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Research Article |
Departments of Reproductive Endocrinology2
Pathology,3 Women's Hospital,
Department of Genetics,4 Zhejiang University School of Medicine, Hangzhou, Zhejiang 310006, China
ABSTRACT
Early pregnancy loss is the most common complication of human reproduction. Given the complexities of early development, it is likely that many mechanisms are involved. Knowledge of differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying early pregnancy loss. To identify proteins with different expression profiles related to early pregnancy loss, we applied a proteomic approach and performed two-dimensional gel electrophoresis (2-DE) on six placental villous tissues from patients with early pregnancy loss and six from normal pregnant women, followed by comparison of the silver-stained 2-DE profiles. It was found that 13 proteins were downregulated and 5 proteins were upregulated significantly (P < 0.05) in early pregnancy loss as determined by spot volume. Among them, 10 downregulated and 2 upregulated spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anomalies of these proteins, including three principal antioxidant enzymes (copper/zinc-superoxide dismutase, peroxiredoxin 3, and thioredoxin-like 1 protein), S100 calcium binding protein, galectin-1, chorionic somatomammotropin hormone 1, transthyretin, fas inhibitory molecule, eukaryotic translation elongation factor, RNA-binding protein, ubiquitin-conjuating enzyme E2N, and proteasome beta-subunit, indicate widespread failure in cell regulations and processes such as antioxidative defense, differentiation, cell proliferation, metabolism, apoptosis, transcription, and proteolysis in early pregnancy loss. This study has identified several proteins that are associated with placentation and early development, shedding a new insight into the proteins that may be potentially involved in the pathophysiological mechanisms underlying early pregnancy loss.
early development, placenta, pregnancy
INTRODUCTION
Human reproduction is described as being extremely inefficient compared to that of other mammalian species. The precise incidence of early pregnancy loss (EPL) at different periods of gestation has been more clearly defined with the routine use of transvaginal ultrasound and urine tests for human chorionic gonadotrophin (hCG). The incidence of pregnancy loss prior to completion of the first trimester is high, estimated at 50% to 70%. Loss rates drop rapidly with increasing gestational age, and the fetal loss rate after 11 wk is <3% [1].
The causes of EPL include chromosomal defects, endocrine diseases, anatomical abnormalities of the female genital tract, infections, immunologic factors, chemical agents, hereditary disorders, trauma, maternal diseases, and psychological factors [1]. Given the complexities of early development, it is likely that many mechanisms are involved in the pathophysiology of EPL.
In EPL, the trophoblastic shell is thin and fragmented, and trophoblastic infiltration of both the lumen of the endometrial vessels and the decidua is reduced in two thirds of cases. This failure in placentation can be a primary event, as a result of major chromosomal abnormality, or a secondary event in an early fetal demise due to a major developmental abnormality. The degree of placentation defect and trophoblastic apoptosis is increased in cases of miscarriage, independently of the presence or absence of a chromosomal abnormality [2–5]. There is also increasing evidence showing a correlation between miscarriage and an anomaly of some of the principal enzymes involved in the metabolism of reactive oxygen species (ROS) [6–8]. In addition, cellular endocrinological and proliferative impairments may be temporary and secondary to the increase in placental oxygen tension [2], and contribute partly to the complex pathophysiological features of EPL [5, 9]. The above investigations focused on detection of mRNA by PCR-based techniques and Northern blotting and of proteins by Western blotting and other immunological methodologies, resulting in a limited understanding of the molecular mechanisms underlying EPL. Thus identification of putative proteins affected in EPL is indispensable to our understanding of the complex biomolecular background associated with this multifactorial event. The proteomic tools enable a systematic analysis of the proteins involved on a much larger scale than is being currently undertaken.
Proteomics, the large-scale study of proteins, contributes greatly to our understanding of gene function in the postgenomic era. High-resolution two-dimensional gel electrophoresis (2-DE) is an excellent tool for proteomic analysis and can be used to compare patterns of protein expression in cells under various physiological and pathological conditions. More importantly, mass spectrometry, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides a powerful tool to rapidly identify protein spots on 2-DE gel [10–12]. Thus proteomics, in conjunction with high throughput polymorphism analysis, may enable us to unravel particular molecular complexes or pathways in the pathogenesis of EPL.
The overall aim of this study was to obtain more comprehensive information on the alterations of protein expression in parallel profiling of the placental villous tissues of EPL with different etiologies by using 2-DE and MALDI-TOF MS. The identification of these proteins will facilitate a better understanding of the molecular mechanisms underlying EPL.
MATERIALS AND METHODS
Subjects and Sample Collection
During the period October 2004 to December 2005, 36 pregnant women with a diagnosis of EPL attending the Department of Obstetrics and Gynecology, Women's Hospital, China, were recruited as the study group. These pregnant women had vaginal bleeding and/or lower abdominal pain for the first time in the previous few days (0–2 days). The diagnosis of EPL was based on the clinical history, clinical examination, and transvaginal ultrasound (TVU) results. In cases where pregnancy structures (a gestational sac without fetal heart rate) were identified by TVU, the final diagnosis of EPL was made. Inclusion criteria were a gestational age at 8 wk (based on the first day of the last menstrual period) and no history of recurrent spontaneous abortions, chromosomal abnormalities, endocrine diseases, anatomical abnormalities of genital tract, infections, immunologic diseases, trauma, internal diseases, or any chemical agent intake before their elective terminations. Sixteen age-matched women with a normal pregnancy who were undergoing terminations of pregnancy for psychological reasons at the same gestational age were designated as the control group. Placental villous tissues were taken through the cervix during dilatation and aspiration according to strict clinical procedures. Informed consent was obtained from each woman for the use of placental villous tissue, and the study was approved by the Ethical Review Committee of Women's Hospital, Zhejiang University School of Medicine.
Sample Preparation
Tissues were collected in ice-cold and sterile phosphate-buffered saline, dissected using a microscope to remove endometrial tissues and fetal membranes, and finally stored at –80 °C until further analysis.
Among all the placental villous tissues collected, six from the EPL group and six from the control one were randomly selected for proteomic analysis. A total of 300 mg of each placental villous sample was homogenized and solubilized in 1000 µL of lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% immobilized pH gradient (IPG) buffer (pH 4–7), and 65 mM dithiothreitol (DTT) and kept on ice for 1 h. After sonication, the lysed cells were centrifuged at 15 000 g at 4°C for 60 min to remove debris. Protein concentrations were determined using the Bradford assay [13]. All samples were stored at –70°C prior to electrophoresis. For each sample, three separate experiments were run independently on three analytical 2-DE gels.
Two-Dimensional Gel Electrophoresis
2-DE was performed using the Amersham Bioscience 2-DE system according to the manufacturer's instructions (Amersham Biosciences, Uppsala, Sweden). For the first dimension, 300 µg of total protein was mixed with a rehydration solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer (pH 4–7), 18 mM DTT, and a trace of bromophenol blue to a total volume of 400 µl and analyzed for both the control and the EPL groups. Rehydration for 12 h and isoelectric focusing (IEF) were conducted automatically on the IPGphor (Amersham) platform at 20°C, and the total Vh of IEF is 65 000–70 000 Vh. Following IEF separation, the strips were equilibrated for 15 min in a buffer (50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue) containing 1% DTT and subsequently in the same equilibration buffer containing 2.5% iodoacetamide for 15 min. The second separation was carried out at 15°C on 12.5% SDS slab gels using an ETTAN DALT II electrophoresis system (Amersham), with the IPG strips sealed on the top of the gels with 0.5% agarose. SDS-PAGE was run at constant power of 2.5 W/gel for 30 min, then switched to 15 W/gel until the bromophenol blue marker reached the bottom of the gel.
Gel Staining, Image Analysis, Data Normalization, and Statistical Analysis
Silver staining was performed using the method described by Swain and Ross [14], except that the treatment with glutaraldehyde (a cross-linking and sensitizing agent) was omitted [15]. The silver-stained 2-DE gels were scanned, and the digitized images were analyzed with PDQuest (v 7.0, Bio-Rad). Image analysis included the following procedures: spot detection, spot editing, background subtraction, and spot matching. Spot detection was controlled by four parameters, including sensitivity, operator size, noise factor, and background. The same parameters were used to detect spots in all of the gels in order to guarantee comparability between the gels. One of the gels in the control group was chosen as a reference gel before matching. To avoid biological variation, the spots unmatched with the reference gel were not added to the reference gel. The amount of a protein spot was expressed as the volume of the spot, which was defined as the sum of the intensities of all the pixels that made up the spot. In order to correct for variations due to silver staining and quantify protein spots, the individual spot volume was normalized as a percentage of the total volume of all the spots detected on the gel.
Enzymatic In-Gel Digestion
Selected protein spots were excised from the gel, and in-gel digestion with trypsin was performed according to published procedures with slight modification [16, 17]. The gel pieces were washed twice with water and destained in freshly prepared destaining solution (30 mM posassium hexacyanoferrate: 100 mM sodium thiosulfate, 1:1[v/v]) for 1–2 min until the brown color disappeared. They were then rinsed sequentially with water and 100 mM ammonium bicarbonate, respectively. The gel pieces were shrunk by dehydration in acetonitrile (ACN) and dried in a vacuum centrifuge (SpeedVac, Thermo Savant, Holbrook, NY). Then the gel pieces were swollen in a digestion buffer containing 40 mM ammonium bicarbonate, 10% ACN, and 20 µg/ml of trypsin (Sigma, proteomics sequencing grade) in an ice cold bath. After 30 min, the supernatant was removed and replaced with 30 µL of the same buffer but without trypsin. The gel pieces were kept wet during enzymatic cleavage (37°C, overnight). The supernants were collected and peptides were extracted twice by two changes of 5% trifluoroacetic acid (TFA)/50% CAN (15 min for each change) at room temperature (RT) and dried out.
MALDI-TOF Mass Spectrometry Analysis and Database Searching
The peptide extracts dissolved in 5 ul of 0.1% TFA were mixed with 5 µl of saturated 
-cyano-4-hydroxycinnamic acid (HCCA, Applied Biosystems) in 50% ACN/0.05% TFA. Aliquots of 2 µl were applied onto a target plate and allowed to air-dry. Mass analysis of peptide mixtures was performed using a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems) with a 337-nm N2 UV laser. The instrument was operated in the reflector/delayed extraction mode with an accelerating voltage of 20 kV, grid voltage of 67%, and delay time of 200 ns. In general, 200 shots were applied for each sample, with a mass range from 900 to 4000 Da. P14R (Mr 1532.8582) and ACTH fragment 18–29 (Mr 2464.1989) were used for external calibration. The raw spectra files were analyzed using the Data Explorer v 4.0 software (Applied Biosystems). After baseline correction, noise filter, and peak deisotoping, the raw peaks were converted into isotopic peaks. The mass list was put in the MASCOT search engine (Matrix Science, London, UK). For protein search the National Center for Biotechnology Information (NCBI) Homo sapiens protein database (December 2004) was used with the following parameters: using monoisotopic masses, a maximum ± 0.3 Da mass tolerance, cysteine in carboxyamidomethyl form and methionine in oxidization form, and an allowance for up to one missed cleavage per peptide. Proteins matching more than four peptides and with a MASCOT score higher than 64 were considered significant (P < 0.05).
Immunohistochemistry
Immunohistochemistry (IHC) was performed on seven representative proteins, namely copper/zinc-superoxide dismutase (Cu/Zn-SOD), peroxiredoxin 3 (PRDX3), thioredoxin-like 1 protein (TXNL1), S100 calcium binding protein (S100A11), galectin-1 (LGALS1), chorionic somatomammotropin hormone 1 (CSH1), and transthyretin (TTR). These proteins were selected based on the availability of suitable antibodies against them. Thirty placental villous tissues from the EPL group and 10 from the control one (none of them were analyzed by 2-DE) were used to measure the distribution and abundance of these proteins in the placental villous tissues by a two-step IHC procedure. Paraffin blocks were cut into 4-µm-thick sections. Then the sections were deparaffinized in xylene and rehydrated in graded alcohol concentrations. Nonspecific binding was blocked by preincubation with blocking solution for 5 min, and then the sections were incubated for 1 h at RT with antibodies against Cu/Zn-SOD (US Biological, 1:100 dilution), PRDX3 (USBiological, 1:500 dilution), TXNL1 (US Biological, 1:200 dilution), S100A11 (Abcam Ltd., UK, 1:200 dilution), LGALS1 (BioVision, 1:100 dilution), CSH1 (Gene Tech Biotechnology, China), and TTR (DAKO Co., Denmark, 1:200 dilution). After incubation with primary antibodies, sections were incubated with horseradish peroxidase (HRP) conjugated goat anti-chicken IgG (Sant Cruz Biotechnology, 1:400 dilution) or goat anti-rabbit IgG (USBiological, 1:200 dilution) for 0.5 h at RT. Substrate-chromogen DAB reagent was then added to each section following rinse, and finally hematoxylin solution was used to stain nuclei. The slides were washed in PBS (three times for 5 min each) between each incubation step. Negative controls, which omitted incubation with the primary antibody, were processed along with all the samples.
Western Blot Analysis
Western blot on LGALS1, SOD, and TXNL1 was performed on 12 pairs of placental villous tissues from the EPL and the control groups (six pairs of them were analyzed by 2-DE, while others were randomly selected from the rest). For each, 100 mg of tissue was lysed in sample buffer (50 mM Tris-HCl, pH 6.8, 1% NP-40, 0.25% sodium desxycholate, 4% SDS, 20% glycerol, 0.2 M DTT, 0.02% bromophenol blue). Portions of the cell lysates that contained 30 µg of protein were subjected to 12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked at RT for 1 h in TBST (50 mmol/l Tris-Cl, pH 7.6, 150 mmol/l NaCl, 0.1% Tween 20) containing 5% nonfat milk, and then incubated with anti-LGALS1 (BioVision, 1:200 dilution), anti-SOD (USBiological, 1:4500 dilution), anti-TXNL1 (USBiological, 1:1000 dilution), and anti-ß-Actin antibody (Santa Cruz Biotechnology, 1:500 dilution) at 4°C overnight. The membranes were washed in TBST for 15 min and incubated with goat anti-rabbit (USBiological, 1:5000 dilution ) or anti-chicken HRP conjugated IgG (USBiological, 1:10 000 dilution) for 1 h at RT. The membranes were washed three times in TBST and processed for chemiluminescence with ECL detection kits according to the manufacturer's instructions (Amersham). Equal protein loading was confirmed by exposure of the membranes to the anti-ß-Actin antibody.
Statistical Analysis
Data from 2-DE image and Western blot analysis were exported into Microsoft Excel 2003. Statistical analysis was performed using the paired t-test. A value of P < 0.05 was considered significant. Only proteins that demonstrated significant and consistent changes (increased or decreased) were used for further proteomic analysis. Data were expressed as mean ±SD.
RESULTS
Differential Analysis of 2-DE Protein Maps of Disease and Control Groups
Three pairs of gels from the EPL and the control groups were analyzed for quantitative spot comparisons with image analysis software from PDQuest. 1735 ± 121 protein spots were detected in the EPL group and 1552 ± 153 spots in the control group. A total of 30 protein spots were decreased or increased 
2-fold in the EPL group compared to the control one. Among them, 13 proteins were downregulated and 5 proteins were upregulated significantly in the EPL group, determined by spot volume (P < 0.05) (Fig. 1). The relative volumes of these protein spots and the spot volume ratios between the EPL and the control groups for the up- and downregulated spots are shown in Tables 1 and 2, respectively.
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Proteins with Altered Expression Identified by MALDI-TOF Mass Spectrometry and Database Searching
The 18 protein spots with altered intensity in the EPL group were excised from the gels, digested with trypsin, and subsequently analyzed by MALDI-TOF MS. The resultant spectra were used to identify the proteins. Of the 18 spots excised from the gels, 12 spots were identified to consist of 10 downregulated and 2 upregulated proteins (Table 3). The identified proteins represented a heterogeneous group that included several antioxidant enzymes relevant to oxidative stress, such as Cu/Zn-SOD, PRDX3, and TXNL1; RNA-binding protein and eukaryotic translation elongation factor involved in transcription; fas inhibitory molecule (FAIM) associated with apoptosis; proteins mediating cell differentiation such as S100A11 and LGALS1; hormone such as CSH1; TTR involved in vitamin A and thyroid hormone metabolism; members of ubiquitin-proteasome complex such as proteasome ß-subunit and ubiquitin-conjugating enzyme E2N (UBE2N), which mediate proteolysis; and other proteins with unknown functions. The remaining six protein spots were not identified by peptide mass fingerprint, mainly because there was no satisfactory spectrum available.
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Verification of Differential Expressed Proteins by IHC
To validate the proteomic findings, we performed IHC on seven proteins, namely SOD, PRDX3, TXNL1, S100A11, LGALS1, CSH1, and TTR. In these cases, there were moderate decreases in the degree of protein staining in the cytoplasm of syncytiotrophoblastic and/or cytotrophoblastic cells in placental villous tissues from the EPL group compared to the control one (Fig. 2).
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Verification of Differential Expressed Proteins by Western Blot Analysis
From the candidates, LGALS1, SOD, and TXNL1 were selected for Western blot analysis. As shown in Figure 3, the expression changes of these selected proteins were consistent with the 2-DE and silver-staining results.
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DISCUSSION
In contrast to conventional biochemical approaches that monitor one or a few specific proteins at a time, proteomics, combining 2-DE and mass spectrometry, is the large-scale study of gene expression at the protein level, which can measure protein expression levels directly and provide insights into the activity state of all relevant proteins under different physiological or pathological conditions. In this study, among those proteins detected that either increased or decreased in the placental villous tissues from the EPL group, 12 proteins were identified by peptide mass fingerprinting. Immunohistochemistry on seven proteins (SOD, PRDX3, TXNL1, S100A11, LGALS1, CSH1, and TTR) and Western blot analysis of LGALS1, SOD, and TXNL1 validated that the differential expressions of proteins obtained from proteomic analysis were convincing. The remaining six proteins were not identified successfully, because the abundance of the protein was too low to produce a good spectrum or because the confidence of database searching through peptide mass fingerprint was not sufficient to yield unambiguous results.
The relationship of gestational age, fetal heart rate, and TVU was previously studied. It has been indicated that fetal heart rate can be detected with TVU as early as 46.9 ± 6.0 gestational days, suggesting a gestational sac with no fetal heart rate by 41 days after the last menses and thereafter may imply, but did not prove, death of conceptus [18]. In this respect, only those pregnant women with EPL at 8 gestational weeks when the diagnosis was first made were included in this study.
In early pregnancy, the trophoblastic cells are extremely sensitive to oxidative stress because of extensive cell divisions and the concomitant exposure of their DNA. Measurement in vivo supports the concept of a physiologically low O2 environment inside the human gestational sac during most of the first trimester of pregnancy [19]. In the early placenta the main antioxidant enzymes also appear to be directly regulated by O2 tension [20]. By contrast, in EPL, the premature entry of maternal blood into the intervillous space at this stage of pregnancy disrupts the placental architecture and is likely to be the mechanical cause of most miscarriages [2]. In this study, our data showed that placental villous tissues possessed lower concentrations of three antioxidant enzymes, Cu/Zn-SOD, PRDX3, and TXNL1, at 8 wk gestational age of EPL compared to those of normal pregnancy, suggesting that the depletion of placental antioxidant defenses is a key factor in EPL. In these circumstances indiscriminate damage to proteins, lipids, and DNA severely impairs normal trophoblastic functions and may even lead to cell death.
SOD, as one of the principal antioxidant enzymes against ROS, was thought to offer cell protection against the damage caused by the increased oxidative stress associated with pregnancy. In placental villous tissues the concentration of the enzyme increases with gestational age [21]. Our observation on the decrease in Cu/Zn-SOD level in the placental villi of EPL further supports the previous findings that Cu/Zn form of the enzyme is downregulated in missed or spontaneous abortion early in the first trimester of periphery circulation and decidual tissues [6–8]. Recent evidence has further shown that Cu/Zn-SOD knockout mice have a high abortion rate [22], suggesting that Cu/Zn-SOD is important in the maintenance of early pregnancy by preventing the accumulation of ROS.
The above result was associated with the significant decreases of PRDX3 and TXNL1 in this study. Both proteins are part of the cell defense mechanism against ROS under biological or pathological status during pregnancy [23–25]. PRDX3 was found to decrease in a human choriocarcinoma cell line cultured under hyperoxia [24], while a similar effect of hypoxia was also observed on cytotrophoblastic cells [8]. Another study has demonstrated that the expression of 1-Cys PRDX increased in response to mild oxidative insult induced by H2O2 in a human lens epithelial cell line, and then decreased below the control level at high doses of H2O2 [26]. Together, the above data indicate that when cell viability is decreased, the level of antioxidant would decrease in parallel and fail to offer protection against the damage caused by hypoxia or hyperoxia, raising the possibility of employing the similar regulatory pathways in EPL. On the other hand, the trophoblastic cells burdened with ROS may also induce mitochondrial damage. The changes of these enzymes provide strong evidence on widespread trophoblastic oxidative damage in placental generation due to the pathological burst of oxidative stress occurring in EPL.
It has been reported that superoxide radicals induce apoptosis [27–29]. In this study, FAIM was decreased in EPL compared to normal pregnancy. It is of interest to speculate that EPL may be associated with apoptosis, and the superoxide radicals due to the oxidative stress may stimulate placental villi apoptosis. On the other hand, two proteins, RNA-binding protein and eukaryotic translation elongation factor involved in transcription, were also found to be reduced in this study. However, whether the failure in cell transcription is a primary event correspondent with the concomitant exposure to oxidative stress or secondary to the abnormality of antioxidative defense underlying EPL is unknown. Thus, further studies are needed regarding the molecular interaction in pathological events caused by oxidative stress in the placental villous tissues of EPL.
Suppression of other functional protein pathways was also evident. Our results demonstrate for the first time the relationship between the decreases of S100A11 and LGALS1 and EPL. S100A11 is a calcium-binding protein implicated in a variety of biological functions, including cell differentiation [30–32]. Recently, S100 proteins have received increasing attention because of their involvement in several human diseases such as wound healing, stress, rheumatoid arthritis, Alzheimer disease, and cancer [31, 33]. LGALS1, which belongs to the mammalian lectins, has also been found to be involved in several biological events including cell attachment, differentiation, apoptosis, embryogenesis, and cancer invasion and metastasis [34, 35]. Thus combined with the present observation, the decreased expressions of S100A11 and LGALS1 may partly explain the malfunction of cytotrophoblastic differentiation during early placentation in EPL.
CSH1 was decreased in EPL due to the malfunction of trophoblastic cells. CSH1 is an important hormone secreted by placental trophoblastic cells. In early gestation, CSH1 may be secreted preferentially into the fetal circulation, exerting fetal growth-promoting effects and acting as the "growth hormone of pregnancy" [36, 37]. TTR, which is involved in vitamin A and thyroid hormone metabolism [38, 39], was found to be downregulated in the placental villous tissues of EPL in this study. Thus the decreased expression of this regulatory compound may play an important role in the pathological development of EPL.
Unexpectedly, we found that the levels of two members of ubiquitin-proteasome complex, proteasome ß-subunit and UBE2N (a ubiquitin-conjugating enzyme), both of which mediate intracellular proteolysis [40, 41], were upregulated in placental villous tissues of EPL. The ubiquitin-proteasome complex is thought to be responsible for the degradation of short-lived intracellular mammalian transcription factors and signal transduction molecules [41], further indicating regulation of general cell processes in EPL. However, the functional significance of these modulations remains to be determined in the pathophysiology of EPL.
In conclusion, we performed a proteomic analysis on the placental villous tissues of EPL with a combination of 2-DE and MALDI-TOF MS. The analysis led to identification of 12 proteins with significantly altered abundance in human placental villous tissues of EPL. These proteins seem to be related to the processes involved in the wide signaling network and the regulation of cellular activities such as cell defense against ROS, differentiation, proliferation, metabolism, transcription, apoptosis, and proteolysis. Significant anomalies of some of these proteins have for the first time been demonstrated in the pathophysiological mechanisms of EPL. This study has also identified several proteins associated with placentation and early mammalian development by using proteomic techniques, providing new insight into the proteins potentially and functionally involved in the pathophysiological mechanisms underlying EPL and also providing a deeper understanding of the mechanisms of normal early pregnancy. However, follow-up experiments (e.g., confirming other proteins identified by Western blot or identifying the remaining six protein spots by MALDI-TOF MS) are suggested and should provide valuable additional data. In addition, more work is needed to evaluate the physiological relevance of particular proteins at different stages of early pregnancy.
ACKNOWLEDGMENTS
The authors would like to thank Professor Ying-Nian Yu for helpful discussion and Run-Liu Yu for technical support during MALDI-TOF MS measurement.
FOOTNOTES
1 Correspondence: He-Feng Huang, Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University School of Medicine, 2 Xue Shi Rd. Hangzhou, Zhejiang 310006 China. FAX: 86 571 8721 7044; huanghefg{at}hotmail.com 
Received: 22 November 2005.
First decision: 16 December 2005.
Accepted: 17 May 2006.
REFERENCES
in the deciduas of failed pregnancy. Mol Hum Reprod 2000 6:642-647This article has been cited by other articles:
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  K. E. Burnum, S. Tranguch, D. Mi, T. Daikoku, S. K. Dey, and R. M. Caprioli Imaging Mass Spectrometry Reveals Unique Protein Profiles during Embryo Implantation Endocrinology, July 1, 2008; 149(7): 3274 - 3278. [Abstract] [Full Text] [PDF]
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