Mol. Hum. Reprod. Advance Access originally published online on July 22, 2005
Molecular Human Reproduction 2005 11(7):489-494; doi:10.1093/molehr/gah187
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Fibromodulin is expressed in leiomyoma and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-ß through Smad and MAPK-mediated signalling
Department of OB/GYN, University of Florida, Gainesville, FL, USA
1 To whom correspondence should be addressed at: Department of OB/GYN, University of Florida, Box 100294, Gainesville, FL 32610, USA. E-mail: cheginin{at}obgyn.ufl.edu
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Abstract |
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Microarray gene expression profiling revealed fibromodulin (FMOD) is among differentially expressed genes in leiomyoma (L) and myometrium. Using realtime PCR, western blotting and immunohistochemistry, we validated the expression of FMOD in paired leiomyoma and myometrium (N = 20) during the menstrual cycle, from women who received gonadotropin-releasing hormone analogue (GnRHa) therapy (N = 7) and in leiomyoma and myometrial (M) smooth muscle cells (SMC) due to transforming growth factor (TGF)-ß and GnRHa treatment. The results indicated that FMOD is expressed at significantly higher levels in leiomyoma as compared to myometrium from proliferative phase (two- to three-folds; P < 0.05), but not the secretory phase of the menstrual cycle, whereas GnRHa therapy reduced FMOD expression to levels detected in myometrium from proliferative phase (P = 0.05). By using western blotting and immunohistochemistry immunoreactive FMOD was detected in leiomyoma and myometrial tissue-extract and in LSMC and MSMC, connective tissue fibroblasts and arterial walls. In a time- and cell-dependent manner, TGF-ß1 (2.5 ng/ml) increased the expression of FMOD in MSMC, whereas GnRHa (0.1 µM) inhibited that in MSMC and LSMC (P < 0.05). The effect of TGF-ß and GnRHa on FMOD expression was reversed following pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126 (MEK1/2 inhibitor), respectively. In summary, menstrual cycle-dependent expression of FMOD and suppression following GnRHa therapy in leiomyoma and myometrium, as well as differential regulation by TGF-ß and GnRHa in vitro suggests that FMOD, a key regulator of tissue organization, plays a critical role in leiomyoma fibrotic characteristics.
Key words: fibromodulin/GnRH/leiomyoma/MAPK/myometrium/Smad/TGF-ß
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Introduction |
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Leiomyomas are benign uterine tumors histologically consisting of smooth muscle cells (SMC) and connective tissue fibroblasts. It is well established that excess accumulation of extracellular matrix (ECM) is among the key features of fibrotic disorders, including leiomyoma. However, our current understanding of ECM components and their contribution to the pathogenesis of leiomyoma has been limited to a few studies (Stewart et al., 1994
, 1998; Arici and Sozen, 2000; Berto et al., 2003; Ding et al., 2004; Leppert et al., 2004). Recent gene expression profiling, including our own, has provided evidence that a number of specific genes functionally categorized as cell and tissue structural modulators, including members of the ECM and cytoskeletal families such as proteoglycans are expressed in leiomyoma (Tsibris et al., 2002; Chegini et al., 2003a; Wang et al., 2003a; Weston et al., 2003; Luo et al., 2005a,b). Proteoglycans consist of a large family of highly anionic glycoproteins ubiquitously expressed in connective tissues (Yanagishita, 1993; Hildebrand et al., 1994; Bartold and Narayanan, 1998; Melrose et al., 2001; Kinsella et al., 2004). Proteoglycans are subdivided into two groups, large molecules such as aggrecan, versican and perlecan, and the relatively small molecules consisting of five distinct members fibromodulin (FMOD), biglycan, decorin, lumican and chondroadherin, containing leucine-rich repeat motifs in their protein cores (Blochberger et al., 1992; Noonan and Hassell, 1993; Neame et al., 1994). The large proteoglycans serve to maintain tissue hydration and contribute to overall structural scaffolding in the ECM, whereas small molecules play important roles in binding to other matrix molecules to either aid fibrillogenesis or act as bridging molecules between various tissue elements (Yanagishita, 1993).
FMOD is a collagen-binding protein widely expressed in many connective tissues (Antonsson et al., 1991; Hedlund et al., 1994; Ezura et al., 2000; Gori et al., 2001; Ameye et al., 2002; Chjakravarti, 2002; Burton-Wurster et al., 2003; San Martin et al., 2003). FMOD has a close homology with decorin and biglycan, and like decorin it binds to collagen types I, II and XII influencing the rate of fibrillogenesis and the formation of collagen fibrils network (Hedbom and Heinegård, 1993; Font et al., 1998). FMOD interaction with transforming growth factor (TGF)-ß, a key profibrotic cytokine, is considered to enhance the retention of this growth factor within the ECM thus regulating TGF-ß local action (Fukushima et al., 1993; Hildebrand et al., 1994). Using gene expression profiling, we have identified several components of ECM including FMOD, decorin and biglycan among the differentially expressed genes in leiomyoma and myometrium and in leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) in response to TGF-ß1 action (Chegini et al., 2003a; Luo et al., 2005a,b). We extend these observations and in the present study validate the expression of FMOD in leiomyoma and matched myometrium from proliferative and secretory phases of the menstrual cycle, and from patients who received gonadotropin-releasing hormone analogue (GnRHa) therapy using realtime PCR, western blotting and immunohistochemistry. Because GnRHa therapy suppresses TGF-ßs and TGF-ß receptor expression in these tissues as well as in LSMC/MSMC, we further investigated whether TGF-ß and GnRHa regulate the expression of FMOD in these cells and if Smad and mitogen-activated protein kinase (MAPK) signalling pathways are involved in mediating their actions, respectively.
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Materials and methods |
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The materials for realtime PCR, western blotting and immunohistochemistry were purchased from Applied Biosystem (Foster City, CA, USA), BioRad (Hercules, CA, USA) and Vector Laboratories (Burlingame, CA, USA), respectively, as previously described (Xu et al., 2003
; Ding et al., 2004). Goat polyclonal antibody generated against human FMOD was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Portions of leiomyoma and matched myometrium were collected from premenopausal women (N = 27) who were scheduled to undergo hysterectomy for symptomatic uterine leiomyomas at the University of Florida affiliated Shands Hospital. Of these patients, seven received GnRHa therapy for a period of 3 months before surgery. The untreated patients did not receive any medication during the previous 3 months before surgery and based on endometrial histology and the patient’s last menstrual period, they were identified as being from proliferative (N = 8) or secretory (N = 12) phases of the menstrual cycle. To maintain a standard, leiomyomas of 2–3 cm in diameter and randomly selected portions were chosen with matched myometrium collected distal from the tumors. Prior approval was obtained from the University of Florida Institutional Review Board for the experimental protocol of this study. Following collection, total RNA and protein was isolated from these tissues and subjected to realtime PCR, western blotting or processed for immunohistochemistry and cell culturing as previously described (Xu et al., 2003; Ding et al., 2004).
Realtime PCR
Briefly, total RNA was isolated from leiomyoma and myometrium using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and complimentary DNA was generated from 2 µg of total RNA using Taqman reverse transcription reagent. The newly synthesized cDNA was used for PCR performed in 96-well optical reaction plates with cDNA equivalent to 100 ng RNA in a volume of 50 µl reaction containing 1x Taqman Universal Master Mix, optimized concentrations of FAM-labelled probe and specific forward and reverse primer for FMOD selected from Assay on Demand (Applied Biosystems). Controls included RNA subjected to RT–PCR without reverse transcriptase and PCR with water replacing cDNA. The results were analysed using a comparative method, and the values were normalized to the 18S rRNA expression and converted into fold change based on a doubling of PCR product in each PCR cycle, according to the manufacturer’s guidelines as previously described (Ding et al., 2004; Luo et al., 2005a).
Western blotting and immunohistochemistry
For western blot analysis, small pieces of tissue were lysed in a lysis buffer, centrifuged and the supernatants were collected and their total protein content was determined using a conventional method (Pierce, Rockford, IL, USA) as previously described (Xu et al., 2003; Ding et al., 2004). Equal amounts of sample proteins were subjected to SDS PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and following further processing, the blots were incubated with FMOD antibody for 1 h at room temperature. The blots were washed with washing buffer and exposed to corresponding horse-radish peroxidase (HRP)-conjugated IgG, and immunostained proteins were visualized using enhanced chemiluminescence reagents (Amersham-Pharmacia Biotech, Piscataway, NJ, USA).
For immunohistochemistry, tissue sections were prepared from formalin-fixed and paraffin-embedded leiomyoma and myometrium and following standard processing immunostained using antibodies to FMOD at 5 µg of IgG/ml for 2–3 h at room temperature. Following further standard processing, chromogenic reaction was detected with 3,3'-diaminobenzidine tetrahydrochloride solution (Xu et al., 2003). Omission of primary antibody or incubation of tissue sections with nonimmune goat IgG instead of primary antibody at the same concentration served as controls.
The expression and regulation of fibromodulin in LSMC and MSMC by TGF-ß and GnRHa
LSMC and MSMC were isolated, characterized and cultured as previously described (Chegini et al., 2002). LSMC and MSMC were cultured in 6-well plates at an approximate density of 106 cells/well in DMEM-supplemented media containing 10% fetal bovine serum (FBS). After reaching visual confluence, the cells were washed in serum-free media and incubated for 24 h under serum-free, phenol red-free conditions (Chegini et al., 2002).
To determine whether TGF-ß and GnRHa influence the expression of FMOD, LSMC and MSMC cultured as above were treated with TGF-ß1 (2.5 ng/ml) or GnRHa (0.1 µM) for 2, 6 and 12 h (Xu et al., 2003; Ding et al., 2004). Because TGF-ß mediates its action in part through activation of the MAPK pathway (Ding et al., 2004), we determined whether inhibition of this pathway alters TGF-ß-mediated action in regulating the expression of FMOD. LSMC and MSMC were cultured as above and following pretreatment with U0126 (20 µM), a synthetic inhibitor of MEK1/2, for 2 h, the cells were treated with TGF-ß1 or GnRHa for 2 h (Ding et al., 2004). Activation of Smad also serves as a major signalling pathway for TGF-ß-mediated action including in LSMC and MSMC (Xu et al., 2003). To determine whether TGF-ß-mediated action through the Smad pathway regulates the expression of FMOD, LSMC and MSMC were cultured as above and transfected with Smad3 SiRNA as previously described (Luo et al., 2005c). LSMC and MSMC at 80% confluence were transfected with 200 pmol of SiRNA using transfectamine 2000 reagent (10 µl) according to the manufacturer’s instructions (Inveritogen, Carlsbad, CA, USA) for 48 h. The cells were then treated with TGF-ß1 (2.5 ng/ml) for 2 h. Untreated or cells transfected with scrambled Smad3 SiRNA were used as a negative control. Total RNA was isolated from the treated and untreated control cells and subjected to realtime PCR as described above.
Where appropriate, the results are expressed as mean ± SEM and statistically analysed using unpaired Student t-test and analysis of variance (ANOVA) using Tukey test. A probability level of P < 0.05 was considered significant.
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Results |
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Expression of FMOD in leiomyoma and myometrium
The expression of FMOD in leiomyoma and matched myometrium from proliferative (N = 8) and secretory (N = 12) phases of the menstrual cycle was evaluated using realtime PCR. The results indicated that myometrium from the secretory phase of the menstrual cycle expresses significantly higher levels of FMOD mRNA as compared to tissues from proliferative phase (P < 0.05; Figure 1). In leiomyoma, the level of FMOD expression was higher in tissues from the proliferative phase with a trend towards a lower expression during secretory phase, although these values did not reach statistical significance (P = 0.06; Figure 1). Comparative analysis indicated that FMOD expression in paired leiomyoma and myometrium from the proliferative phase was higher in leiomyoma (P < 0.05) as compared to the relative expression seen in tissues from the secretory phase (Figure 1). The relative expression of FMOD was significantly reduced in both leiomyoma and myometrium in women who received GnRHa therapy (N = 7), reaching the levels observed in myometrium from the proliferative phase (P = 0.05; Figure 1).
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To further assess the expression of FMOD, total protein was isolated from these tissues and subjected to western blot analysis. As shown in Figure 2, leiomyoma and matched myometrium from proliferative and secretory phases of the menstrual cycle contain immunoreactive FMOD and with an apparent higher density in leiomyoma (L) compared with myometrium (M) in tissue from the proliferative phase, which increased in tissues from the secretory phase. FMOD intensity invariably decreased in L and M in GnRHa-treated groups as compared to tissues from the secretory phase (Figure 2). Immunoreactive FMOD was localized in leiomyoma and myometrial tissue sections with staining associated with LSMC and MSMC as well as connective tissue fibroblasts and vasculature (Figure 3). Incubation of tissue sections with nonimmune goat IgGs instead of primary antibody at the same concentration served as control and showed a substantial reduction in staining intensity associated with these cells.
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Expression of FMOD in LSMC and MSMC and regulation by TGF-ß and GnRH
We have recently characterized the gene expression profile of LSMC and MSMC in response to TGF-ß and GnRHa, indicating that the expression of several genes functionally categorized as regulators of cell and tissue structure including FMOD are the target of their regulatory actions (Luo et al., 2005b). To further evaluate the influence of TGF-ß on FMOD expression in leiomyoma and myometrium, we isolated LSMC and MSMC and following treatment with TGF-ß1 (2.5 ng/ml) determined the expression of FMOD in these cells. As shown in Figure 4, TGF-ß1 in a cell- and time-dependent manner significantly increased the expression of FMOD in MSMC with a gradual reduction in expression reaching control levels after 12 h (P < 0.05). TGF-ß had either no effect or inhibited FMOD expression in LSMC after 12 h of treatment (Figure 4; P < 0.05). Treatment of LSMC and MSMC with GnRHa (0.1 µM) for 2 and 6 h had no significant effect on FMOD expression; however, it was inhibited after 12 h of treatment (Figure 4; P < 0.05).
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Inhibition of MAPK and Smad3 pathways on TGF-ß- and GnRHa-mediated actions
TGF-ß recruits and activates several intracellular signalling pathways, specifically Smad and MAPK pathways, in regulating the expression of many genes including fibronectin and collagen in LSMC and MSMC (Xu et al., 2003; Ding et al., 2004; Luo et al., 2005b). To determine whether TGF-ß action on FMOD expression is mediated through these pathways, LSMC and MSMC were pretreated with MEK1/2 synthetic inhibitor, U0126, followed by treatment with TGF-ß1 (2.5 ng/ml) for 2 h. As shown in Figure 4, pretreatment with U0126 increased the basal expression of FMOD in LSMC and MSMC and TGF-ß-mediated action in LSMC, while inhibiting TGF-ß-mediated action in MSMC (P < 0.05). Pretreatment with U0126 also increased the expression of FMOD in MSMC and LSMC treated with GnRHa as compared with GnRHa-only treated and untreated controls (P < 0.05), but not in U0126-treated LSMC cells (Figure 4).
Transfection of LSMC and MSMC with Smad3 SiRNA, but not scrambled SiRNA significantly inhibited the expression of Smad3 in both cell types (figure not shown) and resulted in a trend towards an increase in basal expression of FMOD in MSMC (Figure 4). However, transfection of the cells with Smad3 SiRNA resulted in significant reduction in TGF-ß-induced FMOD in MSMC reaching control levels, without affecting FMOD expression in LSMC (Figure 4; P < 0.05).
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Discussion |
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In the present study, we validated and further demonstrated the menstrual cycle-dependent expression of FMOD in leiomyoma and myometrium. The level of FMOD expression was significantly higher in leiomyoma as compared to myometrium from the proliferative phase, with relatively similar expression detected in tissues from the secretory phase of the menstrual cycle, which was due to increased expression in myometrium. Despite differential expression of FMOD in leiomyoma and myometrium during the menstrual cycle, GnRHa therapy equally affected FMOD expression in both tissues. Because GnRHa therapy creates a hypoestrogenic condition, the menstrual cycle-dependent expression of FMOD and suppression by GnRHa therapy suggest that FMOD expression may be under the control of ovarian steroids in these tissues. Additionally, GnRHa may directly influence the expression of FMOD because GnRH and GnRH receptors are expressed in leiomyoma and myometrium as well as LSMC and MSMC. Several in vitro studies have demonstrated changes in cell growth, cell cycle progression, apoptosis, and expression of several growth factors, proteases and protease inhibitors in endometrial, myometrial and leiomyoma cells and other steroid sensitive cell types due to GnRHa treatment, which are mediated through GnRH receptor-mediated signal transduction (Chegini et al., 1996
, 2002; Dou et al., 1996, 1997; Raga et al., 1999; Everest et al., 2001; Grundker et al., 2001; Xu et al., 2003; Ding et al., 2004; Cheng and Leung, 2005). Our results also suggest that FMOD expression and regulation by GnRH in LSMC and MSMC may involve activation of signalling through MAPK pathway.
We also demonstrated that TGF-ß, a key profibrotic cytokine, through Smad and MAPK signalling pathways differentially regulates the expression of FMOD in LSMC and MSMC. Because elevated expression of TGF-ß system is a common characteristic of fibrotic disorders, including leiomyoma (Dou et al., 1997; Chegini et al., 1999, 2002, 2003b; Arici and Sozen, 2000; Lee and Nowak, 2001; Xu et al., 2003), we expected a correlation between TGF-ß action and FMOD expression. Although FMOD expression was higher in leiomyoma compared to myometrium (proliferative phase only) and in LSMC compared to MSMC at base-line, TGF-ß1 in a time-dependent manner increased (5- to 10-fold) FMOD expression in MSMC, whereas reducing that in LSMC. Our results show that TGF-ß mediated signalling, though MAPK/extracellular signal-regulated kinase (ERK) and Smad pathways are involved in differential regulation of FMOD in LSMC and MSMC; however, the reason for differential regulation of FMOD expression at tissue and cellular levels is unclear from our study. Whether a different concentration of TGF-ß and/or other TGF-ß isoforms would have similar or different regulatory effects on FMOD expression is yet to be investigated. However, considering that TGF-ß isoforms share the same receptors and activate identical intracellular signalling pathways, they may have relatively similar regulatory actions on FMOD expression. TGF-ß1, TGF-ß2 and TGF-ß3 equally effect LSMC and MSMC cell proliferation (Tang et al., 1997; Lee and Nowak, 2001), and a recent study with a detailed comparison of TGF-ß isoform actions in renal mesangial cells, fibroblasts and tubular epithelial cells revealed that TGF-ß1 increased TGF-ß2 and TGF-ß3 production, while TGF-ß2 and -ß3 stimulated TGF-ß1, and 80% of TGF-ß3¢s fibrogenic effect was mediated by TGF-ß1 (Yu et al., 2003).
In a rat model transiting from scarless fetal-type repair to adult-type repair, the expression of FMOD is reported to decrease as compared to TGF-ß and TGF-ß receptor expression and when compared to adult wound healing (Soo et al., 2000). This as well as our observations in leiomyoma and myometrium suggests that FMOD expression may be relevant and targeted by TGF-ß during the course of tissue fibrosis. TGF-ß1 is reported to modulate the synthesis and accumulation of decorin, biglycan and FMOD in cartilage explants cultured under conditions in which aggrecan synthesis remains relatively constant, with FMOD content most rapidly augmented in response to TGF-ß1 (Burton-Wurster et al., 2003). In addition, connective tissue growth factor (CTGF) which is considered to act as a TGF-ß downstream gene in regulating tissue fibrosis, has been reported to increase the expression of FMOD, type I and III collagens and basic fibroblast growth factor, without affecting the expression of decorin, biglycan, and versican in dermal fibroblast (Wang et al., 2003b). Gene expression profiling studies, including our own, indicated a significantly lower expression of CTGF in leiomyoma as compared to myometrium (Weston et al., 2003; Luo et al., 2005a,c). However, under in vitro conditions, TGF-ß1 self-regulated its own expression and increased the expression of CTGF in LSMC and MSMC (Chegini et al., 1999; Luo et al., 2005b,c). As such, the difference in CTGF expression between in vivo and in vitro conditions provides another example of difference in gene expression at tissue and cellular levels as seen with FMOD in our study. Interestingly, similar to regulating FMOD expression, TGF-ß receptor signalling through MAPK and Smad pathways also regulate the expression of type I collagen and fibronectin as well as CTGF in LSMC and MSMC (Ding et al., 2004; Luo et al., 2005c).
FMOD, a collagen-binding protein and a member of the proteoglycan family is widely expressed in many connective tissues (Westergren-Thorsson et al., 1998; Soo et al., 2000; Venkatesan et al., 2000; Strom et al., 2004). FMOD binds to matrix molecules aiding fibrillogenesis or serve as bridging molecules between various tissue elements, that are important in ECM remodelling (Blochberger et al., 1992; Noonan and Hassell, 1993; Yanagishita, 1993; Westergren-Thorsson et al., 1998; Soo et al., 2000; Venkatesan et al., 2000; Strom et al., 2004). FMOD regulates the formation of collagen fibrils network through interaction with collagen types I, II and XII (Font et al., 1998) and like decorin through interaction with TGF-ß regulates the local biological activity and retention of TGF-ß within the ECM (Fukushima et al., 1993; Hildebrand et al., 1994). As such, the expression of decorin and FMOD in leiomyoma and myometrium, and changes in their expression, could influence the organization of collagen and local availability of TGF-ß, thus the outcome of fibrosis in leiomyoma. Interestingly, FMOD expression has been found in mitotic, but not in mitomycin C-induced post-mitotic skin fibroblasts, endothelial cells and keratinocytes, serving as a specific marker for mitotic activity and cellular ageing (Petri et al., 1999). Proteases such as matrix metallproteinases (MMPs), specifically MMP-13, are reported to effectively cleave FMOD in articular cartilage and explant cultures treated with interleukin-1 (IL-1; Heathfield et al., 2004). Several MMPs and cytokines such as IL-1 are expressed by leiomyoma and myometrium that may target FMOD degradation in a manner demonstrated in other tissues (Duo et al., 1997; Tang et al., 1997; Lee et al., 1998; Palmer et al., 1998). FMOD deficiency leads to a significant reduction in tendon stiffness with irregular collagen fibrils and increased frequency of small diameter fibrils, suggesting FMOD is required early in collagen fibrillogenesis (Svensson et al., 1999; Chakravarti, 2002). Thus, altered expression of FMOD would be expected to impact the organization of collagen in various fibrotic disorders such as leiomyoma.
In summary, we demonstrated the expression of FMOD in leiomyoma and myometrium, and provided evidence for direct regulatory action of TGF-ß and GnRHa on its expression in LSMC and MSMC, which was mediated through Smad and MAPK pathways. Although the functional relevance of FMOD in leiomyoma and myometrium remains to be investigated, because of FMOD key role in connective tissue remodelling, specifically fibrillogenesis, cell-cell adhesion and modulation of cytokine autocrine/paracrine actions, the results suggest that FMOD may play a critical role in pathogenesis of leiomyoma and more specifically fibrotic characteristic.
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Acknowledgements |
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Drs Levens and Luo contributed equally towards this work. This work was supported by NIH grant HD37432. Presented in part at the 60th Annual Meeting of the American Society of Reproductive Medicine, Philadelphia PA, October 2004.
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Submitted on March 14, 2005; accepted on May 9, 2005.
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