Identification of genes differentially expressed in menstrual breakdown and repair.
Does the changing molecular profile of the endometrium during menstruation correlate with the histological profile of menstruation.



We identified several genes not previously associated with menstruation; on Day 2 of menstruation (early-menstruation), processes related to inflammation are predominantly up-regulated and on Day 4 (late-menstruation), the endometrium is predominantly repairing and regenerating.



Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regeneration and repair. Perturbations in the regulation of menstruation may result in menstrual disorders including abnormal uterine bleeding.



Endometrial samples were collected by Pipelle biopsy on Days 2 (n = 9), 3 (n = 9) or 4 (n = 6) of menstruation.



RNA was extracted from endometrial biopsies and analysed by genome wide expression Illumina Sentrix Human HT12 arrays. Data were analysed using 'Remove Unwanted Variation-inverse (RUV-inv)'. Ingenuity pathway analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify canonical pathways, upstream regulators and functional gene clusters enriched between Days 2, 3 and 4 of menstruation. Selected individual genes were validated by quantitative PCR.



Overall, 1753 genes were differentially expressed in one or more comparisons. Significant canonical pathways, gene clusters and upstream regulators enriched during menstrual bleeding included those associated with immune cell trafficking, inflammation, cell cycle regulation, extracellular remodelling and the complement and coagulation cascade. We provide the first evidence for a role for glutathione-mediated detoxification (glutathione-S-transferase mu 1 and 2; GSTM1 and GSTM2) during menstruation. The largest number of differentially expressed genes was between Days 2 and 4 of menstruation (n = 1176). We identified several genes not previously associated with menstruation including lipopolysaccharide binding protein, serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), interleukin-17C (IL17C), V-set domain containing T-cell activation inhibitor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/PAF), trefoil factor 3 (TFF3), laminin alpha 2 (LAMA2) and serine peptidase inhibitor, Kazal type 1 (SPINK1). Genes related to inflammatory processes were up-regulated on Day 2 (early-menstruation), and those associated with endometrial repair and regeneration were up-regulated on Day 4 (late-menstruation).



Participants presented with a variety of endometrial pathologies related to bleeding status and other menstrual characteristics. These variations may also have influenced the menstrual process.



The temporal molecular profile of menstruation presented in this study identifies a number of genes not previously associated with the menstrual process. Our findings provide valuable insight into the menstrual process and may present novel targets for therapeutic intervention in cases of endometrial dysfunction.



All microarray data have been deposited in the public data repository Gene Expression Omnibus (GSE86003).



Funding for this work was provided by a National Health and Medical Research Council of Australia (NHMRC) 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 a NHMRC Early Career Fellowship. The authors have no conflict of interest to declare.
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia ppaiva@unimelb.edu.au.
Premila
P
Paiva
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia.
Michelle G
MG
Lockhart
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia.
Jane E
JE
Girling
Bioinformatics Division, Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, VIC 3052, Australia.|Department of Microbiology, Monash University, Wellington Road and Blackburn Road, Clayton, VIC 3800, Australia.
Moshe
M
Olshansky
Pauline Gandel Imaging Centre, Royal Women's Hospital, 20 Flemington Road, Parkville, VIC 3052, Australia.
Nicole
N
Woodrow
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia.
Jennifer L
JL
Marino
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia.
Martha
M
Hickey
Department of Obstetrics and Gynaecology, The University of Melbourne, Gynaecology Research Centre, Royal Women's Hospital, Cnr Flemington Rd and Grattan St, Parkville, VIC 3052, Australia.
Peter A W
PA
Rogers
D004715:Endometriosis / Q000378:metabolism; D004717:Endometrium / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008597:Menstrual Cycle / Q000235:genetics* / Q000502:physiology; D008598:Menstruation / Q000235:genetics* / Q000502:physiology; D005810:Multigene Family / Q000235:genetics; D016133:Polymerase Chain Reaction
RUV-inv; endometrium; gene array; inflammation; menstruation; repair