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Proteomic Analysis of Menstrual Blood*

  • Heyi Yang
    Affiliations
    New York City Office of Chief Medical Examiner, 421 East 26th Street, New York, New York 10016
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  • Bo Zhou
    Affiliations
    New York City Office of Chief Medical Examiner, 421 East 26th Street, New York, New York 10016
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  • Mechthild Prinz
    Affiliations
    New York City Office of Chief Medical Examiner, 421 East 26th Street, New York, New York 10016
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  • Donald Siegel
    Correspondence
    To whom correspondence should be addressed: 421 East 26th Street, New York, NY 10016. Tel.: (212)323-1434; Fax: (212)323-1560
    Affiliations
    New York City Office of Chief Medical Examiner, 421 East 26th Street, New York, New York 10016
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by award 2008-DN-BX-K011 and 2010-DN-BX-K192 funded by the National Institute of Justice, Office of Justice Programs, United States Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice.
    This article contains supplemental Tables S1 to S6.
Open AccessPublished:July 20, 2012DOI:https://doi.org/10.1074/mcp.M112.018390
      Menstruation is the expulsion of the endometrial lining of the uterus following a nearly month long preparation for embryo implantation and pregnancy. Increasingly, the health of the endometrium is being recognized as a critical factor in female fertility, and proteomes and transcriptomes from endometrial biopsies at different stages of the menstrual cycle have been studied for both diagnostic and therapeutic purposes (1 Kao, L. C., et al. 2003 Endocrinology 144, 2870–2881; Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617–630; DeSouza, L., et al. 2005 Proteomics 5, 270–281). Disorders of the uterus ranging from benign to malignant tumors, as well as endometriosis, can cause abnormal menstrual bleeding and are frequently diagnosed through endometrial biopsy (Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617–630; Ferenczy, A. 2003 Maturitas 45, 1–14). Yet the proteome of menstrual blood, an easily available noninvasive source of endometrial tissue, has yet to be examined for possible causes or diagnoses of infertility or endometrial pathology. This study employed five different methods to define the menstrual blood proteome. A total of 1061 proteins were identified, 361 were found by at least two methods and 678 were identified by at least two peptides. When the menstrual blood proteome was compared with those of circulating blood (1774 proteins) and vaginal fluid (823 proteins), 385 proteins were found unique to menstrual blood. Gene ontology analysis and evaluation of these specific menstrual blood proteins identified pathways consistent with the processes of the normal endometrial cycle. Several of the proteins unique to menstrual blood suggest that extramedullary uterine hematopoiesis or parenchymal hemoglobin synthesis may be occurring in late endometrial tissue. The establishment of a normal menstrual blood proteome is necessary for the evaluation of its usefulness as a diagnostic tool for infertility and uterine pathologies. Identification of unique menstrual blood proteins should aid the forensic community in distinguishing menstrual blood from circulating blood.
      Menstrual blood is a complex biological fluid composed of blood, vaginal secretions, and the endometrial cells of the uterine wall as they exist immediately prior to menses. These cells are the end product of a dynamic cyclical process focused on pregnancy and reproduction. Consequently, many of the proteins in these cells are expressed in preparation for blastocyst implantation and nurturing (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • Garrido N.
      • Navarro J.
      • Garcia-Velasco J.
      • Remoh J.
      • Pellice A.
      • Simón C.
      The endometrium versus embryonic quality in endometriosis-related infertility.
      ). Other proteins in the menstrual blood proteome are a consequence of no implantation and include proteolytic enzymes, cytokines, members of apoptotic pathways, and a host of proteins from the diverse types of immune cells that are an integral part of menstruation (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,
      • King A.E.
      • Critchley H.O.
      Oestrogen and progesterone regulation of inflammatory processes in the human endometrium.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ,
      • Li A.
      • Felix J.C.
      • Hao J.
      • Minoo P.
      • Jain J.K.
      Menstrual-like breakdown and apoptosis in human endometrial explants.
      ). Protein expression in all organs is a consequence of function. In the uterus, however, function changes on a near daily basis. Consequently evaluation of the menstrual blood proteome depends on an understanding of the complex uterine cycle.
      The uterus is composed of three main layers: 1) the luminal facing endometrium, 2) the visceral muscle myometrium immediately beneath it, and 3) the perimetrium, a serous membrane facing the abdominal cavity. The endometrium, which undergoes the greatest changes in response to the monthly endocrine cycle and is shed during menstruation, is further divided into a functional layer, the stratum functionalis, which faces the lumen, and a basal layer beneath it, the stratum basalis. By convention the menstrual cycle begins on the first day of menses and has an idealized duration of 28 days. Ovulation occurs at ∼day 14, with the prior 10 days denoted as the proliferative phase of the cycle and the following 2 weeks the secretory phase. At the beginning of the menstrual cycle estrogen and progesterone concentrations are at their lowest levels. Menstruation typically lasts ∼4 days after which the uterus is denuded of the stratum functionalis and much of the stratum basalis (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). However, even as the endometrium is being shed, proliferation from cells of the adjacent cervix and fallopian uterotubal junction, as well as glandular cells deep in the stratum basalis, begin the process of re-establishing the endometrial lining (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). Starting at about the sixth day of the cycle and continuing for about a week, estrogen levels rise. This is a time of increased proliferation and extensive angiogenesis and gland formation. Under the influence of estradiol there are also increases in the formation of intracellular organelles (ribosomes, mitochondria, Golgi apparatus, etc.), as well as the formation of microvilli and cilia with concomitant increases in cytokeratins and other cytoskeletal elements.
      On approximately day 14 ovulation occurs and estrogen levels decline as progesterone levels rise. Significant changes in gene expression are observed as proliferation gives way to differentiation (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • DeSouza L.
      • Diehl G.
      • Yang E.C.
      • Guo J.
      • Rodrigues M.J.
      • Romaschin A.D.
      • Colgan T.J.
      • Siu K.W.
      Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression.
      ). Dramatic morphological changes also occur (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ) including the hallmark formation of spiral arteries in the functional layer and conspicuous appearance of glycogen vacuoles at the base of glandular cells. Giant mitochondria also appear, and the nuclear envelop forms long channels that penetrate the nucleolus forming the nucleolar canal system (NCS), an organelle found only in the mid-secretory stage of the female endometrium (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ,
      • Wang T.
      • Schneider J.
      Origin and fate of the nucleolar channel system of normal human endometrium.
      ). The NCS is believed necessary for the significant amount of protein synthesis required by the maturing endometrium in preparation for blastocyst implantation including the production of glycoproteins and extensive amounts of glycogen (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). Vascular remodeling continues in this phase regulated by a variety of cytokines and matrix metalloproteases (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ). Finally, as the late endometrial tissue transitions to predecidua (in preparation of implantation) cytokines recruit large numbers of lymphocytes including uterine natural killer cells, T-cells and macrophages (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). The endometrial lining more than doubles in thickness between the proliferative and secretory phases.
      The absence of implantation (e.g. no fertilized egg) results in declining levels of progesterone and leads to menses. Vasoconstriction of basal arteries results in ischemia, apoptosis and necrosis (

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ,
      • Li A.
      • Felix J.C.
      • Hao J.
      • Minoo P.
      • Jain J.K.
      Menstrual-like breakdown and apoptosis in human endometrial explants.
      ). Blood pools beneath the epithelial layer and fills with cell debris and inflammatory exudates. Plasmin, activated by released proteases, prevents the blood from clotting, and matrix metalloproteases aid in the digestion of the extracellular matrix (

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). The stratum functionalis cleaved from the basalis is shed over the following 4–5 days even as renewal from basal glandular cells and the edges of the uterus begins.
      The health of the endometrium is a key to successful pregnancy. Implantation failure or the inability to maintain and nourish a developing blastocyst can lead to infertility. Numerous studies have focused on understanding the causes of endometrial receptivity failure and the search for proteins that may be used as diagnostic markers as well as therapeutic agents is ongoing (
      • Kao L.C.
      • Germeyer A.
      • Tulac S.
      • Lobo S.
      • Yang J.P.
      • Taylor R.N.
      • Osteen K.
      • Lessey B.A.
      • Giudice L.C.
      Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.
      ,
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • DeSouza L.
      • Diehl G.
      • Yang E.C.
      • Guo J.
      • Rodrigues M.J.
      • Romaschin A.D.
      • Colgan T.J.
      • Siu K.W.
      Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression.
      ). In addition, abnormal uterine bleeding can be a sign of significant endometrial disorders ranging from benign fibroids to endometriosis to malignancies (
      • Ferenczy A.
      Pathophysiology of endometrial bleeding.
      ). Here too, identification of reliable and easily obtainable markers from menstrual blood could aid in both early detection and follow up monitoring.
      The menstrual blood proteome offers a snapshot of the processes occurring in the endometrium lining immediately prior to menses. Establishing a normal menstrual blood proteome sets a baseline against which pathologies can be evaluated. Identification of unique menstrual blood markers will enable the forensic community to distinguish menstrual blood from circulating blood.

      DISCUSSION

      Menstrual blood is a complex body fluid composed of endometrial and immune cells shed from the uterus, vaginal fluid, and of course, blood. To identify the maximum possible number of proteins present in menstrual blood, five different methods were employed, three using samples depleted of Hb, Alb, and IgGs to reduce dynamic range and consequently unmask low abundant proteins. Although together these methods identified 1061 proteins, only 40 were common to all five methods while 700 were unique to one of the methods. These data demonstrate the importance of multiple separation techniques for identifying the maximum number proteins in a complex sample (Table I, Fig. 3) and are in agreement with other proteomes described in the literature. Schenk (
      • Schenk S.
      • Schoenhals G.J.
      • de Souza G.
      • Mann M.
      A high confidence, manually validated human blood plasma protein reference set.
      ) for example, employed eight different methods to evaluate the human plasma proteome and found only 56 of 697 proteins by all eight methods.
      Of particular interest were results from the 15,000 × g pellet (Method 5) - a sample not infrequently discarded in proteome analyses. The pellet contained the largest number of proteins identified (722) of which 421 were not found in the supernatants from Methods 3 and 4 (Fig. 3). Of the 385 unique menstrual blood proteins identified in this study, 126 (∼33%) were found only in the pellet, e.g. zona pellucida sperm-binding protein 4. These results are not surprising as most solubilizing buffers contain detergents and/or chaotropic salts inadequate to completely disrupt and solubilize nuclei, mitochondria, the plasmalemma, as well as other internal membranes, all of which contain both integral and associated proteins. Many soluble proteins, perhaps trapped within membranous structures (e.g. histones, enzymes, cytokines, immunoglobulins, and cytoskeletal elements from cilia and microvilli), were also identified in the pellet, some found in greater amounts than in the supernatants, whereas some were not found in the supernatants at all, e.g. fetal hemoglobins ζ, θ and μ (Table II). These data suggest that pelleted materials should be routinely analyzed for protein content.
      A comparison of the 1061 proteins identified in menstrual blood with the proteomes of circulating blood (1774 proteins, supplemental Table S2) and vaginal fluid (823 proteins, supplemental Table S3) identified 385 unique menstrual blood proteins (supplemental Table S4). Discerning which proteins from the complete (1,061) menstrual blood proteome are specifically expressed in the shedding endometrium, however, is not as simple as subtracting common proteins found in blood and vaginal fluid. Certainly many of these proteins are also expressed in shedding endometrial cells whether they function in routine housekeeping or specific cellular pathways. Ultimately, absolute quantitation of all proteins in each of these body fluids will be required in order to determine the relative contribution of common proteins in the shedding endometrium. However, despite likely co-expression of some proteins, it is still worth assessing the 385 unique menstrual blood proteins in order to evaluate biological processes occurring in the endometrium immediately prior to and during menstruation. Certainly, the large number of histones, ribosomal proteins, cytoskeletal elements, cytokines, and immunoglobulins identified in the unique menstrual blood proteins (Fig. 7, supplemental Table S4) are consistent with the biological processes known to occur in the late secretory phase of the menstrual cycle. For as the secretory endometrium progresses toward predecidua and prepares for blastocyst implantation, nuclei becomes euchromatic, the number of ribosomes increases and cell cytoplasms fills with additional cytoskeletal elements (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ,
      • Paule S.G.
      • Airey L.M.
      • Li Y.
      • Stephens A.N.
      • Nie G.
      Proteomic approach identifies alterations in cytoskeletal remodelling proteins during decidualization of human endometrial stromal cells.
      ). At this time endometrial granulocytes appear in order to suppress other immune cells and thereby protect a genetically distinct implanting embryo from its mother's immune defense system (
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ).
      Lack of implantation leads to menstruation which today is regarded as an inflammatory response preceded by leukocyte infiltration (eosinophils, mast cells, T-cells, and uterine specific natural killer cells) and mediated through numerous cytokines (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • Lommel A.T.L.V.
      From Cells to Organs.
      ,
      • King A.E.
      • Critchley H.O.
      Oestrogen and progesterone regulation of inflammatory processes in the human endometrium.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ,
      • Weiss G.
      • Goldsmith L.T.
      • Taylor R.N.
      • Bellet D.
      • Taylor H.S.
      Inflammation in reproductive disorders.
      ). These latter changes in the endometrium are observed in the relatively high percentage of unique menstrual blood proteins found to participate in “multi-organism processes” (Fig. 6), a GO category that includes both inter- and intraspecies defense (the uterus, like all mucosa exposed to the external environment secrets mucus with defensive proteins to block pathogen invasion), as well as pregnancy, parturition, and mating (

      DAVID. (Version 6.7) NIH Bioinformatics Resources http://david.abcc.ncifcrf.gov

      ). It is interesting to note that the one biological process found in whole menstrual blood that is not shared by either whole blood or vaginal fluid is locomotion (Fig. 6) which also includes subcategories consistent with the functional processes of the predecidua and menstruation including: “morphogenesis of an epithelium, inductive cell migration, cytokinesis, secretion by cells, germ cell development, reproduction and oogenesis” (

      DAVID. (Version 6.7) NIH Bioinformatics Resources http://david.abcc.ncifcrf.gov

      ).
      Complementary to and expanding on the biological processes identified by the gene ontology analysis discussed above (Fig. 6, Fig. 7) are the specific proteins described in Table II. These proteins are involved in the dynamic processes of apoptosis and cell shedding, (e.g. death-inducer obliterator 1, MAP kinase-activating death domain protein and plexin-A1, D1 which blocks extracellular matrix adhesion and induces cell rounding), as well as endometrial renewal and angiogenesis (e.g. epithelial discoidin domain-containing receptor 1, thrombospondin type I domain containing 7A Wnt and ADAM33 an extracellular protease that acts in concert with plexins and semaphorins in the release of cells from the extracellular matrix, and also function in mucosal remodeling and angiogenesis). Two proteins were also identified that function in female reproduction, zona pellucida sperm-binding protein 4 (ZP4) which is found in the oocyte plasmalemma and human choriogonadotropin (hCG) subunit β. Although hCG is produced by the developing embryo, Zimmermann et al. (
      • Zimmermann G.
      • Ackermann W.
      • Alexander H.
      Epithelial human chorionic gonadotropin is expressed and produced in human secretory endometrium during the normal menstrual cycle.
      ) have demonstrated in tissue biopsies that “Epithelial human chorionic gonadotropin is expressed and produced in human secretory endometrium during the normal menstrual cycle.”
      Of particular interest, however, are the fetal hemoglobins and additional proteins known to function in hematopoiesis. Evidence from the literature documents the rare occurrence of extramedullary uterine hematopoiesis, but it is typically associated with pathology—endometriosis, endometrial polyps, and leiomyomas—or the result of retained products of conception following spontaneous abortion or terminated pregnancy (
      • Gru A.A.
      • Hassan A.
      • Pfeifer J.D.
      • Huettner P.C.
      Uterine extramedullary hematopoiesis: what is the clinical significance?.
      ,
      • Valeri R.M.
      • Ibrahim N.
      • Sheaff M.T.
      Extramedullary hematopoiesis in the endometrium.
      ). Some patients in which extramedullary uterine hematopoiesis is diagnosed are initially referred with symptoms of chronic anemia and are subsequently diagnosed with a myeloproliferative disease. Currently, there is debate as to whether identified uterine hematopoiesis is pathognomonic of an underlying myeloid disorder, and consequently, if it may be used for diagnostic purposes (
      • Gru A.A.
      • Hassan A.
      • Pfeifer J.D.
      • Huettner P.C.
      Uterine extramedullary hematopoiesis: what is the clinical significance?.
      ,
      • Valeri R.M.
      • Ibrahim N.
      • Sheaff M.T.
      Extramedullary hematopoiesis in the endometrium.
      ). Recently Dassen et al. (
      • Dassen H.
      • Kamps R.
      • Punyadeera C.
      • Dijcks F.
      • de Goeij A.
      • Ederveen A.
      • Dunselman G.
      • Groothuis P.
      Haemoglobin expression in human endometrium.
      ) have identified hemoglobins (immunohistochemically by a pan anti-human Hb antibody and molecularly for Hbs α, β, δ, and γ by RT-PCR), as well the heme metabolizing enzyme heme oxygenase 1 within the epithelia and stromal cells of the endometrium. They have postulated that hemoglobin biosynthesis within these nonerythroid cells functions to protect the endometrium against oxidative and nitrosative stress. It should be noted, however, that while these investigators found mRNAs for hemoglobins α, β, δ, and γ in their endometrial samples, neither they, nor other, have reported finding fetal hemoglobins ζ, θ, and μ in endometrial tissue (although Hb γ is a fetal hemoglobin, it has been reported to make up ∼0.4% of adult hemoglobins (
      • Dover G.J.
      • Boyer S.H.
      Quantitation of hemoglobins within individual red cells: asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects.
      ).
      Although it is possible that hemoglobins ζ, θ, and μ were from a volunteer who was unknowing pregnant (pregnancy tests were not performed on volunteers, and erythropoiesis and hemoglobin production can occur in the yolk sac at 18 days post conception (
      • Tavian M.
      • Péault B.
      Embryonic development of the human hematopoietic system.
      ,
      • Pereda J.
      • Niimi G.
      Embryonic erythropoiesis in human yolk sac: two different compartments for two different processes.
      ,
      • Palis J.
      • Yoder M.C.
      Yolk-sac hematopoiesis: the first blood cells of mouse and man.
      ), it seems unlikely that a “late period” (unrecognized spontaneous abortion) was the source of these fetal hemoglobins for several reasons. First, the sample in which hemoglobins ζ, θ and μ (as well as all the other proteins involved in hematopoiesis and hCG) were identified was the same sample in which zona pellucida sperm-binding protein 4 was found. It seems unlikely that ZP4, an oocyte specific protein, was still present 18 days post conception. Second, Dassen et al. (
      • Dassen H.
      • Kamps R.
      • Punyadeera C.
      • Dijcks F.
      • de Goeij A.
      • Ederveen A.
      • Dunselman G.
      • Groothuis P.
      Haemoglobin expression in human endometrium.
      ) identified individual hemoglobins by RT-PCR and chose primers only for α, β, δ, and γ; consequently they may have found ζ, θ, and μ had they looked. Finally, the menstrual blood donor did not report a late period. (It should be noted that hemoglobins α, β, δ, γ, as well as ε were all identified in the 1061 menstrual blood proteome (supplemental Table S1). However, as they were also found in venous blood and/or vaginal fluid they were not included in the 385 unique menstrual blood proteins evaluated in Table II.)
      It is not possible to say whether the hematopoietic markers identified here are the result of pathology or are part of the normal menstrual cycle, although volunteers self-reported as healthy. Only by testing numerous individuals can a basic menstrual blood proteome be established with confidence. Testing of 50 additional women of various ages, ethnicities and with or without use of oral contraceptives is currently underway.
      The menstrual cycle is a complex, finely orchestrated series of cellular, physiological, and biochemical changes under precise endocrine control that routinely builds a rich endometrial lining in constant anticipation of blastocyst implantation. Lack of implantation results in the controlled elimination of the endometrial lining only to be immediately replenished as the monthly cycle begins again. Each phase of the cycle—proliferative, secretory, and menstrual—has a specific function, and the proteins expressed during these phases reflect those functions (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • DeSouza L.
      • Diehl G.
      • Yang E.C.
      • Guo J.
      • Rodrigues M.J.
      • Romaschin A.D.
      • Colgan T.J.
      • Siu K.W.
      Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). It has long been recognized that changes in the biochemistry or cellular composition of the endometrium can be diagnostic of uterine pathology and is often accompanied by abnormal menses (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,

      Ferenczy, A., Mutter, G, . (2008) The endometrial cycle. Glob. Libr. Women’s Med. 10.3843/GLOWM.10293

      ). Table II, for example, identifies several proteins have been associated with endometrioses as well as endometrial cancer. In recent years it has become equally as evident that such changes can influence fertility, or more precisely infertility (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ). In the late secretory phase, as the uterus prepares for implantation, the stratum functionalis transitions to predecidua with concomitant expression of new or different levels of existing proteins, many of which have been proposed as markers of reduced uterine receptivity, including: integrins, matrix metalloproteases (MMPs), galectins (GALs), glucose transporter proteins (GLUT), interleukins (ILs), immune cell regulators, insulin-like growth factor-binding protein 1 (IGFBP-1), and glycodelin A (
      • Strowitzki T.
      • Germeyer A.
      • Popovici R.
      • von Wolff M.
      The human endometrium as a fertility-determining factor.
      ,
      • Kliman H.J.
      • Honig S.
      • Walls D.
      • Luna M.
      • McSweet J.C.
      • Copperman A.B.
      Optimization of endometrial preparation results in a normal endometrial function test (EFT) and good reproductive outcome in donor ovum recipients.
      ). Many of these proteins were detected in the menstrual blood proteome (supplemental Table S1, e.g. IGFBP-1, MMP-9, galectin-3, glycodelin A, GLUT1, IL1, and others) and may prove useful for diagnosis.
      To our knowledge this is the first proteome analysis of menstrual blood. The two samples were chosen from mid-menstrual cycle for consistency as it seems likely that protein content will change during the course of a woman's period. Indeed, only by testing numerous women throughout their periods can a thorough menstrual blood proteome be established with confidence. Toward that end, testing of fifty additional women of various ages, ethnicities and with or without use of oral contraceptives is currently underway.
      Menstrual blood is an easily obtainable body fluid and collection is noninvasive. The data presented here demonstrate that many proposed markers of uterine infertility are present in the menstrual blood proteome which could, consequently, be used for diagnostic purposes. Abnormal menstruation can be a sign of underlying uterine pathology, and here too a comparison against the normal menstrual blood proteome could prove a useful diagnostic tool. Finally, the detection of a distinct subset of 385 menstrual blood proteins not identified in venous blood or vaginal fluid constitutes an important list of candidate markers for the identification menstrual blood in forensic investigations.

      Acknowledgments

      We would like to thank Elisa Wurmbach for careful reading of the manuscript.

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