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• Original Article • Expression of BAFF in the trophoblast and decidua of normal early pregnant women and patients with recurrent spontaneous miscarriage GUO Wen-jing, QU Xun, YANG Mei-xiang, ZHANG Wei-dong, LIANG Lu, SHAO Qian-qian, KONG Beihua GUO Wen-jing Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China; QU Xun Basic Research Institute of Clinic Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China; YANG Mei-xiang Basic Research Institute of Clinic Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China; ZHANG Wei-dong Department of Pathology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, Shandong 250012, China; LIANG Lu Basic Research Institute of Clinic Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China; SHAO Qian-qian Basic Research Institute of Clinic Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China; KONG Bei-hua Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China Correspondence to: KONG Bei-hua Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China (Tel:86531-82169008 Email:kongbeihua @yahoo.com.cn ) QU Xun, Basic Research Institute of Clinic Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, China (Tel: 86-531-82169251. Email: [email protected]. cn) udy was supported by : National Natural Science Foundation of China to Dr. KONG Bei-hua(No. 30571953) Keywords: B lymphocyte stimulator·recurrent spontaneous abortion·maternal-fetal immunology Abstract: Background BAFF, the B cell activation factor, is a member of the tumor necrosis factor (TNF) ligand family that binds to BCMA, TACI, and BAFF-R. Previous studies have shown that members of the TNF family are detected in human placental trophoblast cells, but the expression patterns of BAFF involved in human decidua and the differential expression of BAFF between normal pregnancy and miscarriage are still incompletely documented or unknown. This study was designed to investigate the expression of BAFF and BAFF-R in the trophoblast and decidua of normal early pregnant women and recurrent spontaneous abortion (RSA) patients. Methods Forty-five patients with RSA and 45 normal pregnant women were included in this study. By reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical experiments, we explored the expression of BAFF and BAFF-R in the maternal-fetal interface of normal early pregnant women and RSA patients. Results Analysis by RT-PCR and Western blotting revealed that BAFF was detected in both trophoblast and decidua of all the samples, and the expression level was higher in the tissues of normal early pregnant women (P<0.05) than that of recurrent spontaneous abortion patients under the same gestational weeks. Messages for BAFF-R were absent. Immunohistochemical experiments showed that expression of BAFF was cell-specific which was localized to villous cytotrophoblast and syncytiotrophoblast cells in trophoblast and to stromal cells in decidua. Whereas BAFF was prominent on the trophoblast and decidua of normal early pregnant women, it was decreased in the tissues of RSA patients. Conclusions BAFF might steer maternal leukocytes away from a harmful immune response and toward a favorable one and play a potentially vital role for successful pregnancy. 2008;121(4):309-315 ·LogIn/LogOut ·Fulltext PDF(282K) Free ·Abstract download TXT | XML ·Articles in CMJ by GUO Wen-jing QU Xun ·Articles in PubMed by GUO WJ QU X ·Put into my bookshelf ·Email to Friend ·Email to author ·Visit:2593 ·Download:1343 ·Advanced Search ·Related Articles ·Change font size: ·Cannot read some characters Maternal immune response during pregnancy is thought to be harmful to the survival of the fetus, but in fact it may be beneficial. The successful pregnancy relies on appropriate growth of the placenta and one of the functions of the placenta is to establish a haven in which the fetus can flourish despite intimate contact with an immunologically semiforeign environment. In this task, the trophoblast is the principal player which lines the villous placenta and separates maternal blood from fetal tissue and has a major role in cellular alterations of the maternal immune system.1 Evidence presented in several studies indicates that immune privilege is known to be conferred by members of the tumor necrosis factor (TNF) superfamily,2-5 which have roles in placental cell growth, cell death, cell migration and hormone production.6-10 Earlier studies stated that messages encoding all of the death-inducing TNF superfamily ligands were detected in placental trophoblast cells.11 Subsequently Phillips et al12 demonstrated that expression of some nonapoptosis- inducing ligands in this family (APRIL, BAFF and CD30L/CD153) were also examined in the trophoblast cells. But during the invasive phase of implantation, maternal decidual cells also secrete products and are proposed to supply nutrition to the growing embryo until the formation of a functional placenta, and to shield the embryo from the maternal immune system.13,14 To date, it has not been determined whether any of these TNF superfamily members and their receptors is expressed in maternal decidual cells. BAFF was known as B cell-activating factor belonging to the TNF family, also called B lymphocyte stimulator (BLyS) TNF- and Apop-releated leukocyte expressed ligand 1 (TALL-1), TNF homologue that activates apoptosis, nuclear factor (NF)-κB, and c-Jun NH2-terminal kinase (THANK), and zTNF4.15-19 BAFF plays an important role in immune responses and overexpression or deficiency of BAFF is associated with the development of autoimmune diseases.20 Recurrent spontaneous abortion (RSA), defined as two or more consecutive miscarriages, is a prevalent disorder. The pathogenesis of most cases remains unknown, but immunological factors are considered to play a vital role in the pathogenesis of recurrent spontaneous abortion. The above mentioned experiments revealed that BAFF was readily detected in trophoblasts and deciduas of normal early pregnant women and recurrent spontaneous abortion patients. But the expression of BAFF gene and protein was significantly decreased in trophoblasts and deciduas of recurrent spontaneous abortion patients than that of normal early pregnant women under the same gestational weeks. By immunohistochemistry, localization of BAFF protein to specific cell types was observed. Thus, information on the expression of BAFF in trophoblasts and deciduas may play an important role in maintenance of successful pregnancy or pathogenesis of failure pregnancy. In this study, we analyzed the expression of BAFF and BAFF-R not only in trophoblasts but also in deciduas of normal early pregnant women and recurrent spontaneous miscarriage patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting; on the other hand, immunohistochemical experiments were used to determine cellular locations of the proteins. METHODS Tissues collection The trial, which enrolled 45 clinically elective women for normal pregnancy termination and 45 patients with recurrent spontaneous miscarriage (gestational age, 4−10 weeks), was conducted between January 2005 and October 2006 at the Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University. The 45 patients were divided into three groups according to gestational weeks (group 1: 4−5 (6/7) weeks; group 2: 6−7 (6/7) weeks; group 3: 8−10 weeks; n=15 each group), respectively. The first-trimester human trophoblasts and deciduas were obtained from the above patients. Each patient completed a signed, written consent form. The Shandong University Human Investigation Committee approved the study. The tissues were manually dissected and immediately frozen, using liquid nitrogen, and stored at −80˚C until used for isolation of total RNA. For every tissue collected, small pieces were also placed into Bouin's fixative, processed into blocks, sectioned and stained with hematoxylin. Reverse transcription-polymerase chain reaction analysis Total RNA was isolated using the Rneasy mini kit® (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. Sample quality was assessed with 2100 Bioanalyzer® (Aligent Technologies, Palo Alto, CA, USA) and confirmed by electrophoresis with an agarose gel. All samples contained 18 S and 28 S rRNA peaks with no degradation. The yield of total RNA was determined by absorbance at 260 nm on a DU 640 Spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The 260/280 nm ratios of the samples were >1.8. To correct for variations in mRNA recovery and reverse transcription yield, the amount of BAFF cDNA was normalized with β-actin. One microgram of each RNA sample was treated with DNase I to remove genomic DNA and then was denatured, and reverse transcription was performed for 1 hour at 42ºC with 0.5 µg oligo(dT)18, 1.0 mmol/L 4dNTP, 20 U RNasin ribonuclease inhibitor (Promega, Madison, WI, USA), 200 U Moloney virus-reverse transcriptase (Superscript II; Life Technologies, Paisley, UK), and 5 × reaction buffer in a total of 20 µl. Amplification was performed with 5 µl cDNA, 0.2 mmol/L dNTP, 1 mmol/L MgCl2, 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT, USA), 0.8 mmol/L specific sense and antisense primers, and 10 × reaction buffer in a 50-µl reaction volume. Amplification primers for the human genes were as follows: BAFF, 134 bp, forward: 5'-TCTTTGAACCACCAGCTCCA-3', reverse: 5'GGCACTTCCCCTTTTAAAGCT-3'; BAFF-R, 144 bp, forward: 5'CAAGGTCATCATTCTGTCTCCG-3', reverse: 5'-GTGGTCACCAGTTCAGTGGA-3'; βactin, 101 bp, forward: 5'- TTGCCGACAGGATGCAGAA-3', reverse: 5'GCCGATCCACACGGAGTACT-3'. After 5-minute precycle at 95ºC, the reaction was followed by 35 cycles of 45 seconds at 94 ºC, 30 seconds at 55ºC, and 1 minute at 72ºC. When the final cycle was over, samples were kept at 72ºC for 10 minutes to complete the synthesis. Polymerase chain reaction products were analyzed on 1.5% agarose gels and ethidium bromide-stained bands were photographed. Authenticity of all of the amplified PCR products was confirmed by direct sequencing. Three independent experiments were performed, each in triplicate. Western blotting Tissues were lysed in a buffer containing 10 mmol/L Tris (pH 7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.5% Nonidet P-40 and protease inhibitors, and then the supernatants were collected as the total cell lysates. Protein concentrations were measured using the protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA, USA). The lysates were electrophoresed on a 12% SDS-PAGE gel before being transferred to a polyvinylidene fluoride (PVSD) membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was then blocked in 5% nonfat milk in TBS-T (Tris buffered saline plus 0.05% Tween-20) and incubated with goat anti-human BAFF mAb (1:1000, R&D, MAB1241, System, Inc.) or isotype antibody at 4ºC overnight. Washed the membrane in TBS-T for 4 times, then the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research Laboratory, West Grove, PA, USA). The immunoreactive protein complexes were detected using an enhanced chemiluminescence detection system (ECL, Amersham) according to the manufacturer's protocol. Endogenous β-actin expression served as an internal control. Immunohistochemistry Paraffin-embedded sections of deciduas and trophoblasts of normal early pregnant women and recurrent spontaneous abortion patients were deparaffinized with xylene and rehydrated to water through graded alcohol series. Endogenous peroxidase activity was blocked by incubation with 50:50 mixtures of 3% H2O2/methanol for 10 minutes and rinsed well with water. The sections were pretreated by microwave heating with 10 mmol/L citrate buffer, pH 6.0, for 30 minutes with a 5-minute cool down before rinsing well in running tap water. After incubation for 10 minutes at room temperature with bovine serum albumin in PBS, slides were incubated for 30 minutes in a humid chamber at 4°C with 10 μg/ml rat anti-human BAFF (Buffy-2; Alexis Biotechnology, San Diego, CA, USA), followed by a biotinylated secondary antibody and finally incubated for 30 minutes with the avidin–biotin complex (ABC) reagent (Vector Laboratories, Burlingame, CA, USA). Isotype-matched Ig control (Dako Cytomation, Carpinteria, CA, USA) was used at the same concentration as the matching primary antibody. The sections were counterstained with hematoxylin and observed with light microscopy (Olympus America). Statistical analysis All values are shown as mean ± standard error (SE). Statistical comparison applied the Mann-Whitney U test and the Wilcoxon matched-pairs test with use of Analyse-it software (Analyse-it Software Ltd., Leeds, UK) for Microsoft Excel. Differences were accepted as significant at P<0.05. RESULTS Detection of transcripts for BAFF in human trophoblasts and deciduas of normal early pregnant women and spontaneous abortion patients Our initial approach to determining whether BAFF is synthesized in human trophoblasts and deciduas using RT-PCR. Parallel PCR analysis of RT-negative controls for all samples and primer pairs failed to yield any products, confirming lack of genomic DNA contamination (data not shown). All amplicons were sequenced and found to be authentic. Figure 1 shows that transcripts encoding BAFF were clearly detected in total RNA acquired from first-trimester trophoblasts of normal early pregnant women and spontaneous abortion patients. Using RT-PCR and primers for BAFF, a single band of 134 bp was amplified in cDNAs from different trophoblasts of normal early pregnant women divided into three groups (Figure 1A, Lanes 1, 3, 5) and other three groups' different trophoblasts of spontaneous abortion patients (Figure 1A, Lanes 2, 4, 6). We then measured the ratio of BAFF to β-actin after the images on the UV transilluminator were put into a computer and the band intensity was measured using densitometrical software. The expression level of BAFF was higher in the trophoblasts of normal early pregnant women compared with that of spontaneous abortion patients under the same gestational weeks (P<0.05) (Figure 1B). There was no significant difference (P>0.05) in the trophoblasts of pregnant women accompanied by first-trimester gestation progression. Figure 1. Analysis of mRNAs encoding BAFF by RT-PCR in firsttrimester trophoblasts of normal early pregnant women and spontaneous abortion patients. (A) RT-PCR products are shown on a gel when amplified with specific primer sets for BAFF. Samples of RNA from different trophoblasts were amplified with primers designed for BAFF (Lanes 1, 3, 5: normal early pregnant women; Lanes 2, 4, 6: spontaneous abortion patients; 1, 2: 4−5 (6/7)week pregnancy; 3, 4: 6−7 (6/7)-week pregnancy; 5, 6: 8−10view in a week pregnancy), a single band of 134 bp was detected in both new window normal pregnancy and abortion tissues. (B) RT-PCR data of BAFF cDNA was normalized with β-actin in each group and is shown as relative gene expression ± SE. *Significantly (P<0.05) lower BAFF expressed in the trophoblasts of spontaneous abortion patients compared to that of normal early pregnant women under the same gestational weeks. 1: 4−5 (6/7)-week pregnancy; 2: 6−7 (6/7)-week pregnancy; 3: 8−10-week pregnancy; N: normal early pregnant women; A: spontaneous abortion patients. The expression of BAFF gene in human deciduas of normal early pregnant women and spontaneous abortion patients are shown in Figure 2. Messages encoding BAFF were also detected in first trimester deciduas of normal early pregnant women (Figure 2A, Lanes 1, 3, 6) and spontaneous abortion patients (Figure 2A, Lanes 2, 4, 6). Figure 2B shows that the level of expression of the BAFF gene in the decidual tissues from normal early pregnant women was significantly more than that of spontaneous abortion patients under the same gestational weeks (P<0.05). There was no obvious difference (P>0.05) in the trophoblasts as gestation progressed to 10 weeks. Figure 2. RT-PCR detection of transcripts for BAFF in first trimester deciduas of normal early pregnant women and spontaneous abortion patients. (A) The size of BAFF product, estimated by its migration relative to size markers, is indicated on a gel. Transcripts for BAFF were readily detected in the deciduas of normal early pregnant women (Lanes 1, 3, 5) and spontaneous abortion patients (Lanes 2, 4, 6) (1, 2: 4−5 (6/7) weeks; 3, 4: 6−7 (6/7) weeks; 5, 6: 8−10 weeks). (B) Ratios of BAFF mRNA to view in a β-actin mRNA are expressed as means ± SE. *Significantly new window (P<0.05) lower BAFF expressed in the deciduas of normal early pregnant women than that of spontaneous abortion patients under the same gestational weeks. 1: 4−5 (6/7)-week pregnancy; 2: 6−7 (6/7)-week pregnancy; 3: 8−10-week pregnancy; N: normal early pregnant women; A: spontaneous abortion patients. And then we detected the concentration of BAFF protein in human trophoblasts and decidual tissue samples by Western blot analysis with a monoclonal antibody specific for BAFF. There was no detectable expression of BAFF protein in the human decidual tissues lysis buffer. The concentration of BAFF protein was significantly reduced in the spontaneous abortion groups than in the normal early pregnancy group (Figure 3). Figure 3. Expression of BAFF protein in human trophoblasts and decidual tissues. Total proteins prepared form pregnancy view in a termination patients and the spontaneous abortion patients were new window separated on a SDS polyacrylamide gel and immunoblotted with anti-BAFF mAb. Lane 1: the trophoblasts of the representative normal early pregnant group; Lane 2: the trophoblasts of the spontaneous abortion group; Lane 3: the decidua of the representative normal early pregnant group; Lane 4: the decidua of the spontaneous abortion group. Expression of BAFF-R in human trophoblasts and deciduas To establish the potential for BAFF ligand to influence cells development and function at the maternal-fetal interface, we examined the same RNA samples from early gestation trophoblasts and deciduas for BAFF-R. But we failed to detect specific messages encoding BAFF-R in first-trimester trophoblasts and deciduas of all the normal early pregnant women and spontaneous abortion patients. Localization of BAFF protein to specific cells in human trophoblasts and deciduas by immunohistochemistry To determine which cells were the source of the BAFF proteins, we performed immunohistochemistry on paraffin-fixed tissue sections taken from first-trimester trophoblasts and deciduas of all the samples (n=at least 3 each). And negative controls did not demonstrate positive staining. Figure 4 illustrates the immunostaining patterns. In first-trimester trophoblasts of normal early pregnant women (Figure 4A), BAFF protein was localized to the cytotrophoblast and syncytiotrophoblast cells. In the same cells of spontaneous abortion patients (Figure 4B), immunostaining was weak. These results show a decrease in BAFF at the trophoblasts of spontaneous abortion patients. As illustrated in Figure 4D, BAFF in early deciduas of normal early pregnant women was localized to stromal cells and blood vessel endothelium cells. As in first-trimester trophoblasts, BAFF declined as immunohistochemistry was performed in decidual cells of spontaneous abortion patients (Figure 4E). However, we failed to find the localization of BAFF protein in decidual blood vessel endothelium cells of spontaneous abortion patients (Figure 4E). The protein was identified in thirty first-trimester trophoblasts and deciduas, the positive cells were of the same subpopulation (cytotrophoblast and syncytiotrophoblast cells) as that in trophoblasts, and BAFF protein was in the same location (predominantly stromal cells) in deciduas, either of normal early pregnant women or of spontaneous abortion patients. These observations of BAFF are consistent with the results of gene expression reported in Figures 1, 2 and 3. Figure 4. Immunohistochemical detection of BAFF in firsttrimester trophoblasts (A and B) and deciduas (D and E) of a representative normal early pregnant woman(A and D; n=at least view in a 3 each) and a spontaneous abortion patient (B and E; n=at least new window 3 each), respectively. The cytotrophoblast and syncytiotrophoblast cells were stained well in two samples, with more prominently in trophoblasts of normal early pregnant woman compared to in that of spontaneous abortion patient (A and B). D shows BAFF protein was localized to the stromal cells and blood vessel endothelium cells in deciduas of normal early pregnant woman. E shows lighter staining in decidual stromal cells of spontaneous abortion patient, while the staining in the blood vessel endothelium cells can not be seen. The BAFF antibodies were tested against trophoblast and decidua sections taken from the same patients; first trimester was 4−10 weeks. Negative staining by control IgG is shown in C (in trophoblast) and F (in decidua). This was noticeable in other tissues, as in the one shown here (Original magnification ×200). DISCUSSION The results of this study demonstrate that BAFF, both gene and protein, is well expressed in trophoblasts and deciduas of normal early pregnant women and recurrent spontaneous abortion patients. Additionally, it is expressed at relatively low levels in the tissues of recurrent spontaneous abortion patients. In view of these observations, expression of BAFF in trophoblasts and deciduas confirms that BAFF is an important factor in the implantation of blastocyst and maintenance of pregnancy, the lower expression of BAFF may be involved in the pathogenesis of unexplained recurrent spontaneous abortion. During the process of successful pregnancy, the coordinated development of the implanting trophoblast and the maternal decidua is believed to be a critical event.21,22 Trophoblast cells, constituting the interface between the fetus and maternal blood and tissues, have an important role in successful pregnancy. Additionally, the importance of decidual cells in ensuring appropriate endometrial functions to support pregnancy is beyond doubt. So the study of human trophoblasts and deciduas is thought to be important. Previous studies obtained using commercially available organ blots have shown relatively high levels of BAFF gene expression in term placentas, peripheral blood leukocytes, lymphoid tissues, bone marrow, and several other organs and tissues.15,16,18 Other two recent reports indicate that human placentas are identified as rich sources of TNF family ligand and receptor transcripts.11,12 Here we found transcript encoding BAFF was detected in trophoblasts of gestated women, and BAFF protein was localized to specific types of cells, cytotrophoblast and syncytiotrophoblast cells, agreeing well with the previous studies above. In addition, our studies expanded other reports and added detections in deciduas, and demonstrated the lower expression of BAFF in trophoblasts and deciduas of recurrent spontaneous abortion patients. Recurrent spontaneous abortion is defined as two or more consecutive pregnancy losses. Although the pathogenesis of most cases remains unknown, several studies have indicated that the majority of these cases are caused by immunological disturbances.23,24 Locksley et al25 have reported that some of the TNF superfamily members that do not mediate apoptosis are key regulators of immune cell development. BAFF, B lymphocyte stimulator, is a member of the TNF superfamily members that do not mediate apoptosis and plays a vital role in immune responses. The study investigated the expression of BAFF in recurrent spontaneous abortion patients and found that BAFF was decreased more in those than in normal early pregnant women. This observation confirms the idea that placental cytokines synthesized at the maternal-fetal interface preferentially divert immunity away from the cell-mediated arm of the immune response, which is associated with cytotoxic T lymphocyte activity, toward the humoral or antibody-mediated arm, which is associated with B lymphocyte activity, as originally postulated by Wegmann et al.26 In the previous study, investigators identified multiple downstream genes transcriptionally induced by BAFF, including the cytokine IL-10 and other factors.27 It has been shown that IL-10 can suppress cytokine production and several accessory cell functions by Th1 cells and is regarded as a potent suppressor of the effector functions of Th1 cells.28 As is known that Th2 immunity is beneficial to pregnancy while Th1 immunity is harmful, fetus acceptance has been associated with a bias toward Th2 cytokine production (e.g., IL-4, IL-10, transforming growth factor-β (TGF-β)) and away from Th1type proinflammatory cytokines (e.g., interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α)) at the maternal-fetal interface.26,29,30 Th1/Th2 cytokine ratios are significantly elevated in women with RSA, hence Th1 cytokine production may up regulated in these women.31,32 In this study, we found expression of BAFF gene and protein was significantly diminished in trophoblasts and deciduas of recurrent spontaneous abortion patients than that of normal early pregnant women under the same gestational weeks. The observation is consistent with the above ideas that BAFF possibly induces the signature Th2 cytokine IL-10, so that the lower expression of BAFF during pregnancy might shift the balance from protective Th2 to a harmful Th1 cytokine bias associated with recurrent pregnancy loss. But the intracellular signaling mechanisms of BAFF lower expression in spontaneous abortion patients are not clear. For the phenomenon there is another possible explanation that BAFF signaling initiates the antiapoptotic activity that is associated with the activation of NF-κB at the maternalfetal interface. Earlier study indicates that BAFF is a cytokine that belongs to the TNF family and activates NF-κB through a distinct receptor.18 Thus, the lower expression of BAFF induces the excessive apoptosis of the cells at the maternalfetal interface associated with recurrent fetal loss. By immunohistochemistry, we found expression of BAFF protein was cell-specific. BAFF was mainly localized to stromal cells of deciduas and cytotrophoblast and syncytiotrophoblast cells of villi in both normal pregnant women and abortion patients. Generally speaking, BAFF ligand is believed to be produced mainly in leukocytes.15,16,18 Our detection of BAFF protein present in decidual stromal cells and cytotrophoblast and syncytiotrophoblast cells was somewhat surprising, but this detection agreed with the previous experiment reported by Phillips et al12 that BAFF was detected primarily in villous term cytotrophoblasts cells. The immunostaining pattern of protein light expression in the tissues of abortion patients was very similar to the pattern of gene expression shown by RT-PCR. It is noticeable that the staining of BAFF in the blood vessel endothelium cells of abortion patients can not be seen, while the protein was localized to that of normal pregnant women. It is possible that BAFF play an important role in the procedure of angiogenesis. Angiogenesis, the formation of new blood vessels from pre-existing capillaries, is of importance in pregnancy.33,34 Typically, it involves activation of quiescent endothelial cells, proliferation and differentiation of endothelial cells, and formation of a new lumen and maturation of the endothelium.35-39 In this study, we can not make sure the specific functions of BAFF found in the blood vessel endothelium cells of normal pregnant women, it may be a critical player in the formation and maintenance of endothelial integrity and reorganization of the vascular bed in pregnancy. It should be noted that messages encoding BAFF-R were undetectable in all the samples, which supports the idea that BAFF-R has been detected only in secondary lymphoid organs and not in any other tissues, including placenta.40 It is already known that BAFF also has other two receptors, BCMA and TACI, but we did not investigate expression of the two receptors in our study. 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