DOI: 10.5061/dryad.b8gtht790
title: Uterine scarring leads to adverse pregnant consequence through impairing the response of endometrium to steroids

description: Uterine surgical scarring is an increasing risk factor for adverse
 pregnant consequences that threaten fetal-maternal health. The detailed
 molecular features of scar implantation remain largely unknown. We aim to
 study the pathologic features of uterine surgical scarring and the
 mechanisms of compromised pregnancy outcomes of scar implantation. We
 generated a mouse model of uterine surgical scarring with a uterine
 incision penetrating the myometrium to endometrium to examine the
 pathologic changes and transcriptome profiles of uterine scarring at
 various post-surgery (PS) time points, as well as features of the
 feto-maternal interface during scar implantation. We found that uterine
 surgical scar recovery was consistently poor at PS3 until PS90, as shown
 by a reduced number of endometrial glands, inhibition of myometrial smooth
 muscle cell growth but excessive collagen fiber deposition, and massive
 leukocyte infiltration. Transcriptome annotation indicated significant
 chronic inflammation at the scarring site. At the peri-implantation and
 postimplantation stages, abnormal expression of various steroid-responsive
 genes at the scarring site was in parallel with lumen epithelial cell
 hyperplasia, inappropriate luminal closure, and disorientation of the
 implanted embryo, restricted stromal cell proliferation, and defective
 decidualization. High embryonic lethality (around 70%) before E10.5 was
 observed, and the small amount of survival embryos at E10.5
 exhibited restricted growth and aberrant placenta defects
 including overinvasion of trophoblast cells into the decidua and
 insufficient fetal blood vessel branching in the labyrinth. The findings
 indicate that chronic inflammation and compromised responses to steroids
 in uterine scar tissues are the pivotal molecular basis for adverse
 pregnancy consequences of scar implantation.
description: <b>Materials and
 methods</b>   <i>Mouse model of uterine
 surgical scar</i> Animal experiments were approved
 by the Experimental Animal Welfare Committee in the Institute of Zoology,
 Chinese Academy of Sciences. Female virgin CD1 mice (8 weeks) were
 purchased from Beijing SiPeiFu Animal Center (China) and housed at 25℃
 with 12/12 hours day–night light cycles. Uterine surgery was performed
 under sterile conditions. Briefly, the mice were anaesthetized by
 intraperitoneal injection of 2% 2,2,2-Tribromoethanol (100ul/10g body
 weight), and a low abdominal incision was made to expose uterine horns.
 Under stereoscope, a 1cm-in-length longitudinal incision at the
 mid-portion of anti-mesometrial side of uterine horn was made by using
 ophthalmic scissors. The incision penetrated through myometrium and
 endometrium of anti-mesometrial side, but not harmed tissue of mesometrial
 side (Supplementary Figure S1A (13)). The incision was closed with 8-0
 absorbable sutures using one-layer closure technique with interrupted lock
 stitches. To mark the incision, both ends of the incision edge were
 labeled with 8-0 non-absorbable sutures.<b> </b>The surgery
 was performed on one random horn, allowing the other horn as un-surgical
 control. The uterus was placed back into the abdominal cavity, and the
 abdominal skin was closed using wound clips. The Sham control animals had
 operations of low abdominal incision but no uterine incision.
 Following 3, 7, 20, 60 and
 90 days of surgery, the mice at diestrus stage (at least 10 mice for each
 time point) were sacrificed and the uterine tissues from the scarring site
 (1cm in length) or Sham control were collected under stereoscope for
 histological examinations and/or RNA measurement as described
 below. Following
 surgery, the mice were permitted to recover for 20, 60 or 90 days and
 mated to fertile male mice. The day on which virginal plug appeared was
 recorded as embryonic day 0.5 (E0.5). The pregnant mice were sacrificed at
 E3.5, E4.0, E5.0, E7.5, or E10.5 (at least 5 mice for each time point) to
 obtain uterine tissues from the scarring site (1cm in length) or Sham
 control for histological analysis and RNA measurement as described
 below.   <i>Preparation of paraffin
 section and frozen section</i> For paraffin section, the freshly
 collected tissues were fixed in 4% paraformaldehyde (PFA), followed by
 routine gradient dehydration with ethanol and embedding in paraffin wax.
 Paraffin sections of 5μm in thickness were prepared using paraffin
 microtome (Leica, RM2245). For frozen section, the fresh
 tissues were subjected to fixation in 4% PFA, dehydration with sucrose
 gradient, and embedding in O.C.T. compound (Sakura Finetek, Torrance).
 Frozen sections of 10 μm in thickness were prepared using frozen microtome
 (Leica, CM1950).   <i>Histological analysis
 and immunohistochemistry</i> Paraffin sections were routinely
 deparaffinized and rehydrated, and subjected to Haematoxylin-eosin
 (H&E) staining or Masson’s trichrome staining (Leagene, DC0033
 -50) according to the manufacturer’s instructions. For
 immunohistochemistry, the rehydrated paraffin sections were retrieved by
 citric acid, and incubated in 3%
 H<sub>2</sub>O<sub>2</sub> to inactivate
 endogenous peroxidase. The sections were incubated with primary antibodies
 followed by HRP-conjugated second antibody (Table S1 (13)). Antibodies
 from Abcam (Cambridge, Massachusetts) included α-SMA (ab5694, Abcam)(14),
 CK(ab181598, Abcam)(15), CD45 (ab10558, Abcam)(16), CD3 (ab11089,
 Abcam)(17), Ki67 (ab15580, Abcam)(18), and MUC1 (ab15481, Abcam)(19).
 Antibodies from Cell Signaling Technology (Danvers, Massachusetts)
 included E-cadherin (3195S, CST)(20), COX2 (12282S, CST)(21) and PR
 (8757S, CST)(22). Antibodies from ZSGB-Bio (Beijing) included Rat IgG-HRP
 (PV-9004, ZSGB-Bio)(23) and goat IgG-HRP (ZB-2306, ZSGB-Bio)(24) and
 Rabbit IgG-HRP (PV-9001, ZSGB-Bio)(25). Antibodies from Novus Biologicals
 (Denver, Colorado) included F4/80 (NB600-404, Novus)(26) and NKp46/NCR1
 (AF2225, RD)(27). Antibody from Santa Cruz Biotechnology (Dallas, Texas)
 was ERα (sc-7207, Santa Cruz)(28). Antibody from Sigma-Aldrich (Allentown,
 Pennsylvania) was Laminin (L9393, Sigma)(29). Followed by visualization
 with DAB (ZSGB-BIO, ZLI-9019) as substrate and counterstaining with
 hematoxylin. The results were recorded with light microscope (Olympus) and
 analyzed with Image-Pro Plus software (Media Cybernetics, USA).
 Immunofluorescent analysis
 was performed using frozen sections, with primary antibodies anti-Bmp2
 (bs-1012R, Bioss)(30) followed by FITC-labeled secondary antibody (A21206,
 Life technologies)(31) (Table S1 (13)) and 4',6-diamidino-2-pheny-
 lindole dihydrochloride (DAPI; Sigma, 28718-90-3) for nuclear staining.
 The results were recorded with confocal microscope (Zeiss, LSM780) and
 processed by ZEN 2012 software (Carl Zeiss Microscopy GmbH,
 Germany).   <i>Artificial
 decidualization</i><i> and in vitro
 decidualization</i> Artificial decidualization was
 carried out as previously described(32). In brief, following 20 days of
 surgery, the mice were mated with vasectomized male, and the day on which
 virginal plug appeared was recorded as pseudopregnancy day 1 (Day 1).
 Mechanical stimulation by intraluminal injections of sesame oil
 (20µl/mouse) in one uterine horn was performed in pseudopregnancy mice on
 Day 4. The mice were killed on Day 6, Day 7, or Day 8 to collect uterine
 tissues from the scarring site (1cm in length) or Sham control for further
 analysis. The
 stromal cells isolation and in vitro decidualization analysis were carried
 out as previously described(33) with slight
 modification.<b> </b>In brief, the collected uterine tissues
 were slit longitudinally and disaggregated with HBSS containing 6 mg/ml
 dispase (Gibco, 17105041), 25 mg/ml trypsin (Sigma, T8003), followed by
 further incubation in HBSS containing 0.5 mg/ml collagenase (Gibco,
 17018-029). The supernatants were filtered through 200 mesh gauze filter
 and centrifuged to collect stromal cells. The stromal cells were suspended
 in phenol-red free DMEM/F-12 (Invitrogen, 11039021) with 10% hormone-free
 fetal bovine serum (BSA; SenBeiJia, Sbjser02), and seeded in 6-well plate
 at 4×10<sup>5</sup> cells/well.<b>
 </b>Decidualization of stromal cells were induced by adding 1µM
 medroxyprogesterone 17-acetate (Sigma, M1629), 10 nM β-estradiol (Sigma,
 E2758), and 10 µM 8-bromoadenosine 3′,5′-cyclic monophosphate<b>
 </b>(8-Br-cAMP; Sigma, B5386), and the cultural media were changed
 every 2 days.   <i>In situ hybridization (ISH)</i> ISH was performed as previously described with slight modification(34). In brief, digoxin labelled riboprobe for mouse Bmp2 gene was synthesized according to the manufacturer’s instruction (Roche, 11175025910, Indianapolis, IN, USA). Frozen sections were hybridized with digoxin labelled riboprobe, followed by incubation with AP-conjugated anti-digoxin antibody (Roche, Indianapolis, IN, USA) and visualization with BCIP/NBT (Promega, Madison, WI, USA) as substrate. Fast red for nucleus staining was performed before mounting. The results were recorded with light microscope (Olympus) and analyzed with Image-Pro Plus software (Media Cybernetics, USA).   <i>Transcriptome profiling and bioinformatical analysis</i> At 3, 7, and 20 days following uterine surgery, uterine endometrium and myometrium were separately collected as described previously (35,36). Briefly, mice at diestrus stage were sacrificed and the uterine tissue from scar site or Sham control was collected and dissected under stereoscope. The endometrium specimen was obtained with rubber scraper, with the remaining part being myometrium (Figure.S1B (13)). Considering the limited amount of tissue at scaring site from one individual mouse, endometrial or myometrial tissues from 10 animals in each study group were mixed as one sample.  Total RNA was extracted using Total RNA kit (Omega Bio-Tek, Norcross, GA, USA) and treated with RNase-free DNase (TaKaRa, Otsu, Japan). An amount of 1 μg RNA per sample was subjected to sequencing library generation using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s instruction. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq Xten platform and paired-end reads were generated. Transcriptome sequencing data were quantitated at a protein-coding mRNA level using the RNA-seq quantitation pipeline in SeqMonk software. Differential expression genes were revealed by the DESeq R package (R Foundation for Statistical Computing, Vienna, Austria; http://www.r-project.org/). The differentially expressed genes were determined by statistical analysis of FDR﹤0.05 and fold change ≥2. Heatmaps and KEGG were generated using Omicshare software (https://www.omicshare.com/tools/Home/Soft/ pathwaygsea). GO enrichment were analyzed using the AmiGO2 database (http://amigo.geneontology.org/amigo) and mapped with Cytoscape (http://www.cyto- scape.org/) to illustrate complex network regulation.   <i>Real time quantitative PCR analysis</i> Real time PCR was performed as described(37). Briefly, total RNA was isolated from tissues or cells with Trizol reagent following the manufacturer’s protocol (Ambion, 15596-018). A total of 2 µg RNA was used to synthesize cDNA, and real time PCR analysis was carried out by using Roche LightCycler 480 system. The primers were used in this study listed in Table S2 (13).   <i>Statistical analysis</i> Statistical analyses were performed with GraphPad Prism 6.0 software (GraphPad Software Inc, USA). Data were expressed as Mean±S.E.M. based on at least three independently repeated experiments. Statistical comparison between groups was performed using non-paired t-test, and P-values of less than 0.05 were considered statistically significant.