2D-DiGE Analysis of the Human Endometrial Secretome Reveals Differences between Receptive and Nonreceptive States in Fertile and Infertile Women Natalie J. Hannan,*,†,‡ Andrew N. Stephens,† Adam Rainczuk,† Cassandra Hincks,† Luk J. F. Rombauts,‡,§ and Lois A. Salamonsen† Prince Henry’s Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, and Monash IVF Clayton, Victoria, 3168, Australia Received May 16, 2010
Endometrial secretions in the uterine cavity contain mediators important for endometrial receptivity and embryo implantation. Unbiased analysis of uterine fluid from a receptive versus nonreceptive time of the menstrual cycle and in fertile and infertile women will provide new insights into uterine receptivity. We hypothesized that proteomic analysis of human uterine lavages would identify proteins important for the establishment of pregnancy in humans. Lavages collected from fertile (n ) 7) and infertile (n ) 8) women during the midsecretory (MS) phase, and from fertile women during the midproliferative (MP) (n ) 7) phase, were assessed using 2D-differential in gel electrophoresis (2D-DiGE) over a pI 4-7 range. Statistical analysis revealed 7 spots that were significantly decreased in the MP compared to the MS phase, while 18 spots showed differential expression between fertile and infertile women. A number of proteins were identified by mass spectrometry, including antithrombin III and alpha-2macroglobulin, whose production was confirmed in endometrial epithelium. Their staining pattern suggests roles during embryo implantation. Assessment of the human endometrial secretome has identified differences in the protein content of uterine fluid with respect to receptivity and fertility. Keywords: 2D-DiGE • human endometrial secretions • infertility • antithrombin III • alpha-2-macroglobulin • uterine lavage
Introduction The human endometrium is an extremely complex and dynamic tissue that constantly undergoes controlled remodeling and differentiation on a cyclical basis throughout reproductive life.1 The normalized human menstrual cycle is of 28 days duration and can be divided into three main phases: menses (breakdown and degradation), proliferative (regeneration) and secretory (differentiation). The purpose of this remodeling is to provide an environment that is conducive to implantation of the conceptus, but only at a time when the conceptus is appropriately developed. The human endometrium is receptive to blastocyst implantation during a few days in the midsecretory (MS) phase of each menstrual cycle. Deficiencies in endometrial receptivity are thought to be responsible for approximately two-thirds of implantation failure and early pregnancy loss (Simon et al., 1998) and thus considered to be a major cause of infertility and the limited success rates observed with in vitro fertilization (IVF). However, oocyte quality, chromosomal abnormalities, embryo quality and immunological problems must also be acknowledged as causes * To whom correspondence should be addressed. Dr. Natalie J. Hannan, PHIMR, PO Box 5152, Clayton, Victoria 3168. Australia. FAX: +61-3-9594 6125. E-mail:
[email protected]. † Prince Henry’s Institute of Medical Research. ‡ Department of Obstetrics and Gynaecology, Monash University. § Monash IVF Clayton.
6256 Journal of Proteome Research 2010, 9, 6256–6264 Published on Web 10/06/2010
of infertility. Whether implantation failure or early miscarriage results from abnormal maternal-fetal interactions, an inadequate uterine microenvironment or defective placentation is not yet known. During the secretory phase, progesterone levels rise dramatically following ovulation, causing endometrial glandular epithelial cells to transform from relatively inactive to highly secretory polarized cells that synthesize and secrete an amorphous material into the uterine lumen.2,3 These secretions, which include binding and nutrient transport proteins, ions, glucose, cytokines, enzymes, hormones, growth factors, proteases and their inhibitors and other substances,2-4 are likely to be important mediators of blastocyst development and endometrial function. In particular, these secreted proteins are believed to provide important nutritional factors during the early stages of implantation and placentation and may play broader roles in regulating endometrial receptivity, immunomodulation and placental morphogenesis. A variety of growth factors and cytokines have been identified in the uterine glands.3,5,6 The importance of secretions from uterine glands is emphasized in a sheep model where uterine gland formation is inhibited7 and pregnancy cannot be established. Deficient glandular activity, usually described as a “secretory phase defect”, has been hypothesized to be an underlying cause of early pregnancy failure in humans.8,9 However, there is no 10.1021/pr1004828
2010 American Chemical Society
Changes in Abundance of Endometrial Secreted Proteins Table 1. Summary of Patient Details; Samples and Histological Dating endometrial tissue and lavage samples
Fertile
age
histological day of cycle
number per group
Midproliferative 36.7 ( 2.3 Days 8-11 Midsecretory 36.4 ( 2.9 Days 19-23
7 7
33.7 ( 4.4 Days 19-23
8
Infertile Midsecretory
direct evidence to support this claim in the human. Disrupted secretion of individual soluble factors including cytokines and growth factors into the uterine lumen has been correlated with infertility.9-11 In addition, the concentration of glycodelin A in uterine lavages (containing those proteins secreted into the lumen) on menstrual cycle days 24-26 are lower in women who subsequently miscarry than in those with successful pregnancies.12 Recently proteomic assessment of pooled human endometrial fluid aspirates collected during the secretory phase was described13 revealing the presence of a number of proteins that may have roles in the endometrium including matrix metalloproteinase 9, tissue inhibitor of matrix metalloproteinase and MUC1. However, studies identifying the products of glandular secretions, their bioactivity and function remain limited and there are currently no definitive markers of endometrial receptivity to facilitate identification of receptive endometrium. Such markers would be of significant clinical value, especially for women undergoing IVF procedures. Previously we assessed differences between the midproliferative and midsecretory proteome of human endometrial tissue, revealing a number of differentially expressed proteins and important functional networks enriched in the receptive endometrium.14 However, the proteome was dominated by abundant structural proteins; in addition, a less invasive sampling procedure is desirable for a clinically useful diagnostic marker. In the present study we have applied 2D DiGE to analyze the secreted proteins present in uterine flushings from women at different stages of their cycle (nonreceptive versus receptive) and according to fertility status. We hypothesized that this approach, using a less complex sample not dominated by the abundant structural proteins found in tissue, would help to identify soluble mediators essential to endometrial receptivity and may also provide novel diagnostic and treatment targets.
Experimental Section Sample Collection and Patient Details. Ethical approval was obtained from appropriate Institutional Ethics Committees for all sample collections. Written informed consent was obtained from all subjects. Human endometrial lavages and biopsies (n ) 7/8 patients group/phase of the cycle; Table 1) were obtained from women who were undergoing hysteroscopy, dilatation and curettage. Sampling was performed during the nonreceptive, midproliferative (MP; days 8-11) and receptive, midsecretory (MS; days 19-23) phases of the menstrual cycle from women with proven fertility, or from the MS phase only of women with unexplained infertility. All infertile women presented with primary infertility and were screened for nonendometrial causes of their infertility, including ovarian dysfunction, tubal patency, endometriosis and male factor. The fertile women all had proven parity and were presenting for tubal ligation or assessment for reversal of tubal ligation. Patients with uterine abnormalities such as endometrial polyps,
research articles endometriosis, endometritis or who had received steroid hormone therapy in the last 6 months were excluded. All patient groups were age matched (see Table 1) and cycling. In brief, prior to the insertion of any surgical instruments, a uterine lavage was collected by gently infusing 5 mL of sterile saline trans-cervically into the uterine cavity through a fine flexible catheter. The saline solution was recovered by aspiration, centrifuged at 1000 rpm to remove cellular debris and immediately stored at -80 °C in 0.5 mL aliquots. Endometrial biopsies obtained by curettage (performed following lavage) were formalin-fixed overnight (16 ( 1 h) at 4 °C, washed three times in Tris buffered saline (TBS, pH 7.6) and stored at 4 °C until wax embedding. Self-reported menstrual cycle stage was confirmed by routine histological dating of the tissue.15 Preparation of Uterine Lavage Samples for Proteomic Analysis. As previously described uterine lavage samples were prepared for proteomic analysis.16 In brief lavage samples were thawed on ice, and protease inhibitors added (Calbiochem; Protease inhibitor cocktail set (#539134); EMD Biosciences, San Diego, CA). Samples were filtered (0.22 µm filter), and then immunodepleted of the six most abundant serum proteins (human serum albumin (HSA), transferrin, immunoglobulin G (IgG), IgA, antitrypsin, and haptoglobin) using a MARS spin cartridge (Agilent Technologies, Santa Clara, CA). Each depleted sample was diluted 1:1 with solubilization buffer#1 [40 mM Tris, 7 M urea, 2 M thiourea, 1% dimethylammoniopropanesulfonate (C7BzO) w/v], and concentrated to ∼800 µL using a centrifugal spin concentrator with a 3 kDa molecular weight cutoff (Macrosep UF, PALL Life Sciences, NY). The concentrated, depleted samples were snap-frozen and stored at -80 °C until required. Prior to use, each sample was reduced and alkylated at room temperature (RT), and then precipitated in 10 volumes of acetone. The precipitated protein was air-dried and resuspended in CyDye labeling buffer [30 mM Tris, 7 M urea, 2 M thiourea and 1% C7BzO w/v]. Protein content was measured using a Bradford Assay kit (Bio-Rad; Hercules, CA). Expression Analysis and Protein Identification. Fluorescent labeling of proteins using minimal CyDye labeling (GE Healthcare Bioscience, Uppsala, Sweden), image analysis and protein identification were carried out as described previously.14,16 In brief, 25 µg of protein from each patient was individually labeled using 200 pmol of either Cy3 or Cy5 (fertile proliferative, midsecretory and infertile midsecretory phase samples). A mixed (equal pool of all samples) internal standard labeled with 200 pmol of Cy2 was included for spot normalization and to allow comparison across all gels within the analysis as published previously.14,17,18 On completion of the labeling reaction, the Cy3 and Cy5 labeled samples were combined pair wise and 25 µg of Cy2-labeled internal standard was added to each (see Table 2), as recommended by the manufacturer. To account for any labeling bias by the different dyes, reciprocal labeling experiments were carried out; any proteins showing differential effects specific to the use of the Cy3/Cy5 dyes were eliminated from the analysis. Each combined labeled sample was precipitated in acetone as above, and resuspended in solubilization buffer without tris prior to isoelectric focusing (IEF). IEF was carried out using a 24 cm immobilized pH gradient strip covering the pH range 4-7, previously shown to optimally resolve proteins in human endometrial lavages.16 IEF parameters were as follows; constant 60 µA per strip, 100 V/1.5 h, 300 V/1.5 h, 500 V/3 h, gradient to 1000 V/4 h, gradient to 8000 V/3 h, constant 8000 V until reaching 60 000 Vh. Second dimension separation was carried Journal of Proteome Research • Vol. 9, No. 12, 2010 6257
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Table 2. CyDye Labeling Scheme and Gel Setup for 2D-DiGE Analysis gel #
1 2 3 4 5 6 7 8 9 10 11
CyDye
sample type
Cy3 Cy5 Cy5 Cy3 Cy3 Cy5 Cy5 Cy3 Cy3 Cy5 Cy3 Cy5 Cy3 Cy5 Cy3 Cy5 Cy5 Cy3 Cy3 Cy5 Cy5 Cy3
Fertile Prolif 1 Fertile MS 1 Fertile Prolif 2 Fertile MS 2 Fertile Prolif 3 Fertile MS 3 Fertile Prolif 4 Fertile MS 4 Fertile Prolif 5 Fertile MS 5 Fertile Prolif 6 Fertile MS 6 Fertile Prolif 7 Fertile MS 7 Infertile MS 1 Infertile MS 2 Infertile MS 3 Infertile MS 4 Infertile MS 5 Infertile MS 6 Infertile MS 7 Infertile MS
out in a 4-20% acrylamide gradient overnight at a constant 50 V using a BioRad Dodeca electrophoresis tank. Differential expression analysis including image alignment, spot detection, background removal, expression and statistical analysis based on normalized spot volumes, where the internal standard was used to perform normalization, was performed using PG240 SameSpots software (Nonlinear Dynamics, Newcastle-uponTyne, U.K.). All proteomic analysis was performed using individual patient samples. Protein spots of interest were excised from the gel using a ProPicII robotic spot picker (Genomic Solutions, MI) based on X-Y coordinates exported directly from PG240 SameSpots. Protein identification by MALDI TOF MS and MS/MS was also as described.14 Monoisotopic peak masses were automatically extracted using GPS Explorer software (v 3.0 build 311; Applied Biosystems, CA) and peak lists searched against the nonredundant UniProtKB/Swiss-Prot database (release 56.2; 20407 human sequence entries; http://www.uniprot.org) using the MASCOT search engine (updated 03-01-2007; http://www. matrixscience.com). Species was restricted to Homo sapiens, carbonylamide-cysteine (CAM - fixed modification) and oxidation of methionine (variable modification) were taken into account, a parent ion mass tolerance of 60 ppm and 1 missed cleavage (trypsin) was allowed. Up to fifteen of the most intense peptides detected in each MS scan were automatically selected for MS/MS analysis. Peak lists were extracted using Data Analysis software version 3.4 (Bruker Diagnostics, Germany). The parameters used to create the peak lists were as follows: mass range 100 to 3000 Da; signal-to-noise threshold of 5; minimum compounds length of 10 spectra. Combined peptide mass (MS) and fragmentation (MS/MS) data were searched using in-house MASCOT search engine (version 1.1, Matrix Science) against the UniProtKB/Swiss-Prot database as above, with fragment mass tolerance of 0.1 Da. Protein identities were assigned using the following criteria to evaluate the search; statistically significant identity as indicated by MOWSE score (>56), minimum number of peptides matched (>4); and direct correlation between the identified protein and its estimated molecular mass and pI determined from the 2D gel. 6258
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Proteins that were not identified by MALDI TOF MS were submitted for further analysis by LC-MS/MS. The peptide mixture was fractionated by nanoflow reversed-phase liquid chromatography using a 1200 series Capillary HPLC (Agilent Technologies, Santa Clara CA) online equipped with a nanoAcquity C18 150 mm ×0.15 mm I.D. column (Waters, Milford, MA). Fractionation was performed at a flow rate of 0.5 µL/min at 45 °C in a linear 60-min gradient from 100% solvent A (0.1% formic acid) to 100% solvent B (0.1% Formic acid, 60% acetonitrile). Mass spectra were acquired using an LTQ mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) for automated MS/MS. Data dependent MS analysis was performed by acquiring one FTMS scan followed by MS2 on the top five most intense ions. Dynamic exclusion was enabled at repeat count 1, exclusion list size 500, exclusion duration 180s, and exclusion mass width ( 1.5 m/z. Collision induced dissociation was performed by setting the ion isolation width at 2 m/z, normalized collision energy at 35%, activation Q at 0.25 and an activation time at 30 ms. Spectra were exported in mascot generic file format (.mgf) and analyzed using the Mascot search engine as detailed above. Standard search parameters included a peptide mass tolerance of 1.5 Da, peptide fragment tolerance of 0.8 Da, peptide charge of +2 or +3 and up to 1 missed cleavage allowed. Immunohistochemistry for Proteomic Validation and Cellular Localization. Immunohistochemistry was performed using endometrial biopsies from the same patients (fertile midproliferative (MP; n ) 7), midsecretory (MS; n ) 7) and infertile MS (n ) 8); Table 1) as has been previously described in detail.19 Primary goat polyclonal antibody against antithrombin III (AT III (C-18): sc-32453; Santa Cruz Biotechnologies, Santa Cruz, CA) or mouse monoclonal antibody raised against alpha-2-macroglobulin (A2M) (R-2 M (2D9): SC-69750; Santa Cruz) were used to visualize protein localization. Negative controls used matching concentration of goat IgG (Dako) substituted for primary antibody. All individual tissue samples were stained at one time for consistency. Analysis of Immunostaining. Staining was examined with an Olympus CH30 microscope and images captured with a Fujix Hc-2000 digital camera. Positive immunostaining was scored semiquantitatively by two independent observers blind to the nature of the tissue. Intensity of staining within the endometrial glandular and luminal epithelium was routinely analyzed. Tissue sections are analyzed for all cellular compartments. The secretory glandular and luminal epithelial cells were assessed and allocated a score from 0 to 3, where 0 is no staining, 1 is minimal staining, 2 is strong staining and 3 is intense staining. Statistical Analysis. Statistical analyses (including principal components analysis (PCA) and analysis of variance (ANOVA)) of proteomic data were performed automatically by the PG240 SameSpots software. Data are provided as mean normalized spot volume ( standard deviation. Hierarchical clustering and protein abundance heat maps were generated using open source R software (http://www.r-project.org/). Statistical analysis of immunohistochemistry was not performed due to the semiquantitative nature of the analysis.
Results Quantitative Proteomic Analysis of Uterine Fluid. Protein abundance in endometrial lavages collected from fertile women
Changes in Abundance of Endometrial Secreted Proteins
Figure 1. (A) Unsupervised hierarchical clustering and abundance mapping of proteins observed by 2D DiGE. Normalized protein spot volumes were used to perform unsupervised hierarchical cluster analysis. Green indicates decreased abundance; red indicates increased abundance. Patient groups (fertile; MP F, MS F or infertile MS IF) are indicated. Infertile uterine fluid signatures are highlighted (green box). (B) Representative 2D PAGE image (pH 4-7) of proteins isolated from uterine lavage. Differentially expressed spots that were picked and analyzed by mass spectrometry for protein identity are shown. Green circles demonstrates spots differentially expressed between fertile MP vs MS phase samples, blue circles demonstrates spots with altered expression between MS phase fertile vs infertile samples and yellow spots demonstrates differentially expressed spots common to both comparisons.
during the MP and MS phases, or from infertile women in the MS phase, were compared by 2D DiGE. Only proteins displaying significant (p e 0.05; ANOVA) expression changes across all gel images were considered to be present at altered levels. Biological variation between individuals within each group, as assessed by coefficient of variance (CV) in normalized protein spot volumes, was consistently between ∼30-40% suggesting significant heterogeneity between individual uterine lavage samples. Unsupervised hierarchical cluster analysis (HCA) was then conducted using log-normalized spot volumes from all experiments. HCA failed to separate patients based on cycle stage (Figure 1A); however highly similar signature profiles for two clusters representing the infertile population were observed, discriminating between samples from fertile versus infertile women. The HCA demonstrates the high degree of
research articles biological variability between the clinical samples that precludes differentiation based on total protein abundance profile. Differentially abundant protein spots (as shown in Figure 1B) were then submitted for identification by mass spectrometry. Proteins that were not successfully identified using MALDI-TOF were further analyzed by LC-MS/MS. The nature of the changes in protein abundance identified are discussed below. (i) Protein Abundance in the Midproliferative versus Midsecretory Phase of Fertile Women. Comparison of the MP and MS phase uterine lavage proteome of fertile women revealed 7 protein spots that were significantly lower (p < 0.05) in the MP versus the MS phase (Table 3); 4 of these were unique proteins, while the remaining 3 spots were identified as multiple isoforms of alpha-2-macroglobulin (A2M). Human serum albumin, targeted for immunodepletion prior to analysis, was also identified in one of the significantly altered protein spots; this is likely to be due to incomplete depletion and not a bona fide change in protein abundance between women. Overall there were no increases in protein abundance identified in the MP relative to the MS phase, which likely reflects the low secretory activity of the uterine glands during the MP phase20,21 and is consistent with the previously described reduced protein production in the MP phase and higher abundance of MS phase proteins.14 (ii) Protein Abundance in the Midsecretory Phase of Fertile versus Infertile Women. A total of 18 changes in protein spot abundance were observed between fertile and infertile women in the MS phase of their cycle (Table 4), including 6 proteins present at lower abundance and 12 at higher abundance in infertile compared to fertile women. Two spots identified as apolipoprotein-A4 (APOA4) were increased 1.4-1.5-fold in lavages from infertile compared to fertile women (Table 4). By contrast, apolipoprotein-A1 (APOA1) was significantly reduced (-1.5-fold) in infertile women. Three different spots of the serine protease inhibitor (serpin) alpha1-antichymotrypsin (AACT) were differentially expressed; two were reduced (-1.2 and -1.6-fold) in infertile women while one was increased (+1.8-fold) (all p < 0.05) (Table 4). This suggests possible isoform differences or post-translational modification of these proteins with respect to fertility. Multiple forms of inhibitory serpins have previously been identified in biological fluids.22 (iii) Protein Abundance Changes Common between Fertile and Infertile Women Across the Cycle. Three proteins were significantly different between both the MP versus MS and the fertile versus infertile comparisons (Tables 2 and 3). Activin receptor type-2B was higher in the MS versus MP phase and was further increased (+1.5-fold) in infertile versus MS fertile lavages. Similarly interalpha-trypsin inhibitor heavy chain H4 (ITIH4) was significantly increased in the MS phase and further increased (+1.4-fold) in the infertile versus the fertile lavages. By contrast A2M, which was higher in the MS than MP phase, was reduced in lavages from infertile (-1.5-fold) compared to fertile women. Immunolocalization of Antithrombin III and Alpha-2Macroglobulin in the Endometrium. To validate the observed protein abundance changes and determine the cellular localization of proteins, immunostaining was carried out on individual endometrial biopsies taken immediately following uterine lavage. Immunoreactive ANT3 protein was detected in endometrium from fertile and infertile women during all cycle phases of the cycle (Figure 2A-C). ANT3 was predominantly Journal of Proteome Research • Vol. 9, No. 12, 2010 6259
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Table 3. Differentially Expressed Proteins in Uterine Fluid Collected during the Mid-Proliferative versus Mid-Secretory Phase of the Normal Menstrual Cycle
1 2 3 4 5 6 7
a
entry name
protein description
p-value
fold changea
MOWSE score
matched peptides
% coverage
A2MG_HUMAN A2MG_HUMAN ALBU_HUMAN AVR2B_HUMAN A2MG_HUMAN A1AT_HUMAN IHRP_HUMAN
Alpha-2-macroglobulin Alpha-2-macroglobulin Serum albumin Activin receptor type-2B Alpha-2-macroglobulin Alpha-1-antitrypsin Interalpha-trypsin inhibitor family heavy chain-related protein (IHRP)
0.02 0.02 0.02 0.04 0.03 0.04 0.03
-1.7 -1.6 -1.5 -1.5 -1.4 -1.4 -1.3
74 69 85 61 73 113 87
5 8 9 6 6 10 9
4 7 11 19 5 33 10
Negative fold change indicated that the protein was decreased in the midproliferative phase compared with the midsecretory phase.
Table 4. Differentially Expressed Proteins between Fertile versus Infertile Uterine Fluid Collected during the Mid-Secretory Phase of the Menstrual Cycle data aquired from
entry name
protein description
p-value
fold changea
MOWSE score
matched peptides
% coverage
1 2 3
LC-MS/MS MALDI-TOF-MS spectra LC-MS/MS
TTHY_HUMAN ANT3_HUMAN Q53F31_HUMAN
0.03 0.04 0.03
+1.9 +1.8 +1.8
184 89 119
8 16 11
53 44 10
4 5 6 7 8 9 10 11 12 13
MALDI-TOF-MS MALDI-TOF-MS MALDI-TOF-MS MALDI-TOF-MS MALDI-TOF-MS LC-MS/MS MALDI-TOF-MS MALDI-TOF-MS MALDI-TOF-MS MALDI-TOF-MS
spectra spectra spectra spectra
AACT_HUMAN A1AT_HUMAN AACT_HUMAN APOA1_HUMAN ANT3_HUMAN B2RMS9_HUMAN APOA4_HUMAN AVR2B_HUMAN A2MG_HUMAN ITIH4_HUMAN
0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.04 0.03 0.05
+1.8 -1.6 -1.6 -1.5 +1.5 +1.5 +1.5 +1.5 -1.5 +1.4
84 113 104 86 56 308 56 61 66 87
9 10 11 11 4 5 7 6 4 9
39 33 23 31 15 6 19 19 21 10
14 15 16
MALDI-TOF-MS spectra LC-MS/MS MALDI-TOF-MS spectra
APOA4_HUMAN A1A508_HUMAN B4DP56_HUMAN
0.01 0.04 0.05
+1.4 +1.3 +1.3
77 64 66
9 4 7
23 7 25
17 18
LC-MS/MS MALDI-TOF-MS spectra
ANGT_HUMAN AACT_HUMAN
Transthyretin Antithrombin-III (SERPINC1) Vitamin D-binding protein (DBP) variant Alpha-1-antichymotrypsin Alpha-1-antitrypsin Alpha-1-antichymotrypsin Apolipoprotein A-I Antithrombin-III (SERPINC1) Interalpha (Globulin) inhibitor H4 Apolipoprotein A-IV precursor Activin receptor type-2B Alpha-2-macroglobulin Interalpha-trypsin inhibitor heavy chain H4 Apolipoprotein A-IV precursor PRSS3 Unnamed protein product similar to Creatine kinase B-type Angiotensinogen Alpha-1-antichymotrypsin
0.03 0.03
-1.2 -1.2
250 88
9 8
22 20
spectra spectra spectra spectra spectra
a Positive fold change indicated that the protein was increased in the infertile lavage collected in the midsecretory (MS) phase when compared to fertile MS phase. A negative fold change indicated that the protein was reduced in the infertile lavage (MS) when compared to fertile MS phase.
localized to glandular and luminal epithelium (Figure 2A-C), with additional strong staining in the decidualising stromal cells (Figure 2B) surrounding the spiral arterioles and in leukocytes (Figure 2C). Analysis of glandular and luminal epithelial immunoreactive ANT3 demonstrated higher levels in the MP versus MS phase endometrial tissue of fertile women (Figure 2D). Similarly, the intensity and localization of ANT3 immunostaining varied with fertility status (Figure 2B, C and E). Increased epithelial staining for ANT3 was also observed in the endometrium of infertile compared to fertile women (Figure 2E), consistent with the elevated ANT3 levels in uterine fluid from infertile versus fertile women detected by 2D-DiGE (Table 4). Immunoreactive A2M was detected in all endometrial samples and was localized to glandular and luminal epithelium, decidualized stromal cells and leukocytes (Figure 3A-C). Staining intensity for A2M was increased in endometrial tissue from the MS phase versus the MP phase in fertile women (Figure 3D), consistent with higher abundance of A2M observed in uterine fluid (Table 3). By contrast to the increase in A2M abundance observed by DiGE between fertile and infertile women, no difference in the intensity of A2M immunostaining between fertile and infertile women was observed in endometrial tissue (Figure 3E). 6260
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Discussion This paper describes the application of 2D-DiGE to analyze the proteome of uterine fluid from which the major serum proteins were depleted. Comparison of protein abundances between either different cycle stages, or fertile and infertile women, revealed that several proteins are present at altered levels dependent on cycle stage or fertility status. Interestingly many of these proteins are protease inhibitors, more commonly associated with serum. However, immunohistochemical analysis confirmed their expression in the luminal and glandular epithelium of endometrial tissuessuggesting that their presence in uterine fluid is due to secretion from the endometrial epithelium rather than transudation from blood. Our previous 2D DiGE analysis of the protein abundance changes between MP and MS phases in fertile women used endometrial tissue, and identified predominantly structural proteins.14 Comparison of these proteomic changes versus corresponding genomic analysis of the endometrium revealed that many of the observed protein abundance changes were not reflected at a genomic level.14 While useful, these findings highlighted the complexity of working with a highly dynamic tissue such as endometrium whose cellular composition and phenotype is constantly changing. By contrast, uterine lavage
Changes in Abundance of Endometrial Secreted Proteins
Figure 2. Immunohistochemical localization of antithrombin III (ANT3) in human endometrium. (A) Fertile midproliferative (MP) phase endometrium showing diffuse immunostaining for ANT3 in glandular epithelium (GE). Less intense staining was observed in fertile midsecretory (MS) phase endometrium GE, with patchy heterogeneous staining. A similar staining pattern was observed for ANT3 in endometrium from infertile women (C) although the staining was of a higher intensity. In addition to GE staining ANT3 was localized to subpopulations of leukocytes (2) and decidualising stroma (Dec) surrounding the spiral arterioles. Semiquantitative scoring of ANT3 immunostaining intensity in glandular and luminal epithelium in human endometrium from fertile women during the midproliferative (n ) 7) (MP) and midsecretory (MS) (n ) 7) phase and from infertile (n ) 8) women during the MS phase. Results are demonstrated by scatter plot (D and E). Comparison of ANT3 staining in MP and MS endometrium revealed ANT3 immunostaining intensity was higher in the MP phase compared to the MS phase (D). Increased ANT3 immunoreactivity was detected in infertile versus fertile endometrium (E). Scale bars: A ) 100 µm, B ) 25 µm, C ) 250 µm. No staining was observed when primary antibody was substituted with IgG, see inset in (A).
is a less complex sample type lacking the abundant structural proteins commonly found in tissue. Affinity depletion of serumassociated proteins,16 as used in our study, is essential to reduce the complexity of this sample type prior to analysis23 Proteomic analysis of uterine lavage offers a less invasive sample for analysis that could potentially be translated directly for clinical sampling and diagnostic testing. The current study identified 7 protein spots that were significantly higher in the MS than the MP phase, while 12 spots were elevated and 6 reduced in infertile versus fertile MS phase. While HCA analysis failed to completely separate the three patient groups assessed by DiGE, two clusters of infertile patients were observed. Given that unexplained infertility is a multifactoral problem the separation revealed by HCA analysis could reflect common protein changes within two distinct groups in the population. In addition, the biological variability observed with these samples suggests there is significant heterogeneity between individual uterine lavage samples as we have observed in tissue previously24 and may explain why HCA could not separate the groups completely. A key group of proteins identified with altered abundance in uterine lavages from women in this study were the protease inhibitors A2M, alpha-1-antitrypsin (A1AT), interalpha-trypsin inhibitor family heavy chain-related protein (IHRP), ANT3, angiotensinogen (ANGT), alpha-1-antichymotrypsin (AACT) and interalpha-(Globulin) inhibitor H4 (ITIH4). In addition to
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Figure 3. Alpha-2-macroglobulin (A2M) localization in human endometrium, immunostaining was observed in all glandular and luminal epithelium examined; fertile MP phase (A), fertile (B) and infertile (C) MS phase endometrium. Enhanced A2M immunostaining was observed in GE from fertile MS (B) compared with both MP phase tissue (A) and MS infertile tissue (C). Staining in GE appeared to be in “vesicles” (V) in all endometrial tissue examined. Scale bars: A 100 µm, B ) 250 µm, C ) 25 µm. No staining was observed when primary antibody was substituted with IgG, see inset in (A). Semiquantitative scoring of A2M immunostaining intensity in glandular and luminal epithelium from fertile midproliferative (MP) (n ) 7) and midsecretory (MS) (n ) 7) and from infertile (n ) 8) women during the MS phase. Results are demonstrated by scatter plot (D & E). Comparison of MP and MS tissue revealed A2M immunostaining intensity was higher in the MS phase compared to the MP phase (D). A2M staining in the fertile versus infertile group collected during the MS phase showed no difference in staining intensity in endometrial epithelium (E).
protease inhibitors the serine protease, PRSS3, was also found to be among the differentially expressed proteins along with apolipoprotein (APO) AI and AIV, transthyretin (TTHY), Activin receptor type-2B (AVR2B) and vitamin D-binding protein (DBP). Many of these proteins had not been previously identified in human uterine fluid, including ANGT, AVR2B ITIH4, PRSS3 and IHRP. Immunolocalization of the protease inhibitors ANT3 and A2M in endometrial tissue demonstrated their presence in glandular and luminal epithelium in MP and MS phase endometrium, suggesting that they could be secreted into the uterine lumen. Furthermore, immunoreactive ANT3 was elevated in endometrium from women with unexplained infertility, supporting the different levels of ANT3 in uterine fluid identified by 2D-DiGE. Immunoreactive A2M was lower in MP endometrium when compared to MS, consistent with its levels in uterine fluid where it was significantly (>1.4 fold) reduced in the MP compared to the MS phase. A2M immunostaining levels were not different in the fertile or infertile tissue, inconsistent with the findings in uterine fluid, this may be due to protein processing events within the uterine cavity, or may be due to the antibodies recognition of all forms of the protein. This is the first description of ANT3 and A2M production in nonpregnant human endometrium. ANT3 is a serine protease inhibitor (SERPIN), possessing anti-inflammatory properties and structurally related to other plasma protease inhibitors including AACT. ANT3 has roles in regulating normal blood coagulation and angiogenesis.25 SpeJournal of Proteome Research • Vol. 9, No. 12, 2010 6261
research articles cific ANT3 mutations have been identified in the population, and are linked to thrombophilia.26 Recently another serpin, SERPINA14, was detected in bovine uterine flushings, and immunolocalized to glandular epithelium. It’s secretion was enhanced by estradiol-17 beta and progesterone in vitro.27 SERPINA14 is postulated to assist in preparing the endometrial environment for embryo implantation. Since we observed elevated levels of ANT3 in uterine fluid and endometrial biopsies from women with unexplained infertility, excess ANT3 may be detrimental to pregnancy success. Intact latent and cleaved ANT3 have also been shown to possess potent antiangiogenic activity in mouse. A2M is a potent proteinase inhibitor that binds and deactivates a broad range of proteinases including matrix metalloproteinase’s,28 thus limiting trophoblast invasion.29,30 In interleukin (IL)11RR null mice, which have impaired decidualization and implantation failure, A2M is severely down-regulated.31 Importantly, in human endometrium where IL11 and its receptor (IL11R) are present in the epithelium; IL11 or IL11R are reduced in cohorts of infertile women.9 Crosstalk between A2M and IL11 in woman may therefore be important for embryo implantation. A2M is also localized to intracellular storage vesicles in the decidua basalis of human first trimester implantation sites.32 We likewise observed A2M in ‘storage like’ vesicles in epithelial cells (Figure 3C). The increase of A2M observed during the MS phase in both endometrial tissue and uterine fluid, and its decrease in uterine fluid from infertile women, suggests a role for A2M in endometrial receptivity. In addition to acting as a proteinase inhibitor A2M functions in immune regulation and cytokine binding.33 In particular, A2M has been implicated as a low-affinity binding protein for the TGF-β super family members, inhibin, activin and follistatin,34 known to play important roles in the uterus.35,36 Furthermore, Activin A is secreted into the human uterine cavity and is dysregulated in women with endometriosis37,38 this dysregulation is likely associated with the implantation failure and associated infertility observed in these patients.37 Given that A2M plays important roles in regulating Activin A levels in the circulation and within tissues34 A2M may play important roles in regulating the levels of these important TGF-β family members in the uterine microenvironment. Further studies are needed to determine whether A2M regulates TGF-β members action in the uterine cavity. Two other differentially expressed proteases, AACT and A1AT, previously localized to whole glands and isolated epithelial cells in the human endometrium,39 have been suggested to regulate the protease activity of the implanting blastocyst or the endometrial immunological response. In the current study, their elevation in the fertile MS compared to both fertile MP phase and infertile MS phase uterine fluid, supports this proposal. AACT also appears to undergo post-translational modification or isoform differences in some women with unexplained infertility. One AACT spot was increased in infertile uterine fluid, but two other AACT spots were decreased. Such modification may be important for the bioactivity and function of AACT within the uterine cavity. Isoform changes are likely to be of considerable importance; many proteins elicit different effects depending on their methylation and glycosylation states.40,41 The soluble carrier apolipoproteins APOA1 and APOA4 were also detected as significantly different in uterine fluid of fertile versus infertile women. APOA1 is also reduced in eutopic endometrium from women with endometriosis42 in whom 6262
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Hannan et al. infertility is common. APOA1 is similarly present in mouse uterine fluid,43 where it is proposed to nutritionally support the peri-implantation blastocyst, since it is endocytosed into the trophectoderm of mouse blastocyst.44 APOA1 was also identified in ovine uterine fluid on day 17 of pregnancy and postulated to play a role in lipid transport and/or accumulation during implantation.45 Together, these data support a role for APOA1 in the establishment of pregnancy in humans. APOA4 has previously been detected in human follicular fluid.46 It participates in lipid metabolism and plays key roles in cholesterol homeostasis and steroidogenesis.47 Thus, it may also have nutritional and endocrine roles during implantation. As mentioned previously, ANGT (the precursor to angiotensin) is identified in the current study as a novel component in human uterine fluid and is significantly reduced in women with unexplained infertility. Recently polymorphisms in the angiotensin I-converting enzyme (ACE), have been associated with recurrent spontaneous miscarriage and reduced reproductive success in women delaying childbearing.48 Whether the reduced levels of ANGT observed in the uterine cavity of infertile women is related remains to be investigated. Vitamin D binding protein (DBP), which was elevated in the uterine cavity of infertile women, is multifunctional with roles including transport of vitamin D metabolites, fatty acid binding, actin sequestration and macrophage activation.49 It can induce cellular proliferation and migration.50 Both human endometrial epithelial cells and embryonic tissues express the vitamin D receptor (VDR). Vitamin D can influence endometrial receptivity, at least in part through regulation of HOX genes,51 key genes in embryonic development and endometrial function. Recently the same vitamin D binding protein variant was identified in pooled uterine fluid as found in the current study, increasing with progression from the early secretory to the MS phase.52 The elevated levels of DBP in the uterine cavity of infertile women shown here call for further investigation of the roles of vitamin D in the human uterine cavity during the window of implantation. Abundant serum proteins comprise approximately 90% of the total protein content of human uterine lavages (Hannan et al., 2009). Human serum albumin, although targeted for immunodepletion prior to analysis was identified as reduced in MP versus MS lavages. As such, the significant difference observed for albumin should be interpreted with caution. No cytokines or growth factors were identified in this study. This is likely to reflect the limited sensitivity of the 2D DiGE approach, rather than their absence, particularly for proteins of lower molecular mass. Further studies including fractionation of the lower molecular weight range and specific multiplex analysis of cytokines and growth factors in the uterine fluid are needed to identify such regulatory proteins, and whether their abundance changes in uterine fluid during the menstrual cycle. While most of the changes identified were of modest fold change given their change in abundance with endometrial receptivity we anticipate that they may play important roles in human endometrial receptivity and blastocyst implantation, further studies are required to investigate this.
Conclusions This study demonstrates the complexity of the human uterine secretome in the context of endometrial receptivity during the menstrual cycle. Analysis by 2D DiGE identified several soluble mediators that contribute to, and have potential
Changes in Abundance of Endometrial Secreted Proteins roles in, endometrial receptivity and support of the periimplantation conceptus. We have identified a previously unknown repertoire of proteins in the uterine cavity during the crucial peri-implantation stage; importantly, the uterine secretory profile was significantly altered in women with unexplained infertility compared to normal, fertile women. Further functional studies will seek to define those proteins important for establishing endometrial receptivity or of importance for supporting the peri-implantation blastocyst. These may provide diagnostic markers of endometrial receptivity, critical for improving current assisted reproductive technologies.
Acknowledgment. We thank Dr. Jenny Chen for technical assistance and Prof. David Robertson and Dr. Peter Stanton for discussion of the data. We also thank the patients for sample provision. Sister Judi Hocking for her assistance with endometrial biopsy collection and doctors at Monash IVF and Monash Medical Centre (Southern Health sites). This study was supported by NHMRC (Australia) Project/Program Grant 494802 and by the Victorian Government’s Operational Infrastructure Support Program. L.A.S. was supported by an NHMRC fellowship No. 388901. A.N.S. and A.R. were supported by Ovarian Cancer Research Foundation Fellowships. Supporting Information Available: Supplementary data section S1 showing additional MS details for identified proteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Salamonsen, L. A.; Kovacs, G. T.; Findlay, J. K. Current concepts of the mechanisms of menstruation. Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 1999, 13 (2), 161–79. (2) Dockery, P.; Li, T. C.; Rogers, A. W.; Cooke, I. D.; Lenton, E. A. The ultrastructure of the glandular epithelium in the timed endometrial biopsy. Hum. Reprod. 1988, 3 (7), 826–34. (3) Hempstock, J.; Cindrova-Davies, T.; Jauniaux, E.; Burton, G. J. Endometrial glands as a source of nutrients, growth factors and cytokines during the first trimester of human pregnancy: a morphological and immunohistochemical study. Reprod. Biol. Endocrinol. 2004, 2, 58. (4) Kane, M. T.; Morgan, P. M.; Coonan, C. Peptide growth factors and preimplantation development. Hum. Reprod. Update 1997, 3 (2), 137–57. (5) Jones, R. L.; Hannan, N. J.; Kaitu’u, T. J.; Zhang, J.; Salamonsen, L. A. Identification of chemokines important for leukocyte recruitment to the human endometrium at the times of embryo implantation and menstruation. J. Clin. Endocrinol. Metab. 2004, 89 (12), 6155–67. (6) Hannan, N. J.; Salamonsen, L. A. Role of chemokines in the endometrium and in embryo implantation. Curr. Opin. Obstet. Gynecol. 2007, 19 (3), 266–72. (7) Gray, C. A.; Burghardt, R. C.; Johnson, G. A.; Bazer, F. W.; Spencer, T. E. Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation. Reproduction 2002, 124 (2), 289–300. (8) Burton, G. J.; Jauniaux, E.; Charnock-Jones, D. S. Human early placental development: potential roles of the endometrial glands. Placenta 2007, 28 (Suppl A), S64–9. (9) Dimitriadis, E.; Stoikos, C.; Stafford-Bell, M.; Clark, I.; Paiva, P.; Kovacs, G.; Salamonsen, L. A. Interleukin-11, IL-11 receptoralpha and leukemia inhibitory factor are dysregulated in endometrium of infertile women with endometriosis during the implantation window. J. Reprod. Immunol. 2006, 69 (1), 53–64. (10) Mikolajczyk, M.; Wirstlein, P.; Skrzypczak, J. Leukaemia inhibitory factor and interleukin 11 levels in uterine flushings of infertile patients with endometriosis. Hum. Reprod. 2006, 21 (12), 3054–8. (11) Boomsma, C. M.; Kavelaars, A.; Eijkemans, M. J.; Amarouchi, K.; Teklenburg, G.; Gutknecht, D.; Fauser, B. J.; Heijnen, C. J.; Macklon, N. S. Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity. Reprod. Biomed. Online 2009, 18 (1), 85–94.
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PR1004828
