Temporal Proteomic Analysis of Human Cervicovaginal Fluid with Impending Term Labor Yujing J. Heng,†,‡ Megan K. W. Di Quinzio,†,‡ Michael Permezel,†,‡ Mustafa Ayhan,§ Gregory E. Rice,†,| and Harry M. Georgiou*,†,‡ Department of Obstetrics & Gynaecology, University of Melbourne, Parkville, Victoria, Australia, Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia, Proteomics Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia, and Translational Proteomics, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia Received October 5, 2009
Keywords: human pregnancy • cervicovaginal fluid • two-dimensional electrophoresis • MALDI mass spectrometry • serine proteases • annexin A3 • collagen
Introduction Term labor is characterized by a final pathway involving cervical ripening, myometrial activation and fetal membrane rupture. The precise biochemical mechanisms involved and the timing of these dynamic processes continue to be elucidated through the investigation of a variety of reproductive tissues and body fluids. Owing to the proximity to the cervix and overlying fetal membranes, exploration of the biochemical pathways surrounding human labor may be achieved by investigation of human cervicovaginal fluid (CVF). Understanding the biochemical mechanisms of human labor may provide the foundation for the discovery of biomarkers predictive of labor, both term and preterm. The CVF proteome in pregnant and nonpregnant women has been described by our laboratory1 and others.2–6 CVF is a complex biological fluid consisting of water, electrolytes, low molecular weight organic compounds (glucose, amino acids and lipids), and a vast array of proteins and proteolytic enzymes arising from plasma transudate, cervical/vaginal epithelial cells, the endocervix, chorion, vaginal microflora, as well as mucus produced by cervical crypts.1–6 We hypothesize that temporal changes in CVF proteins with impending term labor may reflect the biochemical changes occurring in the cervix and overlying fetal membranes. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), our laboratory has previously identified nine differentially expressed protein spots in a select part of the CVF proteome (12-29 kDa) in pregnant women 26-30 days before labor onset, 1-3 days before labor onset and during spontaneous term labor.7 These spots, representing eight different proteins, are involved in protease inhibition (cystatin-A), antiinflammatory cytokine activity (interleukin-1 receptor antagonist), fatty acid metabolism (fatty acid-binding protein), oxi* To whom correspondence should be addressed. University of Melbourne, Department of Obstetrics & Gynaecology, Mercy Hospital for Women, 163 Studley Road, Heidelberg, Victoria 3084, Australia. E-mail:
[email protected]. Phone: 61 (03) 8458 4368. Fax: 61 (03) 8458 4380. † University of Melbourne. ‡ Mercy Perinatal Research Centre. § Proteomics Laboratory, Baker IDI Heart and Diabetes Institute. | Translational Proteomics, Baker IDI Heart and Diabetes Institute.
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dative stress defense (thioredoxin, superoxide dismutase Cu-Zn, peroxiredoxin-2, glutathione S-transferase P) and calciumdependent phospholipid binding (annexin A3), and provide an insight into the timing and biochemical processes occurring in the cervix and fetal membranes before and during term labor. We have validated the utility of one of the proteins, interleukin-1 receptor antagonist, as a predictor of term labor.8 We now expand upon our previous findings by exploring temporal protein changes in the CVF proteome above 25 kDa, in order to identify further potential biomarkers of impending labor. The specific aim of this study is to characterize differential protein expression associated with labor by performing 2D-PAGE proteomic analysis on serial human CVF collected 14-17 days, 7-10 days, 0-3 days before spontaneous term labor and during term labor.
Materials and Methods Subjects and Sample Collection. This study was approved by the Mercy Health Research Ethics Committee (R56/06), Heidelberg, Victoria, Australia. Healthy, multiparous women (mean age ( standard deviation (SD), 37.3 ( 5.2 years; gravidity 3.6 ( 1.5; parity 1.4 ( 0.7) with an uncomplicated singleton pregnancy were recruited from the Mercy Hospital for Women Antenatal Clinic at approximately 36 weeks’ gestation. Multiparous women were recruited because they were more likely to undergo spontaneous labor. Serial CVF samples were collected at weekly intervals, including a sample obtained in spontaneous term labor before the rupture of fetal membranes. Labor was defined as regular uterine contractions and cervical dilatation of >3 cm. Nine subjects who spontaneously labored at term (mean gestation at delivery 40.1 ( 0.5 weeks) were retrospectively selected and included in the study. Each woman provided four serial CVF samples which were grouped into 14-17 days, 7-10 days, 0-3 days before labor and in-labor. Women were excluded if they had bacterial vaginosis or clinical signs of chorioamnionitis, or if a subject had vaginal digital examination in the previous 6 h. CVF was collected and processed according to a previously published protocol.1,7,8 Microbiological assessment of the upper vagina was ascertained once only at the time of recruitment 10.1021/pr900892f
2010 American Chemical Society
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Temporal Proteomic Analysis of Human CVF by obtaining a second swab from the posterior vaginal fornix. Routine microbiological culture and microscopy was performed according to standard laboratory methods (Microbiology Department, Austin Pathology, Austin Health, Heidelberg, Victoria, Australia). Sample Processing and 2D-PAGE. The 2D-PAGE protocol utilized in this study has been previously described in detail.1,7 To improve resolution of proteins above 25 kDa, seconddimension separation was performed on Criterion 10% Tris/ HCl polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA). Gels were stained with Sypro Ruby (Bio-Rad) and scanned with an FX-imager (Bio-Rad). Gel analysis was performed using PD QUEST v7.3.1 software (Bio-Rad). A ‘matchset’ consisting of all 36 gels (cropped and filtered) was generated and a region displaying clearly focused proteins (25-45 kDa) was selected for detailed analysis. Only protein spots common to all gels were subjected to statistical analysis and protein characterization. Statistical Analysis. PD QUEST assigns a volume (area × density) to each spot proportional to the amount of protein. The volume for each common protein spot was exported into Statistics Package for Social Sciences v17.0 (SPSS, Inc., Chicago, IL) for thorough statistical analysis. Data was assessed for normality using the Kolmogorov-Smirnov test. Data that were not normally distributed were square root transformed and homogeneity of variance was confirmed prior to statistical analysis. Two-way analysis of variance (ANOVA; univariate general linear model) was used to compare spot volumes between the groups (in-labor, 0-3 days, 7-10 days and 14-17 days prior to labor onset) where each group was entered as a fixed factor while the subject was included as a random factor to account for multiple sampling. Tukey’s Honestly Significant Difference (HSD) post hoc testing was performed where appropriate. To determine whether spot volumes were linearly changing with approaching labor, regression analysis was performed using two-way ANOVA with days from labor included as a covariate (continuous factor) and subjects included as a random factor. Statistical significance was assumed when p < 0.05. Mass Spectrometry. Protein spots of significance were manually excised, treated with trypsin and resultant peptides were analyzed by Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-ToF) mass spectrometry. Gel plugs were processed using the Ettan spot handling workstation (Amersham Biosciences, Pittsburgh, PA) and applied to the MALDIToF targetplate (AnchorChip, Bruker Daltonics, Bremen, Germany). Briefly, gel plugs were placed into 96-well microtiter plate wells and destained twice with 100 µL of methanol/ ammonium bicarbonate (100%/100 mM (v/v), 30 min) alternating with 3 × 5 min washes with ammonium bicarbonate (100 mM); dehydrated with 100 µL of 75% acetonitrile for 10 min; and left to dry for 23 min. Gel plugs were rehydrated and digested for 3 h at 37 °C with 20 µg/mL sequence grade modified porcine trypsin (Promega, Madison, WI) made in 20 mM ammonium bicarbonate. After adding 100 µL of acetonitrile/trifluoroacetic acid (100%/0.2% (v/v)) into the digested plugs, the peptides were extracted into a new microplate, further dried for 3 h at 40 °C and left overnight at room temperature. The dried peptides were resolubilized with 25 µL of acetonitrile/trifluoroacetic acid (100%/0.2%, v/v). The Ettan robot applied 0.3 µL of the peptide followed by 1 µL of matrix (4HCCA 0.3 g/L (Bruker Daltonics) in ethanol/acetone, 2:1) to
the AnchorChip (600 µm diameter) and was left to dry. Peptide Mass Fingerprint (PMF) data was collected using an Autoflex II mass spectrometer equipped with a 337 nm nitrogen laser, TOF/TOF technology (LIFT) module for ToF/ToF analysis and 2 GHz digitizer (Bruker Daltonics). Spectra were acquired in linear-positive mode under 20 kV of ion acceleration with the ion deflection at mass e800 m/z and time lag focusing at 120 ns. The data collections were done automatically over the m/z range of 800-4000 m/z using the “AutoXecute” function of the software, FlexControl 3.0 (Bruker Daltonics). Typically, 1000 shots (at 25 Hz frequency) were collected to give a comprehensive coverage for each sample spot. Each spectrum represented the average of 1000 laser shots at a fixed laser energy output. Raw MALDI-ToF spectra were processed using the software, FlexAnalysis v2.4 (Bruker Daltonics). BioTools v3.0 software (Bruker Daltonics) was then used to submit the processed spectra to the Swiss-Prot database using the MASCOT search program (Matrix Science). Typical search parameters were as follows: mass accuracy, 100 ppm; missed cleavage, 2; peptide tolerance, 0.2 Da; charge, 1+; enzyme, trypsin; fixed modifications, cysteine modified by carbamidomethyl; variable modifications, oxidation; taxonomy, Homo sapiens. The retrieved database protein hits (protein molar mass and pI values) were compared to those measured experimentally to gain confidence in the correctness of the identification of the protein. A MASCOT probability score is given with each entry retrieved from the database.
Results Microbiology Assessment. Common genital microflora were detected in all nine women including Group B Streptococcus, Ureaplasma spp. or Candida spp.. No subject had bacterial vaginosis and none had clinical signs of chorioamnionitis at delivery. Comparison of Protein Profiles. All 36 gels are displayed in Figure 1. The protein profile in the selected region (25-45 kDa, pI 5.5-7.0) was consistent between all gels with an average of 91.8 ( 17.3 (mean ( SD) spots detected. Thirty spots were confidently matched to all 36 gels (Figure 2). Eleven spots were identified as isomers of squamous cell carcinoma antigen 1 (SCCA1, also known as Serpin B3); nine spots were isomers of monocyte/neutrophil elastase inhibitor (MNEI, or Serpin B1); two spots were identified as albumin; one spot was annexin A3 (AnxA3); one spot was collagen R2 type IV; while the remaining six spots could not be characterized (Supplementary Table 1). Protein identities were verified at least twice. Differential Protein Expression with Labor Onset. Spot volumes were compared between the groups (in-labor, 0-3 days, 7-10 days and 14-17 days prior to labor) using two-way ANOVA. Regression analysis was performed on all 30 spots to determine the linear relationship between protein expression and time from labor. The expression of nine proteins was significantly different between the groups: SPP6901, SPP6904 and SPP7702, MNEI (p ) 0.010, p ) 0.019 and p ) 0.012, respectively); SPP7601, SPP7701 and SPP9403, SCCA1 (p ) 0.030, p ) 0.028 and p ) 0.007, respectively); SPP5301, AnxA3 (p ) 0.002); SPP6903, collagen type IV (p ) 0.011); and unidentified SPP9201 (p ) 0.005). Table 1 displays the mean spot volume ((SD) in-labor, 0-3 days, 7-10 days and 14-17 days prior to labor with Tukey’s HSD post hoc p-values of significant two-way ANOVA Journal of Proteome Research • Vol. 9, No. 3, 2010 1345
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Figure 1. Two-dimensional gels (raw images) of serial cervicovaginal fluid from nine subjects collected in term labor (first column, left), 0-3 days (second column), 7-10 days (third column) and 14-17 days prior to labor onset at term (fourth column).
analysis and percentage (%) change relative to labor. These spots were 62-85% significantly decreased during labor. From the regression analysis, five protein spots, SPP3902, SPP6901 and SPP6904, MNEI (p ) 0.010, p ) 0.003 and p ) 0.034, respectively); SPP6705, SCCA1 (p ) 0.023) and unknown SPP9201 (p ) 0.003) were significantly decreasing with approaching labor, while spot SPP2501 albumin (p ) 0.012) was significantly increasing toward labor (Figure 3).
Discussion Using conventional 2D-PAGE coupled with MALDI mass spectrometry, this comprehensive study has characterized an additional group of proteins in the human CVF that exhibit significant temporal changes in association with spontaneous term labor. This builds upon our previous findings of a group of differentially expressed proteins specifically involved in protease inhibition, anti-inflammatory cytokine activity and oxidative stress defense.7 The differentially expressed proteins presented in this current study are involved in serine and/or 1346
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cysteine protease inhibition (MNEI and SCCA1), calciumdependent phospholipid binding (AnxA3), an extracellular matrix (ECM) structural protein (collagen type IV) and a plasma carrier protein (albumin). Albumin was the only protein that increased with impending term labor. Tang and colleagues observed that CVF with an overabundance of polymorphonuclear leukocytes contained increased quantities of plasma proteins, including albumin.4 The cervical ripening process has been likened to an inflammatory reaction with increased influx of neutrophils and macrophages into the cervix.9 Labor is also an inflammatory process with increased leukocyte density in the myometrium and cervix during labor compared to nonlaboring controls.10,11 Thus, the increasing quantity of albumin in the CVF may indirectly reflect increased plasma transudate associated with an influx of leukocytes and an ongoing inflammation in the cervix and/or supracervical fetal membranes leading to ECM remodeling in preparation for labor.
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Temporal Proteomic Analysis of Human CVF
Figure 2. (A) A representative gel of human cervicovaginal fluid showing the boxed region that was subjected to spot analysis (raw image). (B) An enlarged image of the region (25-45 kDa) in which analysis was performed. (C) Gaussian image of (B) displaying all 30 common protein spots matched to all gels (marked with crosses and labeled with PD Quest generated spot numbers). Table 1. Mean Protein Spot Volumes (Area × Density) ( SD Are Compared Using Two-Way ANOVAa two-way ANOVA p-value
in-labor (n ) 9)
SPP6901 (MNEI)
0.010
4259 ( 4136
11638 ( 7179
SPP6904 (MNEI)
0.019
10279 ( 14162
23364 ( 14847
SPP7702 (MNEI)
0.012
1241 ( 1144
SPP7601 (SCCA1)
0.030
15355 ( 29691
46536 ( 40614
SPP7701 (SCCA1)
0.028
15222 ( 21328
SPP9403 (SCCA1)
0.007
812 ( 1060
SPP5301 (AnxA3)
0.002
7344 ( 4457
SPP6903 (Col IV)
0.011
1614 ( 1428
SPP9201 (Unknown)
0.005
4069 ( 6584
63352 ( 59401 p ) 0.021 (V76%) 5020 ( 5590 p ) 0.004 (V84%) 33673 ( 24653 p ) 0.002 (V78%) 10582 ( 12315 p ) 0.020 (V85%) 8703 ( 5924
protein spot no.
a
0-3 days before labor (n ) 9)
3714 ( 3234
7-10 days before labor (n ) 9)
14-17 days before labor (n ) 9)
20547 ( 23379 p ) 0.036 (V79%) 29367 ( 18354 p ) 0.019 (V65%) 5680 ( 4673 p ) 0.007 (V78%) 49207 ( 38177 p ) 0.043 (V69%) 45620 ( 39155
26517 ( 32995 p ) 0.009 (V84%) 26495 ( 11924
51877 ( 35399
1573 ( 1092
1629 ( 1542
29057 ( 28500 p ) 0.012 (V75%) 9506 ( 8166 p ) 0.017 (V83%) 10661 ( 8452 p ) 0.018 (V62%)
18097 ( 17817
3545 ( 3699 44857 ( 28839
6153 ( 4158 11700 ( 5288 p ) 0.005 (V65%)
Post hoc p-values (Tukey’s HSD) and % change indicate significant differences compared with in-labor samples.
AnxA3 (previously termed lipocortin III or placental anticoagulant protein III) is a 33 kDa protein first identified in the human placenta12 that has been localized to the cytoplasm and membrane of trophoblasts in placental villous tissues.13 AnxA3 is also present in the cytosolic granules of human neutrophils14 and a 36 kDa variant is found in monocyte granules.15 AnxA3 may be released during degranulation of activated neutrophils
or monocytes infiltrating the cervix, thus, explaining its presence in the CVF. The physiologic role of AnxA3 has not been fully determined. AnxA3 has been found to inhibit the activity of phospholipase A2 and act as an anticoagulant in vitro.12 Phospholipase A2 is involved in the first step of prostaglandin synthesis by hydrolyzing arachidonic acid from cell membranes. Free arachidonic Journal of Proteome Research • Vol. 9, No. 3, 2010 1347
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Figure 3. (A-E) Protein spots SPP3902, SPP6901 and SPP6904 (MNEI), SPP6705 (SCCA1) and SPP2901 (unknown) all show a decreasing trend with approaching labor. (F) Protein spot SPP2501 (albumin) significantly increased with approaching labor. The solid line represents the mean regression while the dotted lines represent the 95% confidence interval.
acid is then further metabolized to prostaglandins.16 We observed a significant decrease in AnxA3 during labor. This withdrawal of AnxA3 would theoretically increase the activity of phospholipase A2, thereby facilitating the synthesis of prostaglandin leading to a successful labor. However, Kim et al. studied the effects of AnxA3 on cytosolic phospholipase A2 activity and reported no inhibitory effect.17 More studies are therefore warranted to clarify the physiologic role of AnxA3 and its interaction with phospholipase A2. Recently, AnxA3 was reported to be elevated in the CVF of women with preterm delivery compared to nonlaboring controls and threatened preterm labor.18 We can provide two explanations for this discrepancy. The study by Pereira et al.18 was performed on a small sample size (n ) 5 pooled from each group) using gestation-matched, nonlaboring controls and compared AnxA3 with threatened preterm labor and preterm delivery using multidimensional protein identification technology. By contrast, the current study using 2D-PAGE describes 1348
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the temporal changes of AnxA3 within the same subject in a larger sample set (n ) 9) and stratified according to days from labor onset. Second, it is possible that spontaneous preterm delivery is associated with exacerbated inflammation compared to normal term delivery. Nevertheless, both studies suggest that AnxA3 is involved in labor and its role during term and preterm labor remains to be elucidated. Two additional proteins of interest identified in this study were MNEI and SCCA1. These proteins are members of the superfamily of serine protease inhibitors (serpins) involved in inflammation and extracellular remodeling.19 MNEI (serpin B1) and SCCA1 (serpin B3) are ovalbumin-like serpins generally found in cells that secrete proteases into the external environment.20 Extracellular MNEI and SCCA1 protect neighboring cells and tissues from proteolytic damage.21 MNEI is abundant in neutrophils and monocytes and is a highly efficient inhibitor of the neutrophil granule proteases: neutrophil elastase, proteinase 3 and cathepsin G.22,23 Neutrophil elastase, proteinase
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Temporal Proteomic Analysis of Human CVF 3 and cathepsin G are involved in connective tissue remodeling at the site of inflammation. In addition, these serine proteases play an important role in cell signaling and contribute to the control of the inflammatory process by activating cytokines, growth factors and stimulate various cellular receptors. For example, proteinase 3 can activate tumor necrosis factor (TNF)R, IL-1β and IL-1824 while neutrophil elastase can up-regulate IL-8 via toll-like receptor 4 to contribute to local inflammatory processes.25 Neutrophil elastase is significantly increased in amniotic fluid during term and preterm labor.26 Using immunohistochemistry, Osman and colleagues10 observed higher expression of neutrophil elastase (localized within neutrophils) in the cervix during term labor compared with cervices before labor. In this current study, the expression of selected isoforms of MNEI was significantly decreasing toward and during labor. Reduced MNEI inhibition may, in turn, enhance neutrophil serine proteases to remodel the ECM, leading to cervical ripening, cervical dilation and membrane rupture during labor. As serine protease inhibitors such as MNEI are also capable of suppressing the secretion of TNF-R from activated macrophages,27 the decrease in MNEI may further contribute to inflammatory or proteolytic processes occurring during labor. It is also attractive to speculate the importance of the relative balance between proteases and their inhibitors during ECM remodeling prior to and during labor. The other serpin of interest, SCCA1, is expressed by squamous epithelial cells of the skin, mucosa, lungs, cervix and vagina, and by squamous cell carcinomas.28 SCCA1 is a crossclass inhibitor of papain-like cysteine proteases, such as cathepsins L, K and S.29,30 Like MNEI, a reduction in SCCA1 expression toward and during labor may enhance protease activity, in particular, cathepsin L, permitting ECM remodeling. Cathepsin L is a lysosomal endopeptidase31,32 that degrades laminin, fibronectin, collagen types I, IV and XVIII33–35 and elastin.36,37 These cathepsin L substrates are essential components of the cervix (collagen type I)38 and fetal membranes (collagen type IV).39 The degradation of collagen type IV in the amnion and chorion basement membranes is essential for ECM weakening leading to membrane rupture.40 Not surprisingly, we found a significant decrease in a 39 kDa fragment of collagen type IV, suggesting the enhanced cleavage of collagen IV during labor. This decrease in collagen type IV is further supported by the discovery of increased matrix metalloproteinase-7 in the CVF during labor by our laboratory (manuscript submitted), which also cleaves collagen type IV. In summary, this study has identified temporal changes in MNEI, SCCA1, AnxA3, collagen type IV and albumin expression in association with term labor. Although these findings require validation using traditional immuno-based techniques with a larger sample size, the study has provided further insight into the biochemical pathways intimately involved in ECM remodeling of the cervix and supracervical fetal membranes. The differentially expressed proteins observed in this study reinforce the hypothesis that labor is indeed an inflammatory process and highlight the importance of serine and/or cysteine proteases and their inhibitors in labor. While these findings relate to term labor, the application of these findings in a preterm setting may be useful in developing predictive tools and targeted therapies in the future.
Acknowledgment. The authors thank the Mercy Hospital for Women medical and midwifery staff for
obtaining study samples, in particular, Valerie Bryant, Gabrielle Fleming, Mardi Reeves and Anne Beeston. Karen Oliva provided technical assistance with the Ettan workstation. Microbiological assessments were performed by the Microbiology Department, Austin Pathology, Austin Health, Heidelberg, Victoria, Australia. This work was supported by a National Health and Medical Research Council of Australia (NHMRC) Development Grant (No. 454451) and the Medical Research Foundation for Women and Babies. Y.J.H. is a recipient of the National Health and Medical Research Council Biomedical (Dora Lush) Postgraduate Research Scholarship (No. 454880).
Supporting Information Available: Proteins identified. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Di Quinzio, M. K.; Oliva, K.; Holdsworth, S. J.; Ayhan, M.; Walker, S. P.; Rice, G. E.; Georgiou, H. M.; Permezel, M. Proteomic analysis and characterisation of human cervico-vaginal fluid proteins. Aust. N. Z. J. Obstet. Gynaecol. 2007, 47 (1), 9–15. (2) Dasari, S.; Pereira, L.; Reddy, A. P.; Michaels, J. E.; Lu, X.; Jacob, T.; Thomas, A.; Rodland, M.; Roberts Jr, C. T.; Gravett, M. G.; Nagalla, S. R. Comprehensive proteomic analysis of human cervical-vaginal fluid. J. Proteome Res. 2007, 6 (4), 1258–1268. (3) Shaw, J. L.; Smith, C. R.; Diamandis, E. P. Proteomic analysis of human cervico-vaginal fluid. J. Proteome Res. 2007, 6 (7), 2859– 2865. (4) Tang, L. J.; De Seta, F.; Odreman, F.; Venge, P.; Piva, C.; Guaschino, S.; Garcia, R. C. Proteomic analysis of human cervical-vaginal fluids. J. Proteome Res. 2007, 6 (7), 2874–2883. (5) Klein, L. L.; Jonscher, K. R.; Heerwagen, M. J.; Gibbs, R. S.; McManaman, J. L. Shotgun proteomic analysis of vaginal fluid from women in late pregnancy. Reprod. Sci. 2008, 15 (3), 263– 273. (6) Zegels, G.; Van Raemdonck, G. A.; Coen, E. P.; Tjalma, W. A.; Van Ostade, X. W. Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples. Proteome Sci. 2009, 7, 17. (7) Di Quinzio, M. K.; Georgiou, H. M.; Holdsworth-Carson, S. J.; Ayhan, M.; Heng, Y. J.; Walker, S. P.; Rice, G. E.; Permezel, M. Proteomic analysis of human cervico-vaginal fluid displays differential protein expression in association with labor onset at term. J. Proteome Res. 2008, 7 (5), 1916–1921. (8) Heng, Y. J.; Di Quinzio, M. K.; Permezel, M.; Rice, G. E.; Georgiou, H. M. Interleukin-1 receptor antagonist in human cervicovaginal fluid in term pregnancy and labor. Am. J. Obstet. Gynecol. 2008, 199 (6), 656. (9) Bokstro¨m, H.; Bra¨nnstro¨m, M.; Alexandersson, M.; Norstro¨m, A. Leukocyte subpopulations in the human uterine cervical stroma at early and term pregnancy. Hum. Reprod. 1997, 12 (3), 586–590. (10) Osman, I.; Young, A.; Ledingham, M.; Thomson, A. J.; Jordan, F.; Greer, I. A.; Norman, J. E. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labor at term. Mol. Hum. Reprod. 2003, 9 (1), 41–45. (11) Thomson, A. J.; Telfer, J. F.; Young, A.; Campbell, S.; Stewart, C. J.; Cameron, I. T.; Greer, I. A.; Norman, J. E. Leukocytes infiltrate the myometrium during human parturition: further evidence that labor is an inflammatory process. Hum. Reprod. 1999, 14 (1), 229– 236. (12) Tait, J. F.; Sakata, M.; McMullen, B. A.; Miao, C. H.; Funakoshi, T.; Hendrickson, L. E.; Fujikawa, K. Placental anticoagulant proteins: isolation and comparative characterization of four members of the lipocortin family. Biochemistry 1988, 27 (17), 6268–6276. (13) Zhang, Y.; Zhang, Y. L.; Feng, C.; Wu, Y. T.; Liu, A. X.; Sheng, J. Z.; Cai, J.; Huang, H. F. Comparative proteomic analysis of human placenta derived from assisted reproductive technology. Proteomics 2008, 8 (20), 4344–4356. (14) Ernst, J. D.; Hoye, E.; Blackwood, R. A.; Jaye, D. Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca2+-dependent aggregation of isolated specific granules. J. Clin. Invest. 1990, 85 (4), 1065–1071. (15) Le Cabec, V.; Maridonneau-Parini, I. Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translo-
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