Identification of genes differentially expressed in menstrual breakdown and repair

Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regener- ation and repair. It is a highly regulated process comprising a series of interdependent molecular and cellular events of an in flammatory nature ( Evans and Salamonsen, 2012 ). In countries with ready access to contraception, women may experience over 400 episodes of men- struation during their reproductive life. In contrast, in areas where women are less able to regulate their own fertility, most women will be amenorrheic for a large proportion of their lives due to later puberty, multiple pregnancies and ensuing periods of lactational amen- orrhoea ( Jabbour et al., 2006). A clearer understanding of the regulation of normal endometrial function, including normal menstruation, is important to better under- stand the mechanisms responsible for endometrial dysfunction includ- ing abnormal uterine bleeding, dysmenorrhoea as well as early pregnancy loss and infertility. Several studies have investigated the molecular mechanisms of normal menstruation, particularly the initi- ation of menstruation. These studies have used progesterone antago- nists and/or physiological progesterone withdrawal in vivo ( Catalano et al., 2007) and in vitro (Critchley et al., 2003; Evans and Salamonsen, 2014) and in mouse models of decidualization and menstruation (Cheng et al. , 2007 ; Wang et al. , 2013 ; Xu et al. , 2013 ). Molecular pathways identi fied include those associated with NF-kappa(k) B, prostaglandins (PGs), chemokines, Wnt signalling, matrix metallo- proteases (MMPs, Critchley et al. , 2003; Catalano et al. , 2007; Cheng et al., 2007; Wang et al., 2013; Xu et al., 2013; Evans and Salamonsen, 2014) and haemostasis (the balance between coagulation and fibrin- olysis) ( Livingstone and Fraser, 2002 ). This knowledge has been exploited clinically. For example, PG synthesis inhibitors (mefenamic acid; Cameron et al. 1990) and anti- fibrinolytic therapy with tranex- amic acid ( Gleeson et al. 1993) have shown ef ficacy in the treatment of heavy menstrual bleeding (HMB). However, the molecular pathways underlying such pathologies are still to be fully elucidated and new therapeutic targets are necessary. There is limited information on the molecular mechanisms at play during the course of menstruation due to the dif ficulty of collecting timed endometrial samples during menstrual bleeding. Gaide Chevronnay et al. (2009) compared global gene expression pro files in glands and stroma microdissected from menstrual endometrium. However, this study only included three samples of menstrual endo- metrium. In this study, we collected 36 carefully characterized endo- metrial samples during menstruation. We used gene array analysis to investigate temporal changes in gene expression in human endometrium from Days 2, 3 and 4 of menstruation (where Day 1 is the first day of bleeding). We hypothesized that the changing molecu- lar pro file of the endometrium during menstruation would correlate with the histological pro file of menstruation previously described ( Garry et al. , 2009 ); Day 2 endometrium would be predominantly associated with genes related to breakdown, Day 4 endometrium, predominantly with genes related to repair and Day 3 endometrium with genes common to both. By investigating the changing molecular profile of the endometrium over Days 2, 3 and 4 of menstruation, we aimed to identify genes and pathways that would provide new insights into the menstrual process. The temporal molecular pro file of menstruation identi fied in this study supports the overall view that on Day 2 of menstruation, pro- cesses related to in flammation are up-regulated, whereas on Day 4 of menstruation, endometrial repair and regeneration are actively occurring.

Patient recruitment Premenopausal women were recruited from outpatient clinics of The Royal Women ’s Hospital, a public tertiary women ’s hospital, and from local advertising for a study investigating endometrial gene expression in women with/without self-reported HMB ( Girling et al. , 2016). Participant details are summarized in Table I. Retrospective menstrual histories (frequency, duration, regularity and volume; Munro et al., 2012) were collected and all subjects completed a 3- month prospective menstrual diary. For this study, subjects were included irrespective of comorbidities ( fibroids, adenomyosis and/or ovarian cysts; Table I), bleeding status (the presence/absence of HMB) and menstrual characteristics (frequency, duration, regularity and volume). Participants were excluded if they had known endocrine or haematological abnormal- ities that may cause HMB or were using sex steroids or any medication likely to reduce menstrual blood loss or interfere with vascular stability or coagulation. Endometrial biopsies were obtained using a pipelle suction curette (Pipelle, Cornier, France) on Days 2, 3 and 4 of menstruation (where Day 1 is the first day of menstrual bleeding). Bleeding but not spotting days, as denoted in the menstrual diary, was used to determine day of cycle (Table I). Endometrial biopsies were stored in RNAlater (Ambion, Life Technologies, Carlsbad, CA, USA) overnight before being frozen and stored at −80°C. A total of 36 samples were collected and arrayed. Samples were excluded from analysis if the day of menstruation could not be ascertained from the menstrual diary. Of the 24 samples remaining ( n = 9, Day 2; n = 9, Day 3; n = 6, Day 4), 22 were from women with regular menstrual 899Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 cycles (FIGO de finition, cycle to cycle variation of between 2 and 20 days (Munro et al. , 2012; Table I), whereas 2 samples were from women with irregular menstrual cycles. A separate cohort for validation could not be obtained due to logistical difficulties in obtaining samples during menstrual bleeding. An additional 10 samples were included for PCR validation. These included one Day 2, two Day 3 and seven Day 4 samples (Table I). Ethical approval Ethical approval was obtained from The Royal Women ’s Hospital Human Research Ethics Committee (Project 11/13) and informed consent was obtained from all subjects. Gene array analysis Total RNA was extracted using Trizol (Invitrogen, Life Technologies, Carlsbad, CA, USA) and the Ambion PureLink mini kit (Ambion, Life Technologies, Carlsbad, CA, USA) according to manufacturer ’s instruc- tions. RNA was quanti fied using the Nanodrop ND1000 spectrophotom- eter (Thermo Scientific, Waltham, MA, USA) and stored at -80 ˚C. Three micrograms of RNA were further analysed for quality and hybri- dized to Illumina Human HT12 v4 arrays at the Australian Genome Research Facility (AGRF, Victoria, Australia). Data were then analysed using the ‘Remove Unwanted Variation-inverse (RUV-inv) ’ method ( Gagnon-Bartsch and Speed, 2012 ; Gagnon-Bartsch et al. , 2013 ; Girling et al. , 2016 ). The re-annotation functions of the Illumina Human v4.db Bioconductor R package (Barbosa-Morais et al., 2010) were used to remap the probes according to the current knowledge of the human genome (hg19). Genes were de fined as differentially expressed if the Benjamini – Hochberg corrected P-value was 1 (fold change >2). Ingenuity pathway analysis Gene lists derived from arrays were analysed using Qiagen ’s Ingenuity Pathway Analysis (IPA ®,Q i a g e nR e d w o o dC i t y ,www.qiagen.com/ ingenuity). Canonical pathways were de fined as signi ficant if the Benjamini–Hochberg corrected P-value from right-tailed Fisher exact tests was <0.01. IPA Upstream Regulator Analysis identi fied potential upstream transcriptional regulators. Overlap P-values ( P < 0.05 was con- sidered signi ficant) represented the signi ficance of the overlap between genes in our gene lists and those genes previously identi fied as regulated by a particular factor. Gene cluster analysis Gene lists were also examined using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources v6.7 ( http://david.abcc.ncifcrf.gov/home.jsp)( Huang da et al. , 2009 ). As DAVID analysis does not differentiate between up- vs down-regulated genes, these gene lists were submitted separately. We used the functional annotation cluster (FAC) tool, which combines functionally similar annota- tions derived from various databases into a single cluster. We selected annotations related to Gene Ontology Biological Process Terms, to pro- vide insight into the biological processes associated with our gene lists. For each cluster, DAVID provides an Enrichment Score (geometric mean expressed as –(log [base10]) of the mean of the P-values from each individual annotations within a cluster) and individual P-values or EASE scores calcu- lated using a modi fied Fisher exact test. An EASE score/ P-value (with Benjamini–Hochberg correction) of 0.01 was considered signi ficant; this equated to an enrichment score of 2. Classi fication stringency was set at medium. Quantitative PCR cDNA was generated from 1 ug of RNA using the GoScript Reverse Transcription System (Promega, Alexandria, NSW, Australia) according to manufacturer’s instructions. An RNA spike was also added to the reverse transcription mix ( Fu et al. , 2008 ; Craythorn et al. , 2009 ; Winnall et al. , 2009) to control for variations in cDNA transcription ef ficiency. Real-time PCR was performed using commercially validated TaqMan ® Gene Expression Assays (Applied Biosystems, Life Technologies, Carlsbad, CA, USA, Supplementary Table I) and the 7500 ABI (Applied Biosystems) detection system. Samples were run in duplicate along with appropriate controls (i.e. no template, no RT). Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60°C for 1 min. Data were analysed using the comparative Ct method (2 −ΔΔCT). Several housekeeping genes were examined and compared during the menstrual period as described in Girling et al. (2016). Data were normal- ized to the housekeeping gene RPL13A, which exhibited the lowest stand- ard deviation during the menstrual period, and represented as median fold change relative to the Day 2 group. Statistical analysis was performed using a Kruskal –Wallis test followed by a Dunn ’s post hoc test. P < 0.05 was ............................................................................................................................... .............................................................. Table I Summary of patient characteristics used in array analysis and PCR. Group n Median age [range] Bleeding type Comorbidities Day 2 9 38 [30 –44] n = 4 HMB n = 1 adenomyosis n = 1 ovarian cyst Extras for PCR 1 26 n = 1 HMB Day 3 9 40 [23 –45] n = 6 HMB n = 2 fibroids n = 1 polycystic ovary Extras for PCR 2 29,37 n = 1 HMB Day 4 6 41 [36 –45] n = 1 bleeding type unknown, n = 4 HMB n = 2 fibroid, n = 1 polyp Extras for PCR 7 38 [24 –42] n = 4 HMB n = 1 adenomyosis n = 1 fibroid HMB, heavy menstrual bleeding (self-reported). 900 Paiva et al. Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 considered signi ficant. Statistical analyses were conducted using SPSS for Windows v. 21.0 (2012, Armonk, NY, IBM Corp.)

Differential gene expression Overall, 1753 genes were differentially expressed in one or more com- parisons (P 1) (Figure 1). The top 20 most signi fi- cantly up- or down-regulated genes for each comparison are provided in Table II. The largest number of differentially expressed genes (1176) was observed between Days 2 and 4. Of these, 738 genes were up-regulated, whereas 438 genes were down-regulated in Day 2 com- pared to Day 4. A number of known genes in the context of menstru- ation including fibrinogen A (FGA), fibrinogen B (FGB), complement component 4 binding protein, alpha (C4BPA), nitric oxide synthase 2 (NOS2), chemokine CXC ligand 6 or granulocyte chemotactic protein-2 (CXCL6), colony stimulating factor 3 (CSF3) and solute car- rier family 18 (vesicular monoamine transporter), member 2 (SLC18A2) were identi fied. Genes not previously associated in the context of menstruation were also identi fied (Table III). These included lipopolysaccharide binding protein (LBP) and serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), which were among the top up-regulated genes on Day 2 of menstruation. Vav guanine nucleotide exchange factor 3 (VAV3), major histocompatibility complex, class II, DR beta 1 (HLA-DRB1) and olfactomedin 4 (OLFM4) were among the top up-regulated genes on Day 3 of menstruation. Whereas glutathione-S-transferase mu 1 and 2 (GSTM1 and GSTM2), V-set domain containing T-cell activation inhibi- tor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/ PAF), trefoil factor 3 (TFF3) and laminin alpha 2 (LAMA2) were among the top up-regulated genes on Day 4 of menstruation (Table III). There were 738 genes differentially expressed on Day 2 vs Day 3 (380 up-regulated, 358 down-regulated) and 559 genes on Day 3 vs Day 4 (210 up-regulated, 349 down-regulated). There were 357 (309 + 48) genes with signi ficantly different expression on Day 2 vs Days 3 and 4, 111 (63 + 48) genes on Day 3 vs Days 2 and 4 and 300 (252 + 48) genes on Day 4 vs Days 2 and 3. Forty-eight genes were significantly different in all three comparisons. Ingenuity pathway analysis Day 2 vs Day 3 Significant canonical pathways identi fied using IPA related to the immune and inflammatory response (retinoic and liver X receptor acti- vation), immune cell trafficking (granulocyte and agranulocyte adhesion and diapedesis), extracellular remodelling (inhibition of MMPs) and the coagulation cascade (extrinsic prothrombin activation pathway) (Table IV, Supplementary Table II). The top five upstream regulators (Table V, Supplementary Table III) included cytokines (interleukin-1 beta; IL1B, IL6) and other regulators (v-rel avian reticuloendotheliosis viral oncogene homologue A; RELA, protein kinase D1; PRKD1) also associated with inflammation. Day 2 vs Day 4 IPA analysis identi fied pathways related to the immune and in flamma- tory response (pathogenesis of multiple sclerosis), immune cell traf- ficking (granulocyte and agranulocyte adhesion and diapedesis), the coagulation cascade and glutathione-mediated detoxi fication (Table IV, Supplementary Table II). The top five upstream regulators (Table V, Supplementary Table III) included those associated with in flammation (interleukin-1 beta; IL1B) and growth and cell survival (erb-b2 receptor tyrosine kinase 2; ERBB2, RAB, member RAS oncogene family-like 6; RABL6, transform- ing growth factor beta; TGFB1). Day 3 vs Day 4 Significant canonical pathways identi fied using IPA included glutathione-mediated detoxi fication, the complement and coagulation cascade and immune cell traf ficking (granulocyte adhesion and diape- desis) (Table IV, Supplementary Table II). The top five upstream regu- lators (Table V, Supplementary Table III) included those associated with inflammation (oncostatin M receptor; OSMR, triggering receptor expressed on myeloid cells 1; TREM1 and ILB). Functional cluster analysis Of the genes up-regulated on Day 2 of menstruation, we identi fied four signi ficant clusters, using DAVID analysis (Table VI, Supplementary Table IVA). These included genes related to the posi- tive regulation of cell migration, immune and in flammatory response and the coagulation cascade. These clusters were speci fically asso- ciated with genes up-regulated on Day 2 compared to Day 4. Of the genes up-regulated on Day 3 of menstruation, we identi fied two signi ficant clusters associated with the cell cycle and chemotaxis. These clusters were speci fically associated with genes up-regulated on Day 3 compared to Day 2. We also identi fied one signi ficant cluster related to the negative regulation of cell growth associated with genes up-regulated on Day 3 compared to Day 4 (Table VI, Supplementary Table IVB). Of the genes up-regulated on Day 4 of menstruation, we identi fied six signi ficant clusters (Table VI, Supplementary Table IVC). These 48 318 567 196 309252 63 Day 4 vs Day 3 (559 genes) Day 4 vs Day 2 (1176 genes) Day 3 vs Day 2 (738 genes) Figure 1 Venn diagram illustrating the distribution of differentially expressed genes in menstrual-phase endometrial biopsies from women from Day 2 ( n = 9), Day 3 ( n = 9) and Day 4 ( n = 6) of menstruation. 901Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 ............................................................................................................................... .............................................................. Table II Top 20 genes with the highest fold change (up- and down-regulated) in comparisons between Days 2, 3 and 4 of menstruation. Gene symbol Gene name Fold change Adj. P-value Up-regulated on Day 2 vs Days 3 and 4 Day 2 vs Day 3 GSTT1 Glutathione-S-transferase theta 1 20.3 4.00E-08 XYLT1 Xylosyltransferase I 11.2 6.10E-08 IL17C Interleukin-17 C 5.5 3.90E-09 SCGB2A2 Secretoglobin, family 2A, member 2 4.6 3.30E-06 LBP Lipopolysaccharide binding protein 4.3 1.50E-06 CYP2C9 Cytochrome P450 family 2 subfamily C member 9 4.2 2.30E-08 SERPINB4 Serpin peptidase inhibitor, clade B (ovalbumin), member 4 4.1 6.30E-10 C4BPA Complement component 4 binding protein, alpha 4.0 1.10E-08 C2CD2 C2 calcium-dependent domain containing 2 3.8 8.10E-08 CSF3 Colony stimulating factor 3 3.7 4.40E-08 AGTPBP1 ATP/GTP binding protein 1 3.6 3.90E-08 TMEM233 Transmembrane protein 233 3.6 7.30E-07 SLC18A2 Solute carrier family 18 (vesicular monoamine transporter), member 2 3.6 7.80E-07 DEFB4A Defensin beta 4A 3.5 4.50E-07 FOLR3 Folate receptor 3 3.5 3.90E-05 VCAM1 Vascular cell adhesion molecule 1 3.5 1.10E-08 MROH5 (FLJ43860) Maestro heat-like repeat family member 5 3.4 1.60E-08 FGG Fibrinogen G 3.4 1.20E-07 LIMS3 LIM and senescent cell antigen-like domains 3 3.3 1.40E-07 TCN1 Transcobalamin 1 3.3 1.20E-07 Day 2 vs Day 4 FGA Fibrinogen A 27.1 2.60E-14 FGB Fibrinogen B 19.9 1.50E-10 LBP Lipopolysaccharide binding protein 11.4 5.10E-09 SERPINB4 Serpin peptidase inhibitor, clade B (ovalbumin), member 4 10.2 9.90E-13 PCSK1 Proprotein convertase subtilisin/kexin type 1 8.3 6.50E-12 TCN1 Transcobalamin 1 7.9 1.40E-10 PRKAR1A Protein kinase, cAMP-dependent, regulatory subunit type I alpha 7.6 2.50E-07 HSD17B2 Hydroxysteroid (17-beta) dehydrogenase 2 6.9 1.70E-10 IL17C Interleukin-17C 5.5 8.60E-09 SLC18A2 Solute carrier family 18 (vesicular monoamine transporter), member 2 5.5 5.40E-08 C4BPA Complement component 4 binding protein, alpha 5.5 1.00E-09 CALB2 Calbindin 2 5.1 9.10E-08 EN2 Engrailed homeobox 2 5.0 4.40E-10 SERPINB3 Serpin peptidase inhibitor, clade B (ovalbumin), member 3 4.7 3.50E-09 NOS2 Nitric oxide synthase 2 4.5 5.40E-08 GPX2 Glutathione peroxidase 2 4.3 6.70E-07 CXCL6 Chemokine (C-X-C motif) ligand 6 4.3 8.90E-08 PIP Prolactin-induced protein 4.2 7.70E-08 SPINK1 Serine peptidase inhibitor, Kazal type 1 4.1 5.70E-05 CSF3 Colony stimulating factor 3 4.0 2.80E-08 Up-regulated on Day 3 vs Days 2 and 4 Day 3 vs Day 2 VAV3 Vav guanine nucleotide exchange factor 3 12.0 5.90E-06 Continued 902 Paiva et al. Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 ............................................................................................................................... .............................................................. Table II Continued Gene symbol Gene name Fold change Adj. P-value ODF2L Outer dense fibre of sperm tails 2-like 9.0 4.40E-10 HLA-DRB1 Major histocompatibility complex, class II, DR beta 1 8.0 6.00E-04 SNHG8 Small nucleolar RNA host gene 8 7.0 1.90E-08 OLFM4 Olfactomedin 4 5.3 3.80E-06 HLA-DRB6 Major histocompatibility complex, class II, DR beta 6 (pseudogene) 5.1 6.90E-04 RSAD2 Radical S-adenosyl methionine domain containing 2 5.0 1.90E-07 ZBED2 Zinc finger, BED-type containing 2 5.0 2.20E-09 BBPIFB1 (C20orf114) BPI fold containing family B member 1 4.9 2.10E-03 HLA-DRB1 Major histocompatibility complex, class II, DR beta 1 4.7 8.30E-03 TFF3 Trefoil factor 3 4.6 8.80E-04 GBP5 Guanylate binding protein 5 4.4 1.30E-07 NPR3 Natriuretic peptide receptor 3 4.0 4.60E-08 NMU Neuromedin U 4.0 1.30E-09 TAP2 Transporter 2, ATP-binding cassette, subfamily B (MDR/TAP) 3.9 5.10E-09 APOBEC3A Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A 3.8 4.30E-08 CLECL1 C-type lectin like 1 3.7 2.90E-08 MAP2 Microtubule associated protein-2 3.7 2.30E-09 LOC100132288 3.6 5.00E-09 GSTM1 Glutathione-S-transferase mu 1 3.6 2.50E-03 Day 3 vs Day 4 FGB Fibrinogen B 15.7 1.80E-10 FGA Fibrinogen A 8.8 4.90E-12 HLA-DRB1 Major histocompatibility complex, class II, DR beta 1 7.7 6.70E-04 VAV3 Vav guanine nucleotide exchange factor 3 6.0 2.00E-04 PRKAR1A Protein kinase, cAMP-dependent, regulatory subunit type I alpha 5.1 2.90E-03 CALB2 Calbindin 2 5.1 2.00E-06 HIST1H4E Histone cluster 1, H4e 5.0 3.70E-08 HLA-A Major histocompatibility complex, class I, A 4.6 3.40E-09 SLN Sarcolipin 4.3 3.40E-03 RSAD2 Radical S-adenosyl methionine domain containing 2 4.2 5.60E-09 OLFM4 Olfactomedin 4 4.2 6.70E-07 FOXD1 Forkhead box D1 4.0 2.40E-05 PCDHB2 Protocadherin beta 2 3.8 8.90E-08 PCSK1 Proprotein convertase subtilisin/kexin type 1 3.8 2.30E-06 HIST1H2BG Histone cluster 1, H2b 3.7 1.00E-08 SERPINB3 Serpin peptidase inhibitor, clade B (ovalbumin), member 3 3.7 1.40E-09 IL1RL1 Interleukin-1 receptor-like 1 3.6 1.10E-08 IGFBP1 Insulin like growth factor binding protein 1 3.4 3.20E-05 AI458759 3.4 1.80E-05 SLC16A6 Solute carrier family 16, member 6 3.3 6.00E-07 Up-regulated on Day 4 vs Days 2 and 3 Day 4 vs Day 2 GSTM1 Glutathione-S-transferase mu 1 12.1 9.60E-08 ODF2L Outer dense fibre of sperm tails 2-like 10.3 3.30E-10 GRAMD2 GRAM domain containing 2 7.7 1.10E-08 CFAP77 Cilia and flagella associated protein 77 7.0 9.00E-10 TUBA4B Tubulin alpha 4b 6.6 1.20E-08 Continued 903Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 included multiple clusters associated with cell division, mitosis and cell migration. These clusters were speci fically associated with genes up- regulated on Day 4 compared to Day 2. We also identi fied two signifi- cant clusters related to chemotaxis and complement activation asso- ciated with genes up-regulated on Day 4 compared to Day 3 (Table VI, Supplementary Table IVC). Quantitative PCR To validate the microarray data, the expression of eight candidate genes was examined by qPCR (Figure 2). These included genes that were both up- and down-regulated. These genes were in agreement with the microarray data direction of change. Of these, six were signi fi- cantly altered (Figure 2). These included FGA ( P = 0.008), FGB (P = 0.04), C4BPA ( P = 0.04) and LBP ( P = 0.0001), which were sig- nificantly up-regulated on Day 2 vs Day 4. Whereas VTCN1 (P = 0.004) and GSTM2 ( P = 0.036) were significantly up-regulated on Day 4 vs Day 2. SERPINB4 expression was higher on Day 2 vs Day 4, whereas TFF3 expression was higher on Day 4 vs Day 2. However, these differences were not statistically signi ficant. The expression of GSTM1 was vari- able, being detectable in only 2/9 Day 2 samples, 7/11 Day 3 samples and 6/13 Day 4 samples (data not shown). ............................................................................................................................... .............................................................. Table II Continued Gene symbol Gene name Fold change Adj. P-value UBE2C Ubiquitin conjugating enzyme E2C 6.5 3.40E-09 CR591103 6.4 5.10E-08 VTCN1 V-set domain containing T-cell activation inhibitor 1 5.9 4.90E-09 KIAA0101 (PAF) Proliferating cell nuclear antigen-associated factor 5.9 4.00E-12 CFAP43 (WDR96) Cilia and flagella associated protein 43 5.7 3.50E-08 LAMA2 Laminin subunit alpha 2 5.6 1.00E-09 GSTM2 Glutathione-S-transferase mu 2 5.5 1.30E-08 ARMC3 Armadillo repeat containing 3 5.4 2.10E-07 ADGRG7 (GPR128) Adhesion G Protein-Coupled Receptor G7 5.3 2.00E-04 ATP1A2 ATPase, Na +/K+ transporting, alpha 2 polypeptide 5.2 2.20E-07 CAPSL Calcyphosine-like 5.2 2.50E-08 CDHR4 Cadherin-related family member 4 5.1 9.70E-08 MORN5 MORN repeat containing 5 5.1 7.60E-09 TFF3 Trefoil factor 3 5.1 7.70E-04 ALDH1L1 Aldehyde dehydrogenase 1 family member L1 5.1 9.10E-09 Day 4 vs Day 3 SCGB1D2 Secretoglobin, family 1D, member 2 6.0 6.20E-08 NAPSB Napsin B aspartic peptidase, pseudogene 5.5 1.20E-08 GSTT1 Glutathione-S-transferase theta 1 5.4 3.80E-05 TUBA4B Tubulin alpha 4b 4.9 5.50E-08 C1orf194 Chromosome 1 open reading frame 194 4.8 8.10E-08 GSTM1 Glutathione-S-transferase mu 1 4.7 1.60E-05 CHGA Chromogranin A 4.6 1.90E-05 GPR84 G protein-coupled receptor 84 4.5 9.50E-07 PAGE4 PAGE family member 4 4.4 9.70E-08 LIMS3 LIM and senescent cell antigen-like domains 3 4.4 4.90E-09 HBD Haemoglobin subunit delta 4.3 3.50E-05 CNTN1 Contactin 1 4.2 1.50E-09 HBM Haemoglobin subunit mu 4.2 1.80E-05 DPY19L2 dpy-19-like 2 ( Caenorhabditis elegans) 4.0 8.70E-09 ATP1A2 ATPase, Na +/K+ transporting, alpha 2 polypeptide 4.0 1.00E-06 FAM118A Family with sequence similarity 118 member A 4.0 1.00E-07 LINC00969 (AK128346) Long intergenic non-protein coding RNA 969 4.0 5.30E-09 MSMB Microseminoprotein, beta 3.8 3.60E-05 KIR2DS5 Killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 5 3.8 3.70E-06 HBG2 Haemoglobin subunit gamma 2 3.8 5.50E-04 904 Paiva et al. Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019

In this study, we provide the first comparative global gene expression profile data of human endometrium on Days 2, 3 and 4 of menstru- ation. For the first time, we have demonstrated in detail the temporal, molecular response of the endometrium during menstrual bleeding in vivo. The largest number of differentially expressed genes ( n = 1176) was between Days 2 and 4 of menstruation, demonstrating distinct molecular profiles on these days. We found changes in the expression of several genes not previously associated with menstruation. Other gene clusters and pathways identi fied were consistent with known mechanisms of menstruation including in flammation, immune cell traf- ficking, extracellular remodelling, cell cycle regulation and coagulation. From the molecular pro file of menstruation delineated in this study, our findings suggest that on Day 2 of menstruation (early-menstru- ation), processes associated with in flammation are prominent and by Day 4 of menstruation (late-menstruation) in flammation is reduced and endometrial repair processes take precedence. Pathways and upstream regulators related to the immune and inflammatory response including immune cell traf ficking were enriched in all day-by-day comparisons. This is consistent with the in flammatory nature of menstruation ( Evans and Salamonsen, 2012 ; Maybin and Critchley, 2015 ). Progesterone withdrawal leads to an in flammatory cascade of events characterized by the onset of oxidative stress, NFkB activation, PG synthesis, leucocyte recruitment and the activation of MMPs ( Maybin and Critchley, 2015). ............................................................................................................................... .............................................................. Table III Genes with known and novel biological roles during menstruation. Factor of interest Biological role Biological process associated with menstruation

Genes up-regulated on Day 2 FGA Coagulation Regeneration and repair Davies and Kadir (2012) FGB Coagulation Regeneration and repair Davies and Kadir (2012) C4BPA Complement system Tissue breakdown Blom et al. (2004) NOS2 Vasodilation and tissue breakdown via nitrous oxide production Tissue breakdown Tschugguel et al. (1999), Chwalisz and Garfield (2000) CXCL6 Neutrophil recruitment In flammation and tissue breakdown Wuyts et al. (1997) (GCP-2) CSF3 Neutrophil recruitment In flammation and tissue breakdown Chiang et al. (2013) SLC18A2 Vasodilation via the uptake and release of monoamines Tissue breakdown and bleeding Bottalico et al. (2003) LBP* Activates NFKB signalling In flammation and tissue breakdown Fitzgerald et al. (2003) SERPINB3/4* Activates NFKB signalling In flammation and tissue breakdown Catanzaro et al. (2014) IL17C* Pro-in flammatory In flammation and tissue breakdown Johnston et al. (2013) XYLT1* Enzyme involved in glycosaminoglycan synthesis during cartilage repair Regeneration and repair Venkatesan et al. (2009) Genes up-regulated on Day 3 VAV3* Wound healing Regeneration and repair Sindrilaru et al. (2009) OLFM4* Cell cycle control, apoptosis, cell adhesion, cell migration Regeneration and repair Zhang et al. (2004) HLA-DRB1* Wound healing Regeneration and repair Brown and Bayat (2009) Genes up-regulated on Day 4 GSTM1* ROS deactivation and cell proliferation/ apoptosis Regeneration and repair Kalinina et al. (2014) GSTM2* ROS deactivation and cell proliferation/ apoptosis Regeneration and repair Kalinina et al. (2014) VTCN1* Regulation of the immune response, cell proliferation and migration Regeneration and repair Prasad et al. (2003), Choi et al. (2003), Gao et al. (2015) KIAA0101 (PAF)* DNA synthesis, cell cycle progression Regeneration and repair Yu et al. (2001), Cheng et al. (2013), Zhu et al. (2013), Fan et al. (2015) TFF3* Mucosal repair, cell migration, inhibition of proliferation and apoptosis Regeneration and repair Thim and May (2005), Hoffman (2005), Taupin and Podolsky (2003) LAMA2* Muscle regeneration and repair Regeneration and repair Kuang et al. (1999) SPINK1* Mucousal integrity and repair Regeneration and repair Konturek et al. (1998), Marchbank et al. (1998) *Genes with novel potential roles during menstruation. 905Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 The observation that genes associated with the immune and in flam- matory response were up-regulated on Day 2 compared to Day 4 sug- gests that, on Day 2 (early-menstruation), in flammatory processes signal the onset of tissue breakdown. Consistent with this hypothesis, NOS2, which is induced by the in flammatory milieu post- progesterone withdrawal ( Tschugguel et al., 1999) and stimulates NO production which plays roles in tissue breakdown and vasodilation (Chwalisz and Gar field, 2000), is up-regulated on Day 2. Furthermore, we saw similar up-regulation in the chemokines CXCL6 and CSF3, which stimulate the migration of neutrophils ( Wuyts et al. , 1997 ; Chiang et al., 2013), key mediators of the in flammatory response and the predominant leucocyte population present at the onset of men- struation ( Salamonsen and Lathbury, 2000 ). SLC18A2, the vesicular monoamine transporter, which regulates the uptake and release of monoamines that in fluence vasodilation during menstruation, is also up-regulated ( Bottalico et al., 2003). We are the first to report that the in flammatory genes LBP, IL17C and SERPINB3/4 are associated with menstruation. These genes were among the most up-regulated on Day 2 vs Day 4 of menstruation. LBP (involved in the acute phase response) and SERPINB3/B4 (a member of the serpin family of endogenous serine/cysteine proteinase inhibi- tors) have been shown to activate NFkB pathways by augmenting lipo- polysaccharide signalling through TLR4- ( Fitzgerald et al., 2003) and RAS-mediated signalling, respectively ( Catanzaro et al. , 2014 ). Similarly, a pro-in flammatory pathogenic role for IL17C in association with psoriasiform skin in flammation has been reported ( Johnston et al., 2013). An excessive or prolonged in flammatory response is pos- tulated to lead to excessive tissue damage and may result in HMB (Maybin and Critchley, 2015 ). In support of this notion, PG synthesis inhibitors (e.g. mefenamic acid; Cameron et al. , 1990 ) are a popular treatment for HMB. Our findings point to novel genes associated with inflammation during menstruation. Further investigation of these in the endometrium is warranted. Pathways related to the coagulation and complement cascade fea- tured in all three day-by-day comparisons. FGA and FGB were the top up-regulated genes on Day 2 compared to Days 3 and 4. Fibrinogen biosynthesis is stimulated by acute phase reactants that accompany both acute and chronic in flammatory states ( Ross and Beck, 2014 ). Cessation of menstruation relies on an intact endometrial coagulation system culminating in the formation of fibrin from fibrinogen to achieve haemostasis. Fibrinolysis is key in the regulation of haemostasis ( Davies and Kadir, 2012 ). Disorders that interfere with systemic haemostasis have an impact on menstruation. Von Willebrand disease is the most common of these disorders with a prevalence of 13% in women with HMB ( Shankar et al. , 2004 ). There is evidence that an overactive fibrinolytic system interferes with haemostasis and contri- butes to abnormal uterine bleeding ( Gleeson et al. , 1993 ) consistent with the ef ficacy of tranexamic acid, an anti- fibrinolytic, as a treatment for HMB ( Gleeson et al. , 1994 ). The finding that FGA and FGB are ........................................................................................ Table IV Significant canonical pathways identified using IPA. Ingenuity canonical pathways −log(P-value)* Ratio** Day 2 vs Days 3 Granulocyte adhesion and diapedesis 4.03 0.10 Agranulocyte adhesion and diapedesis 3.23 0.09 Inhibition of MMPs 2.89 0.16 Extrinsic prothrombin activation pathway 2.84 0.25 LXR/RXR activation 2.72 0.09 Day 2 vs Day 4 Pathogenesis of multiple sclerosis 5.79 0.67 Granulocyte adhesion and diapedesis 4.19 0.13 Coagulation system 4.18 0.26 Glutathione-mediated detoxification 3.74 0.29 Agranulocyte adhesion and diapedesis 3.39 0.12 Day 3 vs Day 4 Glutathione-mediated detoxification 5.83 0.29 Coagulation system 3.66 0.17 Complement system 3.59 0.17 Granulocyte adhesion and diapedesis 2.99 0.07 PXR/RXR activation 2.99 0.11 *−log(P-value) after right-tailed Fisher exact tests and Benjamini –Hochberg multiple correction. A P-value of 0.01 equates to a –log(P-value) of 2. **Ratio of genes within our gene list to the total number of genes in the canonical pathway. ........................................................................................ Table V Top five significant upstream regulators identified using IPA. Gene symbol Gene name P-value* Day 2 vs Day 3 IL1B Interleukin-1 beta 1.50E-06 RELA v-rel avian reticuloendotheliosis viral oncogene homologue A 2.80E-05 E2F5 E2F transcription factor 5, p130-binding 1.10E-04 PRKD1 Protein kinase D1 1.20E-04 IL6 Interleukin-6 1.40E-04 Day 2 vs Day 4 ERBB2 erb-b2 receptor tyrosine kinase 2 1.50E-10 TGFB1 Transforming growth factor beta 1 1.60E-10 RABL6 RAB, member RAS oncogene family-like 6 2.20E-09 IL1B Interleukin-1 beta 6.10E-08 RNASE1 Ribonuclease, RNase A family, 1 4.20E-07 Day 3 vs Day 4 OSMR Oncostatin M receptor 5.10E-06 TREM1 Triggering receptor expressed on myeloid cells 1 1.70E-05 IL1B Interleukin-1 beta 4.30E-05 PAF1 PAF1 homologue, Paf1/RNA polymerase II complex component 1.20E-04 FOXO1 Forkhead box O1 2.70E-04 *P-value of overlap: determines whether there is a signi ficant overlap between genes in our gene lists and those known to be regulated by a particular factor. 906 Paiva et al. Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 ............................................................................................................................... .............................................................. Table VI Summary of FAC derived from the DAVID Bioinformatics Resources 6.7: for up- and down-regulated genes between Days 2, 3 and 4 of the menstrual cycle (for speci fic details of each annotation within a cluster, see Supplementary Tables IVA–C).a Annotation cluster GOBPTERM FAT Enrichment score No. % Up-regulated in Day 2 vs Days 3 and 4 Day 2 vs Day 4 Cell migration Regulation of cell migration 3.57 14 3.67 Regulation of locomotion Regulation of cell motion Positive regulation of cell migration Positive regulation of cell motion Positive regulation of locomotion Immune and inflammatory response Response to wounding 2.95 47 12.30 Defence response Immune response Inflammatory response Regulation of inflammatory response Regulation of in flammatory response 2.64 11 2.89 Regulation of response to external stimulus Regulation of acute inflammatory response Coagulation Coagulation 2.26 11 2.89 Blood coagulation Wound healing Haemostasis Up-regulated in Day 3 vs Days 2 and 4 Day 3 vs Day 2 Cell cycle Cell cycle phase 2.62 23 6.91 Cell cycle process M phase Cell cycle Chemotaxis Chemotaxis 2.29 11 3.30 Taxis Locomotory behaviour Day 3 vs Day 4 Negative regulation of cell growth Regulation of cell growth 2.72 11 5.85 Negative regulation of cell growth Regulation of growth Negative regulation of cell size Up-regulated in Day 4 vs Days 2 and 3 Day 4 vs Day 2 Cell division Cell cycle phase 8.71 55 8.04 Mitosis Nuclear division M phase of mitotic cell cycle Cell division Cell migration/cell division Microtubule-based process 5.32 39 5.70 Microtubule cytoskeleton organization Spindle organization Cytoskeleton organization Continued 907Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 up-regulated on Day 2 vs Day 4 is consistent with the up-regulation of inflammatory processes on Day 2 of menstruation (early-menstru- ation) and the cessation of clotting processes by Day 4 (late-menstru- ation). A recent study showed that increased circulating fibrinogen levels were associated with delayed clot formation in women with HMB ( Gebhart et al., 2015). However, further studies to evaluate the role of fibrinogen isoforms in normal menstruation are warranted. C4BPA, a regulatory protein in the complement system also fea- tured among the top up-regulated genes on Day 2 compared to Day 4. C4BPA inhibits the classical pathway of complement activation by limiting DNA release post-cell death thus suppressing in flammation (Blom et al., 2004; Trouw et al., 2005). The up-regulation of C4BPA is again consistent with the up-regulation of in flammatory processes. Interestingly, XYLT1, an enzyme with roles in cartilage repair speci fic- ally via glycosaminoglycan synthesis ( Venkatesan et al., 2009), was also up-regulated on Day 2 of menstruation suggesting some degree of overlap in breakdown and repair processes in Day 2 menstrual endometrium. The glutathione-S-transferases (GSTM1, GSTM2) were among the top up-regulated genes on Day 4 vs Day 2. GSTs are cytosolic or mem- brane bound enzymes involved in the detoxi fication of reactive oxygen species (ROS) via conjugation with glutathione ( Kalinina et al. , 2014 ). GSTs, therefore, play an important role in cellular protection against oxidative stress. They are also involved in redox-dependent cell sig- nalling and regulate cellular processes including proliferation and apoptosis through interactions with kinases that are activated by oxi- dative stress ( Kalinina et al. , 2014 ). Steroid hormone withdrawal is known to decrease mechanisms that protect against ROS-induced damage in decidualized stromal cells ( Sugino et al. , 2002 )a n dt oe l e - vate in flammatory signals such as cyclooxygenase-2 and PG-F-2alpha (Sugino et al.,2 0 0 4). Based on these observations, we hypothesize that by Day 4 of menstruation (late-menstruation) endometrial regeneration is occurring and the GSTs play roles in cell proliferation and the restor- ation of the redox balance post-endometrial breakdown. Genes coding for GSTM1 are polymorphic. The GSTM1 null variant is present in ~45% of healthy individuals ( Seidegard et al. , 1990 ; Brockmoller et al. , 1996 ; Coles and Kadlubar, 2003 ). GSTM1 mRNA was undetectable in a large proportion of our endometrial tissue sam- ples as was observed also in fibroid tissues from women who were poor responders to mifepristone treatment ( Engman et al. , 2013 ). Although inconsistent, several studies have suggested a correlation between endometriosis and the GSTM1-de ficient genotype ( Baranov et al., 1996; Baranova et al., 1999; Hsieh et al., 2004; Hur et al., 2005). The GSTM1 null variant is also associated with an increased risk of ............................................................................................................................... .............................................................. Table VI Continued Annotation cluster GOBPTERM FAT Enrichment score No. % Regulation of mitosis Regulation of mitotic cell cycle 5.27 29 4.24 Regulation of cell cycle process Regulation of mitosis Regulation of nuclear division Positive regulation of mitosis Regulation of mitosis Regulation of mitosis 4.15 19 2.78 Regulation of nuclear division Spindle checkpoint Regulation of mitotic metaphase/anaphase transition Negative regulation of nuclear division Negative regulation of mitosis Cell migration Locomotory behaviour 3.77 47 6.87 Chemotaxis Cell division Chromosome segregation 3.73 17 2.49 Mitotic sister chromatid segregation Sister chromatid segregation Day 4 vs Day 3 Chemotaxis Chemotaxis 2.26 15 4.73 Taxis Locomotory behaviour Complement cascade and immune response Complement activation, classical pathway 2.18 11 3.47 Humoral immune response mediated by circulating immunoglobulin Inflammatory response aFAC, functional annotation cluster. 908 Paiva et al. Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 leiomyoma development ( Huang et al., 2010). Therefore, polymorph- isms in these genes appear to be associated with various endometrial pathologies; however, the mechanisms involved remain to be eluci- dated. Future studies examining the speci fic roles of GSTs during men- struation are warranted. Further evidence for endometrial regeneration by Day 4 of men- struation comes from DAVID analysis which indicates that several genes associated with cell division and mitosis are up-regulated at this time. We are the first to observe associations between these genes and menstruation. VTCN1 (also known as B7-H4, B7X or B7S1) is a FGAABC D GH EF FGB C4BPA LBP VTCN1 GSTM2 SERPINB4 TFF3 Figure 2 Expression of FGA ( A), FGB ( B), C4BPA ( C), LBP ( D), VTCN1 ( E), GSTM2 ( F) SERPINB4 ( G) and TFF3 ( H) in menstrual-phase endo- metrial biopsies from women from Days 2, 3 and 4 of menstruation as determined by quantitative PCR. Median fold change was calculated using ΔΔCT with RPL13A as the housekeeping gene. Data points in red are additional samples that were not in the original gene array. 909Identifying genes involved during the menstrual process Downloaded from https://academic.oup.com/molehr/article-abstract/22/12/898/2354520 by Biomedical Library user on 04 February 2019 B7 family member that plays critical roles in regulating not only the immune response via T-cell activation and cytokine secretion ( Prasad et al., 2003; Choi et al., 2003) but also cell proliferation and migration as demonstrated in tumour models in vitro (Gao et al. , 2015 ). KIAA0101 encodes a proliferating cell nuclear antigen-associated fac- tor (Xie et al., 2014), regulates DNA synthesis and cell cycle progres- sion ( Chang et al. , 2013 ) and has oncogenic roles in tumour progression (Yu et al., 2001; Cheng et al., 2013; Zhu et al. , 2013; Fan et al., 2015). We also identi fied for the first time TFF3 and LAMA2 as potentially associated with endometrial repair during menstruation. TFF3 is a secreted protein, which functions in mucosal repair of the gastrointestinal tract ( Taupin and Podolsky, 2003 ; Hoffmann, 2005 ; Thim and May, 2005 ) through the combined stimulation of cell migra- tion and prevention of apoptosis and anoikis ( Taupin and Podolsky, 2003). Dysregulated TFF3 expression has been implicated in numer- ous cancers ( Regalo et al. , 2005 ; Perry et al. , 2008 ). Moreover, LAMA2, a subunit of the basement membrane protein laminin2/mer- osin, has been demonstrated to play a role in muscle regeneration and repair and defects in its expression are a major contributor to muscu- lar dystrophy ( Kuang et al. , 1999 ). Both TFF3 and LAMA2 are up- regulated on Day 4 vs Day 2, consistent with roles in endometrial regeneration and repair. In contrast, SPINK1, which has roles in gut mucosual repair and integrity, is down-regulated on Day 4 vs Day 2 ( Konturek et al. , 1998 ; Marchbank et al. , 1998 ). These findings merit the further investigation of the roles of these factors in endometrial repair post-menstruation. Identifying the processes occurring during menstruation is dif ficult for a number of reasons. The most signi ficant of these is that shedding and repair can occur simultaneously in menstrual endometrium ( Cheng et al., 2007; Gaide Chevronnay et al., 2009; Garry et al., 2009). Therefore, menstrual endometrium is likely to contain areas of unshed, late secretory phase endometrium together with partially or completely sloughed and/or repaired endometrium ( Garry et al. , 2009). Additionally, these processes proceed at different rates in dif- ferent women, as re flected by variation in the length of the menstrual process. For example, repair in one woman may be complete by Day 3 of the menstrual cycle whereas repair may not be complete until Day 6 in another ( McLennan and Rydell, 1965). The women in this study also presented with a variety of endomet- rial pathologies related to bleeding status and other menstrual charac- teristics. These variations may also have in fluenced the menstrual process. It is also important to note that the current knowledge of canonical gene pathways cannot necessarily be related directly to a tissue as dynamic as the endometrium, which appears to have tissue- specialized processes ( Ponnampalam et al. 2008) possibly also pertain- ing to menstruation. In addition, although endometrial sampling by pipelle biopsy provides a long ribbon of tissue, it is possible we may not have detected subtle changes in endometrial activity that occur very locally. In spite of the practical dif ficulties of obtaining well-characterized clinical samples for this work, we have been able to provide new insights into the fundamental mechanisms underlying menstrual bleeding. Understanding the key regulators associated with menstru- ation is important for the development of new therapies that target aberrations in the expression of factors important for normal endo- metrial function. Supplementary data Supplementary data are available at Molecular Human Reproduction online.

We would like to thank all the women who generously donated tissue to this study. We would also like to acknowledge the expertise of the Research Nurses and Drs Catarina Ang, Wan Tinn Teh and Stefan Kane as well as other clinical staff at the Royal Women ’s Hospital who contributed to this study. We would like to acknowledge Marie Everest from AGRF for her assistance with the expression arrays and RNA quality determination. Authors’ roles P.P. was responsible for conducting all PCR experimentation, data ana- lyses, interpreting the results and preparing the manuscript. M.L. undertook sample processing and PCR analyses, and contributed to data and cluster analysis. J.G. was responsible for study conception and design, data analysis and preparing the manuscript. M.O. under- took bioinformatics analyses and contributed to data interpretation. N.W. performed the pelvic ultrasounds and interpreted clinical results. J.M. assisted with development of the study database. M.H. was responsible for study conception and design, clinical aspects of the study including patient recruitment and biopsy collection, data inter- pretation and manuscript preparation. P.R. was responsible for study conception and design, study coordination, data interpretation and manuscript preparation. All authors made substantial contributions in critically revising the manuscript. Funding Funding for this work was provided by a National Health and Medical Research Council (NHMRC) of Australia Project Grant APP1008553 to M.H., P.R. and J.G. M.H. is supported by an NHMRC Practitioner Fellowship. P.P. is supported by an NHMRC Early Career Fellowship. Conflict of interest The authors declare no con flict of interest.

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