of HbSC pregnant women when compared to controls. In the HbSC group, the following genes were differentially expressed, BCL6, CEBPB, CXCL1, IL1RAP, IL5, RIPK2, TLR3, TLR9, CCL23, CRP, and CXCL6, in comparison to the control group. The upregulated genes suggest an increase in the inflammatory response in this

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a

b

c

d

e

f

Fig. 2 Relative mRNA levels in HbSS group. mRNA expression was normalized to ACTB, B2M, and GAPDH mRNA expression levels. a CXCL10, b CXCR1, c IL1RAP, d BCL6, e C3, and f CXCL1. CON

control group. The lines in each plot show the mean ± standard deviations (SD), n = 7/study group, *p < 0.05, Mann-Whitney U test

a

b

c

d

e

f

Fig. 3 Relative mRNA levels in HbSC group. mRNA expression was normalized to ACTB, B2M, and GAPDH mRNA expression levels. a CXCL10, b IL1RAP, c TLR3, d BCL6, e C3, and f CXCL1. CON

control group. The lines in each plot show the mean ± standard deviations (SD), n = 7/study group, *p < 0.05, Mann-Whitney U test

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tissue. The downregulated genes also may contribute to placental dysfunction. For example, the CXCL10 gene which decreased expression upon interaction with extravillous trophoblast could indicate a decreased induction of the migration of these cells. Obstruction or limited blood perfusion leads to tissue injury and might evolve to necrosis [29]. Placentas from women with SCD have a higher incidence of necrosis in comparison to placentas from women that do not have SCD, suggesting the presence of vaso-occlusive events [9]. Decreased mRNA expression of CXCL10 detected in the HbSC group may indicate an adaptation to the necessity of neovascularization at the placenta, which is fundamental to supplying the fetus with adequate levels of oxygen. Furthermore, previous reports have shown that decidua natural killer cells have the ability to promote and direct human trophoblastic cell invasion by means of production of specific chemokines, such as CXCL10 [30]. A decrease in CXCL10 expression upon interaction with extravillous trophoblast could also suggest a decreased induction of the migration of these cells [30]. Taken together, these findings strongly suggest that SCD can affect placental physiology. The placenta might become an environment with cycling hypoxia, since vessel occlusion is common in the disease, and the alteration of several inflammatory genes indicate that there is an increased inflammatory response that could trigger cellular stress and cause disturbance in trophoblast function, which may lead to improper placental development. It is important to emphasize that even in the less severe HbSC disease, an altered gene expression was identified in the placenta. Although this study has some limitations, such as the small number of patients in each group and the slightly different gestational age in the groups, it is the first study that has assessed the inflammatory response in placentas from pregnant women with SCD. The results provide novel insights that can guide future research in order to understand how SCD can affect placental physiology. Most of the patients in our study were under regular prophylactic transfusion after 28 weeks aiming to reduce the amount of HbS below 30 % [14]. The data presented here indicate that this procedure may not be enough to completely abolish inflammation in the placenta of these patients. Our data suggest that the prophylactic transfusion may not be only important in HbSS and HbSC but possibly should be started earlier, during second or even first trimester of gestation. The knowledge of the pathways that might be altered in placentas from women with SCD could potentially contribute to a better understanding of the clinical complications and therapy of these patients in the future.

This study was supported by São Paulo Research Foundation (FAPESP) grant 2008/57441-0, grant 2014/00984-3 and grant 2014/ 01925-0 to MLC and by the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq). Compliance with ethical standards Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all patients for being included in the study. Conflict of interest The authors declare that they have no conflict of interest.

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