Presence of HIF-1 and related genes in normal mucosa, adenomas and carcinomas of the colorectum

Expression of the transcription factor hypoxia-inducible factor 1 (HIF-1), which plays a key role in cellular adaptation to hypoxia, was investigated in normal colorectal mucosa (ten), adenomas (61), and carcinomas (23). Tissue samples were analyzed for HIF-1α, its upstream regulators, von Hippel–Lindau factor, AKT, and mammalian target of rapamycin (mTOR) and its downstream targets glucose transporter 1 (GLUT1), carbonic anhydrase IX, stromal-cell-derived factor 1 (SDF-1) by immunohistochemistry. In normal colorectal mucosa, HIF-1α was observed in almost all nuclei of surface epithelial cells, probably secondary to a gradient of oxygenation, as indicated by pimonidazole staining. The same staining pattern was present in 87% of adenomas. In carcinomas, HIF-1α was present predominantly around areas of necrosis (78%). Active AKT and mTOR, were present in all adenomas, carcinomas, and in normal colorectal mucosa. GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases, whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells. In conclusion, HIF-1α appears to be physiologically expressed in the upper part of the colorectal mucosa. The present observations support that upregulation of HIF-1α and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia, in close interaction with an active phosphatidylinositol 3-kinases–AKT–mTOR pathway.
Introduction
Colorectal carcinomas arise from normal epithelial cells of the colorectal mucosa through an adenoma stage. The transition from benign adenomas to malignant carcinomas, occurring in about 5% of all adenomas, is only beginning to be explored in appreciable detail. Biological changes involved in this process include important signaling pathways like the mammalian target of rapamycin (mTOR)–AKT pathway while recently also a role for hypoxia-driven signaling was shown [6, 14]. Both pathways are mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) as depicted in Fig. 1. HIF-1 is involved in cellular adaptation to hypoxia in solid tumors, including stimulation of angiogenesis and glycolysis [33]. HIF-1 consists of two subunits of which the β-subunit is constitutively expressed, whereas HIF-1α is regulated in particular by the oxygen level. Under normoxia, the HIF-1α protein is hydroxylated by prolyl hydroxylases [15, 16], which enables the von Hippel–Lindau factor (VHL) to bind, resulting in ubiquination and rapid degradation by the proteosome [27]. HIF-1α is overexpressed in many solid tumors [38] and its presence has been correlated with, i.e., poor prognosis in breast and colorectal cancer patients [4, 36]. Stromal expression of hypoxia-regulated proteins is an adverse prognostic factor in colorectal carcinomas.
Fig. 1Schematic representation of the interaction of the analyzed proteins. The PI3K–AKT–mTOR pathway is inducing HIF-1α. In addition, the absence of hypoxia prevents HIF-1α from degradation mediated by VHL. When HIF-1α is present, it binds to HIF-1 β in the nucleus. The formed complex activates target genes, like GLUT1, SDF1, and CA IX
High protein levels of HIF-1α are not only caused by hypoxia but can also be induced by the absence of wild-type protein 53, mutated VHL and other tumor suppressor genes, as well as by activation of oncogenes like Her2neu (c-erbB-2) [24]. In addition, growth factors play a role. However, both hypoxia and signaling-mediated HIF-1 stimulation are mediated by the phosphatidylinositol 3-kinase (PI3K)–AKT, and the mTOR pathway [8]. Interestingly, both the serine–threonine protein kinase AKT as well as mTOR have been suggested to play a role in an early stage of colorectal carcinogenesis [2, 30]. Furthermore, the presence of a non-hypoxic HIF-1 induction has also been suggested, in colorectal adenomas [9].
Upon HIF-1 activation, multiple target genes become transcriptionally active, including genes involved in anaerobic glycolysis, metabolism, and angiogenesis. This includes glucose transporter 1 (GLUT1), which facilitates cellular glucose uptake [11, 26] and the angiogenesis-associated genes vascular endothelial growth factor and stromal-cell-derived factor 1 (SDF-1) also known as CXCL12 [5]. Furthermore, carbonic anhydrase IX (CA IX) which is prominently induced by hypoxia and is therefore considered to be an intrinsic hypoxia marker, which correlates with a chemical marker of hypoxia, pimonidazole [1, 17, 23, 28]. An additional function of CA IX is in the regulation of the maintenance of the physiological equilibrium in normal gastrointestinal tissue [31].
The present study aims to analyze the role HIF-1 in normal colorectal mucosa, adenomas, and carcinomas. To this end, the presence of HIF-1α as well as of its downstream targets GLUT1, SDF-1, and CA IX were analyzed in normal colon tissue, colorectal adenomas, and carcinomas. To explore the possible cause of HIF-1 overexpression, i.e., hypoxia or oncogenic stimulation, both the presence of the chemical hypoxia marker pimonidazole and of the HIF-1α upstream regulators, VHL, AKT, and mTOR, were investigated.
Materials and methods
Tumor material
A total of 84 tissue samples were studied, consisting of 61 colorectal adenomas, 20 of which contained already a focus of carcinoma and are therefore referred to as progressed adenomas, in contrast to the 41 adenomas with dysplasia only, which are referred to as nonprogressed adenomas. In addition, 23 colorectal carcinomas were analyzed. These samples were obtained from 75 patients, 37 of which were female and 38 male. Mean age was 67.8 years (range 40–89). Normal mucosa samples were obtained from the resection margins of colectomy specimens from ten patients with colorectal cancer. The study was approved by the Institutional Review Board and was in accordance with Dutch medical ethical guidelines.
Determination of hypoxic regions
Hypoxic regions in mouse tissues were visualized using the Hydroxyprobe-1 kit (Chemicon International, Temecula, CA, USA). These experiments were approved by the animal experimentation ethics committee, according to local and governmental regulations.
Briefly, mice were injected intravenously with pimonidazole (60-mg/kg body weight), which forms adducts from reductively activated pimonidazole in hypoxic cells. Mice were killed 90 min later by asphyxiation in CO2. The Hydroxyprobe-1 monoclonal antibody was biotinylated using D-biotinyl-ɛ-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim) and was used to stain pimonidazole adducts in formalin-fixed paraffin-embedded tissues according to recommendations by the manufacturer.
Immunohistochemistry
Immunohistochemistry was performed on 4-μm-thick full-block consecutive sections. Slides were deparaffinized, followed by rehydration, and subsequently staining in batches with the antibodies described in Tables 1 and 2. HIF-1α staining was performed as described previously [3]. In brief, antigen retrieval was performed in antigen retrieval solution (DAKO, Glostrup, Denmark) for 45 min at 96°C. The primary antibody against HIF-1α was incubated at a dilution of 1:500 for 30 min at room temperature and the catalyzed signal amplification system (DAKO) was used for detecting HIF-1α staining.
Table 1Details of the immunohistochemical stainingsAntibodySpeciesCompany and institutesDilutionIncubationAntigen retrievalDetectionHIF-1αMouseBD Pharmingen1/50030′ RTWB TRS 97°C 45′CSAAktRabbitCell Signaling1/50o/n 4°CWB Citr 97°C 10′ABCmTORRabbitCell Signaling1/50o/n 4°CWB Citr 97°C 10′ABCVHLMouseBD Pharmingen1/10060′ RTWB Citr 97°C 20′PowervisionCA IXMouseLab Harris1/5030′ RTnoneEnvision+Glut-1RabbitDAKO1/40060′ RTWB Citr 97°C 20′ABCSDF-1MouseR&D systems1/400o/n 4°CWB Citr 97°C 10′ABCHydroxyprobe-1MouseChemicon International1/20040′RT0.01% pronase in PBS 40°C for 40′ABCBD Transduction laboratories, Mab monoclonal antibody, PAb polyclonal antibody, RT room temperature, WB water bath, TRS target retrieval solution (DAKO), Citr citrate buffer 10 mM pH 6.0, CSA catalyzed signal amplification system (DAKO), ABC avidin–biotinylated peroxidase complex, Powervision from immunologic R&D systemsTable 2Number (and percentages) of HIF-1α, P-AKT, P-mTOR, VHL, CA IX, GLUT1, and SDF-1 expression in nonprogressed and progressed colorectal adenomas and adenocarcinomas in the respective categories of staining intensity Nonprogressed Adenoma, n = 41Progressed adenoma, n = 20Adenocarcinoma, n = 23Kruskal–Wallis test, P-valueHIF-1Neg6 (15)2 (10)5 (22)0.8+0 (0)2 (10)1 (4)++7 (17)3 (15)3 (13)+++28 (68)13 (65)14 (61)AKTNeg0 (0)0 (0)0 (0)0.1+12 (29)7 (35)0 (0)++11 (27)5 (25)10 (43)+++18 (78)8 (40)13 (57)mTORNeg2 (5)0 (0)a0 (0)0.4+2 (5)3 (16)0 (0)++12 (29)6 (32)7 (30)+++25 (61)10 (53)16 (70)VHLNeg0 (0)a1 (5)0 (0)0.9+7 (18)0 (0)1 (4)++12 (30)11 (55)11 (48)+++21 (53)8 (40)11 (48)CA IXNeg10 (24)4 (20)1 (4)0.3+0 (0)0 (0)0 (0)++1 (2)2 (10)2 (9)+++30 (73)14 (70)20 (87)GLUT1Neg0 (0)1 (5)0 (0)0.02+8 (20)6 (30)0 (0)++15 (37)4 (20)6 (26)+++18 (44)9 (45)17 (74)SDF1Neg8 (20)a7 (37)a0 (0)<0.005+4 (10)4 (21)0 (0)++13 (33)2 (11)5 (22)+++15 (38)6 (32)18 (78)aOne case not tested
For CA IX staining, no antigen retrieval step was used. Slides were incubated with a mouse primary antibody to CA IX in a 1:50 dilution for 30 min at room temperature. Detection was performed with the Envision+ system–horseradish peroxidase system for mouse primary antibodies (DAKO).
GLUT-1 staining was performed with a rabbit polyclonal anti-GLUT1 (antibody clone A 3536, DAKO, dilution 1:400) without antigen retrieval and subsequently developed with a standard avidin–biotinylated peroxidase complex (DAKO).
Staining procedures for AKT and mTOR were identical. After antigen retrieval, endogenous peroxidase activity was blocked for 10 min in methanol containing 0.3% hydrogen peroxide. The AKT antibody (Phospho-Akt (Ser473)) and mTOR antibody (Phospho-mTOR (Ser2448)) both obtained from Cell Signaling (Danvers, MA, USA) were incubated overnight at 4°C in a 1:50 dilution and subsequently detected with a standard avidin–biotinylated peroxidase complex.
SDF-1 staining was performed similar to the AKT and mTOR staining, except for the secondary antibody, which was a goat anti-rabbit antibody in this case.
For VHL staining, antigen retrieval was performed and endogenous peroxidase was blocked as the AKT and mTOR staining. Primary antibody against VHL was incubated for 60 min by room temperature, followed by Powervision (Immunologic, Duiven, the Netherlands) incubation for 30 min and subsequently the staining was detected with diaminobenzidine (SIGMA FAST™ 3,3′-diaminobenzidine tablets, Sigma Aldrich, St. Louis, MO, USA).
Before the slides were mounted with cover slips, all sections were counterstained for 30 s with Mayer’s hematoxylin and dehydrated in 70%, 96%, and subsequently 100% ethanol and finally in xylene.
Evaluation and statistics
The intensity of the staining was compared between the normal mucosa, nonprogressed, progressed adenomas, and carcinomas and scored by two investigators. Staining intensity was scored in four classes (0 to 3) taking the negative control as a reference for score 0 and the positive control (clear cell renal adenocarcinomas (Grawitz tumor) as a reference for score 3). For HIF-1α, only nuclear staining was scored, for CA IX, membrane staining, and AKT and SDF-1 cytoplasmic staining, for mTOR predominantly cytoplasmic but also membrane staining was scored. Significance of differences between categories was analyzed by means of the Kruskal Wallis test. P-values < 0.05 were considered to be significant.
Results
HIF-1α expression and its regulators hypoxia and VHL
In normal colorectal mucosa nine out of ten samples showed HIF-1α present in nuclei of the surface epithelium (Fig. 2). To evaluate whether hypoxia played a physiologic role in the induction of HIF-1α in normal colorectal mucosa, we used mice that were injected with pimonidazole, a chemical marker of hypoxia that can be visualized by immunohistochemistry. The presence of tissue hypoxia was demonstrated in the surface epithelium of normal colon mucosa of the treated mice (Fig. 3). The pattern of hypoxic cells in the normal colorectal mucosa in mice matched with the distribution of HIF-1α immunohistochemical staining in human colorectal mucosa.
Fig. 2Serial sections of normal colon mucosa, colorectal adenoma, and adenocarcinoma analyzed for HIF-1α and VHL expression. HIF-1α staining was observed in the nuclei of surface epithelium of normal mucosa and colorectal adenoma, whereas in adenocarcinomas HIF-1α was present near hypoxic regions with necrosis. VHL expression was observed in the cytoplasma of all epithelial cells of normal mucosa, adenomas, and adenocarcinomasFig. 3Detection of hypoxia in murine colorectal mucosa by pimonidazole. a Colorectal mucosa of a non-injected mouse, b colorectal mucosa of a mouse that was injected intravenously with pimonidazole. After 90 min, the mouse was killed and colon tissue harvested and formalin fixed. Immunohistochemical staining against pimonidazole revealed hypoxia in the surface epithelium of the normal colon mucosa
The presence of HIF-1α in normal colorectal mucosa was compared with that in adenomas and carcinomas. Adenomas displayed HIF-1α expression in 53 of the 61 cases, predominantly at the surface, gradually decreasing towards the center of the adenomas. No differences were seen between nonprogressed and progressed adenomas (85% and 90%, respectively), as shown in Table 2. Of the carcinomas, 78% (n = 18) were positive, showing nuclear staining of HIF-1α in epithelial tumor cells. In carcinomas, a decreasing gradient of HIF-1α staining was observed from necrotic areas towards the well-vascularized stroma. Besides the epithelial tumor cells, in carcinomas, inflammatory cells in the tumor-associated stroma, and in five cases B-cells in lymph follicles, were positive for HIF-1α. Statistical analysis showed no significant differences in intensity of HIF-1α staining between normal mucosa, adenomas, and carcinomas.
VHL, the regulator of HIF-1α degradation, was also analyzed by means of immunohistochemistry (Fig. 2). VHL expression was observed in all epithelial cells in normal colorectal mucosa as well as in colorectal adenomas and carcinomas.
AKT and mTOR, oncogenic pathways regulating HIF-1α
To investigate whether, in addition to hypoxia, oncogenes and/or growth factors can contribute to the elevated HIF-1α, we studied the presence of phosphorylated forms of AKT and mTOR, which reflect an active state of the oncogene-driven pathway of HIF-1α activation. Low-intensity cytoplasmic staining for the active form of AKT (P-AKT) was seen in epithelial cells of normal mucosa in all tissues (Fig. 4). Positive staining was also seen in the smooth muscle cells of the muscularis propria and muscularis mucosae and in the walls of blood vessels. In adenomas, staining in epithelial cells was more intense, present throughout the neoplastic tissue, and observed in all cases. Staining patterns in adenocarcinomas were similar and also observed in all samples analyzed.
Fig. 4Typical immunohistochemical staining patterns of the active forms of AKT (P-AKT) and mTOR (P-mTOR) in normal colon mucosa, colorectal adenoma, and adenocarcinomas. Presence of active AKT is seen in the cytoplasm of all epithelial cells with increasing intensities from the normal mucosa towards adenocarcinoma. Presence of active mTOR is restricted to the surface epithelium in normal mucosa and adenomas. In adenocarcinomas, cells surrounding necrotic areas show active mTOR as well. In addition, in adenocarcinomas, intracellular localization of mTOR changes from cytoplasm towards cellular membrane staining
Active mTOR was only present as a cytoplasmatic staining; nuclear staining was not observed in any of the cases (Fig. 4). In normal mucosa, mTOR was detected in the surface epithelium in all tissues. Likewise, in adenoma samples, mTOR was expressed in the surface epithelium but also more diffusely throughout the adenomas. In adenocarcinomas, staining patterns were diffused across the whole tumor, but more intense expression was detected in areas surrounding necrosis. Higher levels of active mTOR were observed at the invasive front of the tumor. In the adenocarcinomas, besides the cytoplasmic staining in some cells, a clear cellular membrane staining was observed. In all normal mucosa samples, adenomas, and carcinomas, mTOR was present, except for 5% of the nonprogressed adenomas, which were negative for mTOR (Table 2).
Expression of the HIF1 downstream targets Glut1 and SDF-1
To investigate to what extent downstream pathways were affected by HIF-1 upregulation, several HIF-1 target genes, including GLUT1 and SDF-1, were analyzed in normal mucosa, adenomas, and adenocarcinomas. Expression of GLUT1, which is involved in glucose metabolism, was closely correlated with HIF-1α in all colon tissues analyzed. In normal colon mucosa, GLUT1 expression showed a gradient from the surface epithelium towards the basis of the crypts (Fig. 5). All normal tissues showed a positive GLUT1 staining. In nonprogressed adenomas, all cases showed GLUT1 expression compared to 95% of progressed adenomas. All adenocarcinomas (n = 23) were positive for GLUT1 as well. Staining was localized at the cell membrane of epithelial cells in normal mucosa, adenomas, and adenocarcinomas (Fig. 5). Most intense staining was seen at the surface epithelium of adenomas and in the vicinity of necrosis in carcinomas. As to staining intensity, a significant difference was detected between the adenomas and the carcinomas (Table 2).
Fig. 5Typical immunohistochemical staining patterns of HIF-1α, GLUT1, SDF-1, and CA IX in normal colon mucosa, colorectal adenomas, and adenocarcinomas. The downstream targets of HIF-1, i.e., SDF-1, GLUT1, and CA IX, have predominantly overlapping staining with the presence of HIF-1. However, in normal mucosa, CA IX is not expressed in the HIF-1α positive surface epithelium but is expressed in the basal of the crypts. In adenocarcinomas, SDF-1 is not only present in HIF-1α-positive cells but in all epithelial cells
In normal colon tissue, SDF-1 expression was comparable to that of HIF-1α, i.e., it was detected in a gradient from the surface epithelium towards the basis of the crypts. However, in all colorectal adenomas and adenocarcinomas, intense SDF-1 expression was observed in the epithelial cells, which was located in the cell membrane and cytoplasm (Fig. 5). Because this epithelial SDF-1 expression was seen in all epithelial cells, it co-localized only partly with HIF-1α presence. In contrast to the comparable intensity of the HIF-1α staining in adenomas and adenocarcinomas, the SDF-1 staining intensity was much higher in adenocarcinomas than in the adenomas (Table 2). In addition to the epithelial expression of SDF-1, endothelial cells of blood vessels stained positive for SDF-1 (Fig. 6).
Fig. 6SDF-1 expression in epithelial and endothelial cells of normal detected by immunohistochemistry
Expression of CA IX
CA IX was originally identified in gastrointestinal tissues and later recognized as a HIF-1 target gene in many tissues. In normal mucosa, the presence of CA IX actually did not co-localize with that of HIF-1α. CA IX was observed in the bottom parts of the crypts, i.e., in the proliferative compartment, in contrast to HIF-1α, which was present in the surface epithelium. Seventy percent of the normal tissues showed CA IX expression. In adenomas and particular adenocarcinomas, CA IX and HIF-1 expressions had more resemblance. In adenomas, CA IX was present in a more diffuse pattern throughout the whole adenoma, including the surface epithelium (Fig. 5). In the adenocarcinomas, CA IX showed a peri-necrotic staining, mainly co-localizing with the HIF-1α expression.
Discussion
The present study demonstrates the presence of nuclear HIF-1α in the surface epithelium of normal colon and adenomas and in areas associated with peri-necrotic areas of colon adenocarcinoma. The data support a hypoxia-induced HIF-1 cascade, both in normal and neoplastic colorectal mucosa, as suggested by pimonidazole and HIF-1α patterns in association with GLUT1 and SDF-1 in normal colorectal mucosa and adenomas, as well as peri-necrotic HIF-1α and CA IX patterns in colorectal carcinomas. The occurrence of hypoxia around the surface epithelium was confirmed by pimonidazole staining of unaffected murine colon. Furthermore, hypoxia and the oncogenic activation of the AKT–mTOR pathway both contributed to HIF-1 expression in adenomas and adenocarcinomas. Moreover, the expression patterns of CA IX in normal mucosa and SDF-1 in neoplastic mucosa pointed to additional regulations independent from HIF-1.
The hypoxia–HIF-1 cascade
The presence of HIF-1α under physiological circumstances in the surface epithelium of normal colorectal mucosa observed in the present study may be due to the anaerobic environment in the lumen of the colon, which contributes to a relatively hypoxic state of especially this surface epithelium. Indeed, we observed accumulation of the chemical hypoxia marker pimonidazole particularly in the surface epithelium, indicating low oxygen levels [19]. The presence of pimonidazole showed a gradient towards the basis of the crypts, suggesting that the cause of hypoxia was indeed related to the anaerobic lumen. Apparently, the capillaries in the surface regions of the colon epithelium were unable to cope with the high oxygen demand of the tissue. This strongly suggests that these hypoxic areas may induce HIF-1α, which contributes to a sustained pressure on the stimulation of angiogenesis and glycolysis to adjust the energy levels of these cells.
Although immunohistochemical analysis of patient samples does not allow a formal analysis of functional relationships, it is highly likely that the HIF-1 detected in normal colorectal mucosa was transcriptionally active because HIF-1α was detected in the nuclei and the HIF-1 downstream targets GLUT1 and SDF-1 were present in the same regions where HIF-1α was found.
In 87% of colorectal adenomas, HIF-1α was present, showing staining predominantly in the nuclei of the surface epithelium. In colorectal adenocarcinomas, the presence of HIF-1α changes from the surface epithelium to epithelium cells surrounding regions of necrosis, indicating severe hypoxia. Presence of HIF-1α in the nuclei of epithelial cells went along with expression of the downstream targets GLUT1 and SDF1 in all different stages of the colon carcinogenesis.
In contrast to SDF-1 and GLUT1, the downstream target CA IX, known as a stringent hypoxia marker [35], did not co-localize with HIF-1α staining in normal colorectal mucosa. It should be noted that CA IX was originally described to have a role in maintaining the physiological equilibrium in the gastrointestinal tract. In fact, CA IX was more abundant in the basal half of the crypts, containing the proliferating compartment, rather than in the upper half of the mucosa where HIF-1α expression was observed. Indeed, co-localization of CA IX with Ki-67, a proliferation marker, has been found in colorectal cancer [31]. The lack of HIF-1α in the CA IX-positive regions indicates an HIF-1α-independent regulation of CA IX in normal colon epithelium. CA IX expression was reported to be also regulated by the transcription factor SP1 [18, 31].
Oncogenic upregulation of HIF-1
Oncogene-dependent regulation of HIF-1 can be mediated by the AKT–mTOR pathway [24, 39]. In the present study, in epithelium of all normal colorectal mucosa, a weak staining of P-AKT was observed, confirming data of Itoh et al. [14]. In all adenomas and adenocarcinomas analyzed, the active (phosphorylated) forms of AKT and mTOR were detected as well. Especially active mTOR showed co-localization with HIF-1α in normal tissue as well as in adenomas and adenocarcinomas. Co-localization of P-AKT with HIF-1α was less evident. Recently, in breast cancer, it has been shown that in the absence of P-AKT, only low levels of HIF-1α could be detected while in the presence of P-AKT high HIF-1α protein levels were observed [10]. In colorectal tumorigenesis, this regulation mechanism may be less relevant because staining was already observed in normal tissue. In addition, the presence of active AKT was not HIF-1 related because in adenocarcinomas P-AKT was present in a higher percentage of cells present than HIF-1α. The downstream target of AKT, mTOR, displayed comparable cellular expression patterns to HIF-1α in normal, adenomas, and carcinomas. This suggests that the AKT–mTOR–HIF-1α axis may be important in tumor growth and development as was seen in prostate epithelial cells [25].
Expression of VHL
An alternative mechanism that can lead to HIF-1α overexpression is loss of VHL expression. In normal colon mucosa, VHL is expressed, while in colorectal tumors VHL has been found to be lost or mutated in 11% of cases [7, 21]. Mutations in the VHL gene have been reported to result in cytoplasmic staining of HIF-1α in colorectal cancer in one study [22], while this could not be confirmed in another study of 80 patients [40]. Also in the present study, no cytoplasmic staining of HIF-1 α was seen at all, and VHL expression was seen in all epithelial cells of normal mucosa, colorectal adenomas, and carcinoma.
Expression of SDF1
SDF-1 was investigated in the present study as a downstream target of HIF-1 [13, 32]. Surprisingly, epithelial cells showed much stronger SDF-1 expression than endothelial cells lining blood vessels. In addition, expression was not merely restricted to areas showing HIF-1α staining but was observed in all epithelial adenomas and adenocarcinomas. SDF-1 staining in tumors cells has also been reported in breast cancer patients [29] and in clear cell–renal cell carcinomas, in which HIF-1α is constitutively expressed [37]. The intense staining of the colon tumor cells suggests that SDF-1 not only has a function in attracting circulating progenitor cells towards the angiogenic areas [12]. Indeed, SDF-1 stimulates intestinal epithelial cell migration and enhances the integrity of the mucosal barrier [34].
The level of expression of SDF-1 might also play a role in tumorigenesis because it is related with prognosis in colorectal cancer. The presence of its coding mRNA and the mRNA of its receptor, CXCR4, have been shown to be a poor prognostic factor [20]. In tumor formation, SDF-1 may bear impact on the recruitment of various types of progenitor cells needed for enhanced growth of the tumor tissue [29]. Our study supports a role of SDF-1 in carcinogenesis because SDF-1 staining becomes more intense with the progression of normal colon tissue toward carcinomas. However, future studies are needed to substantiate such role.
Conclusion
The common expression of HIF-1α in the surface epithelium of the colorectal mucosa suggests that HIF-1 may play a role in the physiology of normal colon tissue. HIF-1α presence does not appear to be related to progression of colorectal carcinogenesis in contrast to GLUT1 and SDF-1, which are expressed more intensely at later stages of this process.


Liver Protective Effects of Morinda citrifolia (Noni)

This study evaluated the protective effects of Noni fruit juice on acute liver injury induced by carbon tetrachloride (CCl4) in female Sprague-Dawley (SD) rats. Liver damage (micro-centrilobular necrosis) was observed in animals pretreated with 20% placebo (drinking water) + CCl4. However, pretreatment with 20% Noni juice in drinking water + CCl4 resulted in markedly decreased hepatotoxic lesions. Furthermore, serum alanine aminotransferase and aspartate aminotransferase levels were significantly lower in the Noni group than the placebo group. In a correlative time-dependent study, one dose of CCl4 (0.25 mL/kg in corn oil, p.o.) in female SD rats, pretreated with 10% placebo for 12 days, caused sequential progressive hepatotoxic lesions over a 24 h period, while a protective effect from 10% Noni juice pretreatment was observed. These results suggest that Noni juice is effective in protecting the liver from extrinsic toxin exposure.
Introduction
Carbon tetrachloride (CCl4) is a common environmental pollutant and liver carcinogen. Due to its detoxifying function in protecting the body, the liver is a target for CCl4 toxicity [1]. Antioxidants and anti-inflammatory agents play a critical role in liver protection by scavenging superoxide anion radicals (SAR) and neutralizing lipid peroxides (LPO). They also suppress the inflammatory response [2–4]. Therefore, there is need for a natural product that protects the liver, but is also cost-effective and safe, without long-term side effects. This study evaluated the liver protective potential of the fruit juice of Morinda citrifolia (Noni), from Tahiti, a strong antioxidant and anti-inflammatory nutritional supplement.
We have previously reported that Noni juice is an effective antioxidant, demonstrating a dose-dependent effect against SAR and LPO in vitro [5]. Based on the data from our laboratory and others, we suggested that Noni juice may be effective in protecting the liver against acute CCl4 toxicity. To test our hypothesis, a classic CCl4-induced liver injury model was chosen to study the liver protective effect in female SD rats. Morphological change is the most direct sign of liver injury. Histopathological changes were evaluated for morphological evidence of the liver protective properties of Noni juice. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assays that measure liver function were performed on the Noni group (Noni + CCl4) and compared to those of the positive control group (placebo + CCl4).
Materials and Methods
Materials
Carbon tetrachloride was purchased from Sigma Chemical Co., St. Louis, MO, USA. Noni juice was made by Morinda Holdings Inc. (Provo, UT, USA) from Noni fruit originating in Tahiti. The placebo was prepared using the same procedures and ingredients for making Tahitian Noni® Juice (TNJ), but without the Noni component.
Animals
Six-week-old female SD rats were purchased from Charles River Inc. (Wilmington, MA, USA). The animals were housed in a room maintained at 25 °C with a 12 h photoperiod. They were fed a laboratory chow diet and water ad libitum. The experimental design for these tests was approved by the Institutional Ethics Review Committee for animal experiments at the National Cancer Center.
Experiment 1
Animal Treatment
Sixteen female SD rats were divided into two groups of eight each. Both groups were provided drinking water for 7 days. Twenty percent placebo was added to the drinking water in one group and 20% Noni juice in the other. On day 8, four animals from each group were fed 0.5 mL/kg CCl4 in corn oil. All animals were sacrificed at 6 h post-CCl4 administration. The liver was removed and fixed in 10% neutral formalin for histological examination. Blood was collected from each animal for serum AST and ALT activity to examine liver function.
LM Examination
Liver samples were preserved in 10% neutral buffered formalin and dehydrated in a graded alcohol series. Following xylene treatment, the specimens were embedded in paraffin blocks and cut into 5 μm thick sections. Consecutive sections were stained with hematoxylin and eosin (H & E) and examined under an Olympus BH2 light microscope. The number of foci/mm2 was counted to assess the degree of necrosis [6]. Digital images were recorded with the Pixera Visual Communication Suite.
ALT and AST Assay
Serum was separated from clotted blood by centrifugation at 1,600 rpm for 10 min at 4 °C. Serum ALT and AST activities were measured using an automatic biochemical analyzer calibrated with quality control standards (E.I. du Pont de Nemours) in a clinical laboratory (Family Medicine Laboratory, UIC College of Medicine at Rockford).
Experiment 2
Animal Treatments
Thirty-six female SD rats were divided into two groups of 18 each. While dose range-finding experiments revealed that maximum protection occurred with 20% Noni juice in drinking water, optimum protection (per amount ingested) occurred with 10%. Therefore, each group was supplied 10% placebo or 10% Noni juice in drinking water for 12 days. On day 13, 15 animals from each group were fed 0.25 mL/kg CCl4 in corn oil. Three animals from each group were sacrificed at 1, 3, 6, 16, and 24 h post-CCl4 administration. The remaining three animals in each group were fed 1 mL/kg corn oil and sacrificed 1 h later, to serve as vehicle placebo and Noni controls. Multiple liver-tissue samples were removed from each rat to be fixed for LM examination.
LM Examination
Liver samples for experiment 2 were prepared and observed for light microscopy as described in experiment 1.
Statistical Analysis
Statistical analysis was done by a Student two-tail T test [7]. Differences were considered as significant at P < 0.05 and highly significant at P < 0.01. Results were expressed as mean ± SD.
Results and Discussion
In experiment 1, the liver lobular structure among rats treated 6 h earlier with 20% placebo or 20% Noni juice did not reveal any noticeable histological changes (Fig. 1a,b), which indicates that high-dose pretreatment with Noni juice or placebo does not produce hepatotoxicity. Hepatic sections, from rats pretreated with 20% placebo followed by CCl4 treatment, showed typical CCl4-induced liver injury (Fig. 1c). Sections revealed extremely vacuolized hepatocytes (balloon liver cells and apoptotic necrotic cells) adjacent to the central vein (arrows and arrowhead, Fig. 1c), indicating acute CCl4-induced hepatotoxicity. Hepatocytes were significantly protected from CCl4-induced toxicity by 20% Noni juice pretreatment (Fig. 1d), and demonstrated a normal appearance, except for slight steatosis (fatty deposition) in hepatocytes pericentral to the vein. In the CCl4 + 20% placebo group, 23.8% of cross-sectional areas contained swollen balloon cells, whereas in the CCl4 + 20% Noni group, only 1.9% contained vacuolized cells (P < 0.001). Furthermore, the apoptotic cells were dramatically decreased by 50% in the Noni pretreated group when compared with the CCl4 + placebo group (P < 0.001).
Fig. 1H & E liver sections (250X), from female SD rats showing protective effect of 20% Noni pretreatment d on acute hepatotoxicity at 6 h post-CCl4 exposure (0.5 mL/kg CCl4 in corn oil). The rats in a (control) and b (noni treated) received no CCl4 exposure, and show no signs of liver-cell damage. With CCl4 exposure, there are significant hepatotoxicity effects: balloon liver cells and apoptotic necrotic cells adjacent to the central vein (arrow and arrowhead (c)). d In contrast, with Noni pretreatment + CCl4 exposure, shows no signs of hepatotoxicity. b and d (noni treated) show cell patterns that are more ordered, and show no signs of hepatotoxicity
In experiment 2, 10% Noni + CCl4 pretreatment resulted in a delayed appearance of damaged hepatocytes with a significantly decreased number of necrotic foci (80%) and apoptosis (50%), compared to the placebo group at 3, 6, and 16 h (arrows and arrowhead, Fig. 2b,d,f). These results indicate that Noni juice protects the liver from acute CCl4 exposure. Cellular damage consisted of vacuolated hepatocytes and inflammatory cells surrounding the central vein in the placebo + CCl4 group (Fig. 2b,d,f). Conversely, liver lobules in the Noni juice + CCl4 group maintained a normal appearance in each of the post-treatment time intervals (Fig. 2a,c,e).
Fig. 2Rat liver sections (100×), showing representative sequential hepatopathology at 3, 6, or 16 h following pretreatments with 10% Noni juice, or placebo, plus 0.25 mL/kg CCl4. Panels: a 3 h post-CCl4 + Noni juice (normal hepatic structure); b 3 h post-CCl4 + placebo, (first stages of hepatocyte swelling pericentral to vein; arrow denotes swollen hepatocytes); c 6 h post-CCl4 + Noni (normal hepatic structure); d 6 h post-CCl4 + placebo, (increased number and degree of swollen hepatocytes); e 16 h post-CCl4 + Noni (balloon cells adjacent to pericentral vein, and delayed response of hepatotoxicity); and f 16 h post-CCl4 + placebo (increase in number of damaged foci, and an extension of damaged area midzonal to vein; arrow indicate swollen hepatocytes, arrowhead denotes inflammatory foci)
Comparative histopathological changes of the Noni juice and placebo-treated groups, at 3 h post-0.25 mL/kg CCl4 treatment, are shown in Fig. 3. The effects of 10% Noni juice on reducing inflammation surrounding the central vein area (Fig. 3a), and the absence of swollen hepatocytes are clearly shown in the H & E sections. In Fig. 3b, a similar liver section from the CCl4 + 10% placebo treatment reveals swollen hepatocytes and a massive influx of inflammatory cells (arrow and arrowheads, Fig. 3a,b).
Fig. 3H & E cross sections (150×), of pericentral vein areas showing inhibitory effect of 10% Noni juice on inflammation at 3 h post-0.25 mL/kg CCl4-induced acute hepatotoxicity (a), versus placebo + CCl4 (b). Arrows denote increased foci of inflammatory cells, arrowheads indicate swollen hepatocytes
Noni Juice Suppresses ALT and AST Activities
Serum ALT and AST activities were determined for enzymatic evidence of liver injury (Table 1). The protective effect of Noni juice against CCl4-induced hepatotoxicity was demonstrated by a significant 50% suppression (P < 0.05) of ALT activity compared to the placebo. AST activity in the Noni juice pretreated group was significantly suppressed by 68% (P < 0.01), compared to the placebo. As noted in the table, the 20% placebo or 20% Noni juice in drinking water for 7 days did not influence liver function compared with the control. In contrast, the 20% placebo + CCl4 treatment produced acute liver damage as measured by elevated serum ALT and AST activities.
Table 1Effect of Noni juice on acute CC14-induced hepatitis in SD ratsTreatmentALT (IU/L)AST (IU/L)Control67 ± 31**222 ± 121**Placebo (20%)67 ± 26**222 ± 99**Noni juice (20%)65 ± 59**184 ± 31**Placebo (20%) + CCl4 0.5 mL/kga136 ± 411135 ± 466Noni juice (20%) + CCl4 0.5 mL/kg68 ± 12**358 ± 69*aData from each group were compared to that of the positive control group (20% placebo + 0.5 mL/kg CC14). Data are expressed as means ± SD (n = 4). Student two-tail T test*P < 0.05, **P < 0.01
The present study supports the protective effects of Noni juice on acute CCl4-induced hepatotoxicity. Necrosis of hepatocytes was minimized in livers of animals pretreated with Noni juice in both experiments. Noni juice prevented free-radical-induced oxidative-pathological events.
Damaged hepatocytes release ALT and AST enzymes following acute exogenous toxin exposure. Therefore these elevated serum enzyme levels are good chemical indicators of liver damage. Noni juice is able to normalize liver function after acute exposure to CCl4. Serum ALT and AST activity, a measure of defense enzyme levels, was significantly suppressed after CCl4 exposure in the Noni juice pretreated animals, compared with the elevated enzyme levels in the placebo pretreated animals. Therefore, pretreatment with Noni juice indeed protected hepatocytes from acute CCl4 exposure.
A few reports have suggested the involvement of Noni juice in the development of chemically induced hepatitis in a limited number of cases in Europe. However, no causal role has been established in any of these instances. Further, an official European investigation of these cases determined that no relationship between Noni juice and hepatitis was evident and that consumption of Noni juice is unlikely to induce adverse human liver effects [8]. Animal and human studies have revealed that Noni juice is not hepatotoxic, even at very high doses [9, 10].
Conclusions
Our investigation suggests that Noni juice exerts effective protection against acute extrinsic chemical induced hepatic injury by inhibiting inflammatory response and suppressing elevated liver enzyme activities; thus preventing consequent cell membrane damage. Histopathological observations revealed morphological evidence, and bioassays revealed functional evidence, that Noni confers resistance to acute CCl4-induced hepatotoxicity. High dosages of Noni and placebo pretreatments did not induce liver damage. Noni may thus be an efficacious natural hepatoprotective nutritional supplement against chemically induced hepatotoxicity. Future studies should focus on how Noni juice influences the inflammatory and cytotoxic cytokines involved in liver injury.

liver protection
morinda citrifolia
noni
carbon tetrachloride


Congenital anomalies of the male urethra

The spectrum of congenital anomalies of the male urethra is presented. The embryologic basis of each anomaly, when known, is discussed. Clinical and imaging features of each entity are presented.
Introduction
The development of the male genitourinary system is complex. Numerous anomalies of the male urethra exist, either as isolated anomalies or in combination with other disorders. The current understanding of the embryology and anatomy of normal male urethral development might help explain the causes of the various anomalies as well as their relationships.
Embryology of the male urethra
Between weeks four and five of fetal life, the cloaca is separated from the amniotic cavity by the cloacal membrane [1]. The cloaca is divided into the urogenital sinus and the hindgut by the urorectal septum, which descends in a rostral-to-caudal fashion (Fig. 1). The urogenital sinus is the precursor of the bladder and posterior urethra. The urorectal septum is formed by proliferation, migration and fusion of mesodermal folds that arise from the lateral walls of the cloaca. When the urorectal septum reaches the caudal aspect of the cloaca, the cloacal folds, which lie on the dorsal portion of the cloacal membrane, fuse to form the rudimentary perineum. The cloacal membrane subsequently undergoes disruption such that the hindgut and phallic cloaca open separately to the exterior, divided by the urorectal septum (Fig. 2).
Fig. 1Embryologic development of the male genitourinary tract. The cloaca is divided into the urogenital sinus (precursor of the bladder and posterior urethra) and the hindgut by the urorectal septum (adapted from reference 2)Fig. 2Embryologic development of the male genitourinary tract. The urorectal septum continues to descend and the cloacal membrane undergoes disruption. The hindgut and phallic cloaca now open separately to the exterior (adapted from reference 4)
The most caudal aspect of the cloaca (phallic cloaca) extends distally through the developing genital tubercle. Proliferation of rostral mesoderm of the genital tubercle displaces the cloaca so that it lies on the caudal aspect of the developing glans (Fig. 3). Failure of this step results in epispadias. The endodermal lining of the phallic cloaca appose to form the urethral plate. Portions of the cloacal membrane also contribute to the urethral plate (Fig. 4). The urethral plate undergoes median cleavage. Following mesodermal proliferation and fold fusion extending proximally to distally, the shaft urethra and proximal two-thirds of the glandular urethra are formed. This is lined by endoderm. The distal urethra at the glans penis is formed from fusion of distal folds that arise from the apical part of the glans distal to the cloaca and subsequent urethral plate (Fig. 4). It is lined by ectoderm [2–4]. Discontinuity in the urethral plate or abnormality of fusion of the urethral folds can lead to hypospadias.
Fig. 3Embryologic development of the male genitourinary tract. The phallic cloaca extends distally through the developing genital tubercle. Proliferation of rostral mesoderm of the genital tubercle displaces the cloaca so that it lies on the caudal aspect of the developing glans (adapted from reference 2)Fig. 4Embryologic development of the male genitourinary tract. Median cleavage of the urethral plate occurs. The urethral plate forms the deep and lateral walls of the proximal urethra. The phallic cloaca opens to the exterior at the base of the glans (adapted from reference 4)
Normal anatomy of the male urethra
The male urethra is divided into the prostatic, membranous and penile (cavernous) urethra (Figs. 5 and 6). The prostatic urethra courses through the prostate and is the widest part of the urethra. On the posterior wall of the prostatic urethra lies the urethral crest, an elevated longitudinal ridge of tissue. The verumontanum (seminal colliculus) is located at the midportion of the urethral crest and is the site of the ejaculatory duct orifices. Additionally, the orifice of the prostatic utricle, a glandular grouping, is located at the summit of the verumontanum. The utricle, a derivative of the Mullerian duct and urogenital sinus, and the male homologue of the uterus and vagina, is a blind indentation that extends dorsally into the prostate and can measure up to 6 mm. The prostatic ducts drain into the prostatic sinus, the fossa on either side of the elevated urethral crest. The intermuscular incisura produces an indentation on the anterior aspect of the prostatic urethra, at the level of the verumontanum, and results from prominent collagenous tissue.
Fig. 5Normal male urethral anatomyFig. 6Voiding cystogram in a 3-year-old with prior urinary tract infection. Normal urethral anatomy is shown (P prostatic urethra, short arrow incisura, long arrow verumontanum)
The membranous urethra is the shortest, least distensible portion of the urethra. It is surrounded by the sphincter urethrae membranaceae (external sphincter), which in addition to vessels, nerves and deep transverse perineal muscle represents the urogenital diaphragm. The internal sphincter is located at the bladder neck.
The penile urethra is the longest portion of the urethra. The proximal portion of the penile urethra, the bulbar urethra, is dilated and is the site of drainage of Cowper’s ducts. The penile urethra is also dilated distally at the glans penis, where it forms the fossa navicularis. The midportion of the penile urethra is tethered superiorly by the suspensory ligament [5].
Congenital anomalies of the urethra
Posterior urethral valves
Posterior urethral valves (PUV), first described in 1717 by Morgagni, occur in one of every 5,000–8,000 male infants [6]. PUV can lead to bilateral renal obstruction, bladder dysfunction, vesicoureteral reflux, hydronephrosis and renal damage. In 1919, Young proposed a classification system consisting of three types of valves. Type I valves (the most common) arise from the verumontanum and extend distally to attach to the lateral walls of the urethra as two leaflets (type Ia) or as a unicuspid leaflet (type Ib). Type II valves extend proximally from the verumontanum to the bladder neck, and type III valves represent a diaphragm attached to the circumference of the urethra with a central hole, distal (type IIIa) or proximal (type IIIb) to the verumontanum [1, 8]. The existence of Young’s type II valves is debated [7]. Type III valves are considered by many to be the same entity as Cobb’s collar and are discussed in the section on congenital urethral strictures [1, 7].
The valves are composed of connective tissue interspersed with smooth muscle, similar to the tissue encasing the ejaculatory ducts, and are lined by stratified squamous epithelium rather than the transitional epithelium that lines the posterior urethra [1].
The embryologic defect leading to the development of PUV is not known. Many authors believe the anomaly is caused by abnormal integration of the Wolffian ducts into the urethra, while others consider it a result of persistence of the cloacal membrane [1, 7].
Autopsy studies have shown that the valves represent a single obliquely oriented diaphragm rather than a bicuspid structure as originally believed [7, 9]. The term congenital obstructive posterior urethral membrane (COPUM) has, therefore, been introduced [10].
Many cases of PUV are detected by prenatal sonography. Normal fetal renal parenchyma and amniotic volume do not necessarily predict good postnatal renal function [11]. Intrauterine intervention, while improving hydronephrosis, does not prevent the development of renal dysplasia [12]. Newborns might present with pulmonary hypoplasia secondary to oligohydramnios, or an abdominal mass caused by hydronephrosis or bladder distension. In older patients clinical signs include failure to thrive, poor urinary stream, sepsis, poor renal function or salt-losing nephropathy [13]. Treatment is by fulguration of the valve, although in infants vesicostomy or pyeloureterostomy might be performed prior to valve ablation. Posterior urethral dilatation persists after fulguration in 20% of patients. Despite therapy, 40% of patients have poor renal growth and another 40% develop end-stage renal disease [12, 14]. A normal or near normal serum creatinine level or GFR at 1 year of life, following decompression of the urinary tract, are useful positive prognostic indicators [12, 14]. In contrast, the development of proteinuria is associated with a poor prognosis [14].
Ultrasonography is usually the initial imaging modality used to evaluate patients with suspected PUVs. US reveals a thick bladder wall and bilateral but asymmetric hydronephrosis. Transperineal sonography can demonstrate dilatation of the posterior urethra (the “keyhole” sign) (Fig. 7) [15]. A perirenal urinoma from forniceal rupture or urinary ascites can also be seen. PUV is the most common cause of urinary ascites in the newborn [16]. Echogenic kidneys might also be seen and are associated with a poor prognosis [14].
Fig. 7Posterior urethral valves. Sonography demonstrates a thick-walled bladder and dilatation of the prostatic urethra (arrow), the “keyhole sign”
Typical findings of PUV on voiding cystourethrography (VCUG) include a trabeculated bladder with diverticula, dilatation of the prostatic urethra and a discrepancy between the caliber of the prostatic urethra and the bulbar and penile portions of the urethra (Fig. 8). During voiding, the contrast stream between the prostatic urethra and bulbous portion of the urethra is more dorsal in position than normal. Vesicoureteral reflux is present in 50% of cases and is more often unilateral than bilateral. Reflux into the prostate might also be seen.
Fig. 8Posterior urethral valves. An image from a VCUG demonstrates a thick-walled trabeculated bladder and dilatation of the posterior urethra (long arrow), and the location of the valve (small arrow)
Prune-belly syndrome
Prune-belly syndrome (PBS) was first described in 1950 by Eagle and Barret as a triad of deficient abdominal wall muscles, urinary tract anomalies and cryptorchidism. Associated abnormalities include cardiac, limb and intestinal abnormalities (30% of patients have malrotation). Although PBS is primarily seen in males, it has been described in females; female patients have genital abnormalities including bicornuate uterus and vaginal atresia [17].
Urinary tract abnormalities, which can be evaluated by sonography and VCUG, include both upper tract disease and lower tract disease. Hydroureter, more pronounced than the coexisting hydronephrosis, is caused by poor ureteral peristalsis and poor bladder emptying rather than true obstruction. Varying degrees of renal dysplasia with or without cystic change might be present. Characteristic findings on VCUG include a large, smooth-walled bladder without trabeculations and marked bilateral vesicoureteral reflux into tortuous and laterally positioned ureters. A urachal diverticulum might be present, resulting in anterior and midline tethering of the bladder dome (Fig. 9). Calcification is rarely identified within the bladder and urachal diverticulum and is secondary to urinary stasis [18].
Fig. 9Prune-belly syndrome. a Images from a VCUG show the characteristic smooth-walled bladder that is tethered anteriorly by a urachal remnant (long arrow). Note the utricle (short arrow) arising from the dilated prostatic urethra, which is positioned dorsal to the wide bladder neck. b Voiding film shows dilatation of the proximal urethra consistent with megalourethra. Megalourethra can coexist in these patients
The prostatic urethra is dilated secondary to or in association with prostatic aplasia or hypoplasia. (Pathologically, the verumontanum is small and the ejaculatory ducts are normal.) Unlike PUVs, the prostatic urethra in patients with PBS is high in position and its posterior wall is elongated relative to its anterior wall. A normal utricle might be present. The penile urethra might be normal, or additional urethral anomalies such as megalourethra, urethral atresia or coexisting posterior urethral valves can be present. Patients with urethral obstruction do not survive [17].
Congenital urethral stricture
Although most urethral strictures in males are post-traumatic, there are rare reports of congenital urethral strictures of the bulbous urethra in neonates and older children [19]. These might be secondary to a failure of canalization of the cloacal membrane during fetal development [1, 20] and have also been referred to as Cobb’s collar, Moormann’s ring and Young’s type III valve [7, 19]. Patients present with urinary tract infection. Older children might have diurnal enuresis. Diagnosis is by VCUG or retrograde urethrography. VCUG will demonstrate focal narrowing of the bulbous urethra, while retrograde urethrography will confirm a normal penile urethra. The site of urethral narrowing in congenital urethral stricture is distal to the external urethral sphincter, which differentiates this entity from PUV (Fig. 10). Vesicoureteral reflux can be seen in up to 53% of patients [20]. Treatment is by transurethral incision. Other causes of urethral stricture, including trauma, must be excluded.
Fig. 10Congenital urethral stricture. A markedly dilated urethra is seen proximal to a congenital stricture in the bulbous urethra. Retrograde urethrography in this patient (not shown) demonstrated a focal narrowing at the bulbous urethra with a normal penile urethra. The site of obstruction is more distal than that seen with PUV
Congenital urethral polyps
Congenital urethral polyps are benign and arise from the prostatic urethra near the verumontanum. They are composed of vascular connective tissue, although glandular and nerve tissue have been described. Because they have a stalk, these polyps are mobile and can move proximally into the bladder or distally into the bulbous urethra. They can be a cause of urethral obstruction or bleeding. VCUG is diagnostic and demonstrates a mobile filling defect in the bladder neck or below the verumontanum (Fig. 11). Endoscopic resection is the treatment of choice [21, 22].
Fig. 11Urethral polyp. a Image from a VCUG demonstrates a polypoid filling defect arising from the prostatic urethra (arrow). b Note change of position of the filling defect (arrow) during the examination (images courtesy of J. Michael Zerin, MD)
Mullerian duct remnants: enlarged prostatic utricle and Mullerian duct cyst
The normal prostatic utricle (Latin for “pouch of the prostate”) is a minute, blind opening located at the verumontanum [23, 24]. It is a glandular grouping lined with epithelial cells with differentiation similar to those lining the main prostatic ducts and acini [23]. It has no function and is the male homologue of the female uterus and vagina (Fig. 12).
Fig. 12Utricle. Voiding cystourethrogram in a child with urinary tract infection demonstrates a normal utricle that was incidentally detected (arrow). The ventral urethra is not filled with contrast material
Midline cystic structures arising at the dorsal aspect of the prostatic urethra represent two distinct categories of Mullerian duct remnants. They are described as enlarged prostatic utricles when they communicate with the urethra and as Mullerian duct cysts when they do not. These terms have been used interchangeably, contributing to confusion in nomenclature.
An enlarged prostatic utricle is a congenital abnormality. Although its embryogenesis is not certain, theories include its derivation from the Mullerian duct or urogenital sinus [23] or secondary to an error in the production or sensitivity to Mullerian regression factor [25]. Unlike normal prostatic utricles, enlarged prostatic utricles are lined by squamous epithelium. Patients present with urinary tract infection, epididymitis, and postvoid dribbling [23–26]. Diagnosis is made by VCUG. During voiding, filling of a structure arising from the dorsal aspect of the prostatic urethra is seen (Fig. 13). Retrograde urethrography is also diagnostic. In some cases, urethroscopy demonstrates absence of the verumontanum [23].
Fig. 13Enlarged prostatic utricle. There is filling of a large utricle (arrow) that arises from and communicates with the posterior urethra in this patient with intersex abnormality. The patient has had penile urethral reconstruction (asterisk bladder)
Enlarged prostatic utricles are classified according to a grading system. In grade 0, the opening is located in the prostatic urethra but the utricle does not extend over the verumontanum. In grade I the utricle is larger but does not extend to the bladder neck. In grade II, it extends over the bladder neck, and in grade III, the opening of the utricle opens into the bulbous urethra rather than the prostatic urethra [23]. Enlarged prostatic utricles are often seen in patients with intersex, hypospadias (11–14%), and cryptorchidism. There is a direct relationship between the degree of hypospadias and utricular size [25].
In contrast to enlarged prostatic utricle, Mullerian duct cysts are acquired abnormalities. They present later in life, are not associated with abnormal genitalia and do not communicate with the urethra. Histologically, they are lined by columnar or cuboidal epithelium identical to that of a normal prostatic utricle. Kato et al. [23, 24] have proposed that Mullerian duct cysts develop at a later stage than enlarged prostatic utricles, secondary to narrowing or obstruction of the communication between the normal utricle and the urethra. Patients present with an incidental rectal mass, constipation, urinary retention, hematuria or obstructive azospermia [23–26]. On US, CT or MRI a cystic mass is identified dorsal to the prostatic urethra (Fig. 14). This may contain debris. VCUG is not helpful, since these cysts do not communicate with the urethra. Surgical excision is curative. Of note, there is a 3% incidence of malignancy in Mullerian duct cysts, most commonly occurring in the fourth decade [25].
Fig. 14Mullerian duct cyst. Transverse sonographic image of the bladder in this 15-year-old boy with recurrent epididymitis demonstrates a cystic mass with internal debris (arrow) posterior to the bladder (asterisk)
Cowper’s syringocele
Cowper’s glands are small paired glands located dorsal to and on either side of the membranous urethra. They secrete a mucous substance during ejaculation that acts as a lubricant. The main duct draining Cowper’s glands drain below the urogenital diaphragm into the ventral aspect of the bulbous urethra [27, 28]. During VCUG, the main duct and Cowper’s glands can fill with contrast material and appear as a tubular channel paralleling the ventral aspect of the undersurface of the bulbous membranous urethra and ending at the urogenital diaphragm (Fig. 15) [29]. This finding is usually of no clinical significance.
Fig. 15Cowper’s gland. During voiding there is filling of a structure at the ventral aspect of the bulbourethra (arrow)
Cowper’s syringocele, a rare anomaly, occurs when there is dilatation of the main draining duct. Maizels et al. [30] described four types of Cowper’s syringocele: simple syringocele, in which there is reflux into a minimally dilated duct; imperforate syringocele, in which the orifice draining the dilated duct is closed and there is cystic dilatation of the distal duct at the level of the bulbourethra; perforate syringocele, in which the orifice draining the duct is patulous and there is free reflux into the duct resembling a diverticulum; and ruptured syringocele, in which the distal portion of the duct is dilated but is not in communication with the more proximal portion of the main duct [30]. Cowper’s syringocele can also be classified as open or closed, open if it communicates with the urethra and closed if it does not [31].
Clinically, patients present with frequency, urgency, dysuria, post-void incontinence, hematuria or urinary tract infection. Diagnosis is made by VCUG, retrograde urethrography or urethrocystoscopy. Treatment requires marsupialization of the syringocele.
Anterior urethral valves and diverticula
Anterior urethral valves and diverticula are rare. Most anterior valves (40%) are located in the bulbous urethra, but they can also be located at the penoscrotal junction (30%) and penile urethra (30%) [32]. They are composed of folds located on the ventral aspect of the urethra that rise during voiding, resulting in urethral obstruction [33]. Anterior urethral diverticula communicate with the urethra and are found on the ventral aspect of the urethra between the bulbous and mid-penile urethra [32].
Although many authors distinguish between anterior urethral valves and diverticula, others consider them the same entity. It has been proposed that valves cause proximal urethral dilatation with the formation of a saccular diverticulum [32]. Conversely, progressive enlargement of a diverticulum can result in a distal valve-like flap. Embryologic theories of anterior urethral diverticula formation include a developmental defect in the corpus spongiosum leading to formation of a diverticulum, cystic dilatation of urethral glands, and sequestration of an epithelial rest [33]. McLellan et al. [28] have demonstrated connections between an anterior urethral diverticula and cowperian ducts, proposing that anterior urethral diverticula and valves arise from the anterior lip of a ruptured cowperian duct syringocele.
Clinical presentation depends on the patient’s age and the degree of urethral obstruction and includes difficulty voiding, incontinence and recurrent urinary tract infections. VCUG is diagnostic (Fig. 16). Some authors describe a difference in appearance between anterior diverticula and valves; in valves, the urethral dilatation forms an obtuse angle with the ventral floor of the urethra, while in a urethral diverticulum, the angle is acute [31]. Vesicoureteral reflux can be found in up to one-third of patients with anterior urethral valves and 20% of patients with diverticula [32]. Sonography has also been used for diagnosis [34]. Stone formation within a diverticulum has been reported [35]. Treatment consists of transurethral valve ablation or, in the case of diverticula, transurethral or open diverticulectomy and urethroplasty [32].
Fig. 16Anterior urethral diverticulum. Arrow points to a diverticulum arising from and communicating with the ventral aspect of the urethra
Anterior urethral valves occurring in the fossa navicularis, the most distal aspect of the urethra, are referred to as valves of Guerin. These are described as a septum partially separating the lacuna magna (a widening at the dorsal aspect of the distal urethra at the glans penis) from the urethra. Valves of Guerin might have a different embryologic basis than other types of anterior urethral valves. Some propose a failure of alignment between the glans urethra and penile urethra while others believe they represent an anatomic variant, as many patients are asymptomatic [36]. Valves of Guerin have been associated with urethral bleeding. VCUG is diagnostic and will demonstrate a small collection of contrast material at the dorsal aspect of the distal urethra (Fig. 17). Marsupialization of the valve into the urethral lumen is the treatment of choice [37].
Fig. 17Valves of Guerin. A small contrast-filled diverticulum (arrow) is present, arising from the dorsal aspect of the glans urethra. There is no urethral obstruction
Megalourethra
Megalourethra is caused by defective formation of the corpus spongiosum and corpora cavernosa secondary to a mesodermal defect. There are two types of megalourethra: (1) scaphoid, in which there is ventral urethral dilatation and hypoplasia of the corpus spongiosum, and (2) fusiform, in which there is circumferential urethral dilatation and hypoplasia of the corpus spongiosa and corpora cavernosa. Megalourethra is often associated with other congenital abnormalities including cryptorchidism, renal agenesis, hypospadias, primary megaureter, PBS, VACTERL complex [38] and severe gastrointestinal anomalies [39]. These patients have a functional rather than anatomic urethral obstruction, causing stasis and back pressure into the upper urinary tracts. Diagnosis is by VCUG (Fig. 18). Reconstructive surgery is required.
Fig. 18Megalourethra. Single view from a VCUG in a boy with partial sacral agenesis demonstrates focal dilatation of the urethra (arrow). Note the wide bladder neck
Urethral duplication
Urethral duplication is a rare anomaly frequently seen in association with other anomalies including hypospadias, epispadias, cleft lip and palate, congenital heart disease, tracheoesophageal fistula, imperforate anus and musculoskeletal anomalies [40].
Duplication commonly occurs along the sagittal plane. The ventral urethra is the more functional urethra and contains the verumontanum and sphincters [40]. When urethral duplication is present along the coronal plane, bladder duplication is always present [41].
Urethral duplication can be classified into three types using Effmann’s classification [42]. In type I, there is partial duplication of the urethra. In type II, there is complete duplication of the urethra. Type II urethral duplication can be subclassified as IIA1 if both urethras arise from separate bladder necks, type IIA2 if one channel arises from the other, and type IIB if there is duplication with one meatus. IIA2 Y-duplication occurs when one urethra arising from the bladder neck or posterior urethra opens to the perineum. This type of urethral duplication often coexists with stenosis of the anterior portion of the normally positioned urethra [43] and other severe congenital anomalies [44]. Type III urethral duplication consists of complete duplication of the urethra and bladder.
Embryologically, urethral duplication can be caused by abnormal Mullerian duct termination and growth arrest of the urogenital sinus [40] or misalignment of the termination of the cloacal membrane with the genital tubercle [41].
Depending on the type of duplication, patients might be asymptomatic. Symptoms include urinary tract infection, epididymitis, and incontinence [43]. Diagnosis can be made by VCUG or retrograde urethrography (Figs. 19 and 20). Urodynamic studies are helpful to confirm the position of the functional urethra in order to distinguish it from congenital urethroperineal fistula (see below) [44]. One-third of patients have associated vesicoureteral reflux [41].
Fig. 19Complete urethral duplication (type II). Image from a voiding cystourethrogram demonstrates contrast agent filling a dorsal and ventral urethra (arrows). Vesicoureteral reflux is presentFig. 20Partial urethral duplication (type I). There is partial duplication of the urethra in this patient with a history of hypospadias repair. The two channels (arrows) join to a single channel, which is demarcated by the catheter
Congenital urethroperineal fistula
Although congenital urethroperineal fistula (CUF) resembles Y-type urethral duplication, it should be considered a separate entity. In Y-type urethral duplication the ventral urethra opens to the perineum and, as in all urethral duplications, is the functional urethra. In contrast, in CUF, the dorsal urethra is the functional urethra and the ventral urethra (fistula) is hypoplastic [41, 45]. The differentiation between Y-type duplication and CUF is particularly important in the surgical management of these patients. In Y-duplication the functional ventral channel should not be resected, while in the CUF resection of the ventral channel is curative [45].
The embryology of CUF is not clear. Diagnosis is made by retrograde urethrography or VCUG to determine the dominant urethra. In the case of congenital urethroperineal fistula, voiding will be predominantly through the dorsal urethra. Urodynamics is important to determine the position of the external urethral sphincter [39].
Anorectal malformations
Congenital anorectal malformation represents a complicated spectrum of anomalies that range from anal stenosis to imperforate anus with communication with the urogenital system [46]. In males, the anomaly is characterized by gastrointestinal anomalies, imperforate anus and frequently other congenital anomalies including tracheoesophageal fistula, heart disease, limb anomalies and radial and renal abnormalities (VACTERL complex) [47]. The malformation can be classified as low or high/intermediate. Fistulous communications between the blind-ending rectum and the urinary tract are more common in males with high/intermediate anomalies. Rectobulbocutaneous fistulous tracts have also been described in which a fistula arises from the rectum, communicates with the bulbous urethra at its mid-portion and continues ventrally to terminate at the scrotum wall [48, 49]. A concurrent rectourethral fistula to the posterior urethra or, less commonly, the bladder, has been described in 28% of patients of imperforate anus [50] and there is a high incidence of other urethral anomalies as well [39, 40].
Embryologically the normal sequence of cloacal division by the urorectal septum into the urogenital sinus and rectum is disrupted. Preexisting fusion of mesoderm at the dorsal aspect of the cloacal folds might be present, which prevents normal caudal descent of the urogenital septum and results in a urorectal septal defect. Recent reports describe a possible genetic link between abnormalities of the Sonic Hedgehog gene (Shh) and midline developmental abnormalities such as those seen with the VATERL association [40, 47, 51].
Diagnosis is made by VCUG or distal loop study following colostomy (Figs. 21 and 22).
Fig. 21Anorectal malformation. Image from a VCUG demonstrates an anourethral fistula (arrow) in a newborn. This is also referred to as an H-type fistula because of its configurationFig. 22Anorectal malformation. Distal loop study in an infant with a high imperforate anus demonstrates a fistula extending from the rectum to the posterior urethra
Conclusion
The development of the male urethra is complex. Although understanding its embryology can help explain certain anomalies, there are others in which the embryologic defect has not been elucidated. Imaging, including VCUG and retrograde urethrography, is helpful in defining the anatomy and identifying associated findings.

congenital
anomalies
male urethra
embryology


The Generation R Study Biobank: a resource for epidemiological studies in children and their parents

The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health from fetal life until young adulthood. In total, 9,778 mothers were enrolled in the study. Prenatal and postnatal data collection is conducted by physical examinations, questionnaires, interviews, ultrasound examinations and biological samples. Major efforts have been conducted for collecting biological specimens including DNA, blood for phenotypes and urine samples. In this paper, the collection, processing and storage of these biological specimens are described. Together with detailed phenotype measurements, these biological specimens form a unique resource for epidemiological studies focused on environmental exposures, genetic determinants and their interactions in relation to growth, health and development from fetal life onwards.
Introduction
The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood. The background and specific research projects of the study have been described in detail previously [1, 2]. Briefly, the Generation R Study is designed to identify early environmental and genetic causes of normal and abnormal growth, development and health from fetal life until young adulthood. The study focuses on four primary areas of research: (1) growth and physical development; (2) behavioural and cognitive development; (3) diseases in childhood; and (4) health and healthcare for pregnant women and children. The main outcomes and determinants are presented in Tables 1 and 2. The general aims of the study are:To describe normal and abnormal growth, development and health from fetal life until young adulthood;To identify biological, environmental and social determinants of normal and abnormal growth, development and health from fetal life until young adulthood;To examine the effectiveness of current strategies for prevention and early identification of groups at risk.Table 1Main outcomes per research areaGrowth and physical developmentFetal growth patterns and organ developmentPregnancy complicationsPostnatal growth patternsObesityRisk factors for development of cardiovascular diseaseRisk factors for type 2 diabetesBehavioural and cognitive developmentMaternal and paternal psychopathologyFetal and postnatal brain developmentBehaviour, psychopathology and cognitionNeuromotor developmentChronic painAttachmentStress reactivityDiseases in childhoodInfectious diseases in childhoodDevelopment of the immune systemAsthma and asthma related symptomsParoxysmal neurological disordersHealth and healthcareQuality of lifeHealth care utilizationEffectiveness of screening programmesTable 2Main determinantsBiological determinantsParental anthropometrics and blood pressureFetal and postnatal growth characteristicsEndocrine and immunological factorsGenetic variantsEnvironmental determinantsMaternal and childhood dietParental life style habits (including smoking, alcohol consumption)Housing conditionsSocial determinantsParental education, employment status and household incomeParental marital statusEthnicity
For these aims, a detailed and extensive data collection has been conducted in the prenatal phase of the study and is currently conducted in the early postnatal period [2]. This data collection comprises also biological specimens including DNA, blood for phenotypes and urine samples. These biological specimens form the Generation R Study Biobank, which enables future epidemiological studies focused on environmental exposures, genetic determinants and their interactions in relation to growth, health and development from fetal life onwards. In this paper, the design, collection, processing and storage of these biological specimens in the prenatal phase of the study are described.
Design and biological samples collection
The Generation R Study is a population-based prospective cohort study from fetal life until young adulthood [2]. Mothers with a delivery date between April 2002 and January 2006 were eligible. Enrolment was aimed in early pregnancy but was allowed until birth of the child. Extensive assessments were carried out in mothers and their partners in pregnancy and are currently performed in their children. Assessments in pregnancy were planned in early pregnancy (gestational age <18 weeks), mid-pregnancy (gestational age 18–25 weeks) and late pregnancy (gestational age ≥25 weeks) and at birth. The individual time scheme of these assessments depended on the specific gestational age at enrolment [2]. The partners were assessed once in pregnancy. All children form a prenatally recruited birth-cohort that is currently being followed until young adulthood.
Additionally, more detailed assessments of fetal and postnatal growth and development are conducted in a randomly selected subgroup of Dutch children and their parents, referred to as the Generation R Focus Cohort. Studies conducted in this subgroup examine etiological associations using more time-consuming methods that cannot be applied in the whole cohort.
Biological materials have been collected in early, mid- and late pregnancy and at birth. Figure 1 shows the design of the collection of biological materials in this phase. In early, mid- and late pregnancy, biological materials were collected at the visits to one of our dedicated research centres. The planned amounts of blood taken by ante-cubital venous puncture were 35 ml in early pregnancy and 20 ml in mid-pregnancy from the mother and 10 ml from her partner. When mothers were enrolled in mid- and late pregnancy, 35 ml was taken at the first visit. Urine samples of mothers have been collected in early, mid- and late pregnancy from February 2004 until November 2005. Directly after delivery, midwives or obstetricians collected 30 ml cord blood from the umbilical vein.
Fig. 1Design data collection biological specimens
The Generation R Study is approved by the Medical Ethical Committee of the Erasmus Medical Center, Rotterdam. Mothers and their partners received written and oral information about the study. Participants were asked for their written informed.
Study cohort
In total, 9,778 mothers were enrolled in the study. Of these mothers, 91% (n = 8,880) was enrolled in pregnancy. Only partners from mothers enrolled during pregnancy were invited to participate. In total, 71% (n = 6,347) of all partners were enrolled. The general characteristics of the mothers and their partners are presented in Table 3. Of all participating mothers, enrolment was in early pregnancy in 69% (n = 6,748), in mid-pregnancy in 19% (n = 1,857), in late pregnancy in 3% (n = 275) and at birth of their child in 9% (n = 898). The cohort comprises various ethnic groups. The largest ethnic groups are the Dutch, Surinamese, Turkish, Moroccan, Dutch-Antilles and Cape Verdian mothers.
Table 3Characteristics of mothers and their partners MothersPartners(n = 9,778)(n = 6,347)Gestational age at enrolment (%)    Early pregnancy69–    Mid-pregnancy19–    Late pregnancy3–    Birth9–Pregnancy number in study (%)    1st pregnancy 94–    2nd pregnancy 6–    3rd pregnancy 0.1–Age at enrolment (years)a29.7 (5.3)32.7 (5.8)Parity (%)    055–    131–    ≥214–Ethnicity (%)    Dutch, other-European 5870    Surinamese96    Moroccan 74    Turkish 96    Dutch Antilles 43    Cape Verdian 42    Others 99Education (vocational training) (%)    Lower 138    Intermediate4541    Higher 4251Values are percentages aMean (standard deviation)
Logistics
All biological samples were bar coded with a unique laboratory number. Blood or urine samples collected at one visit have the same bar code with an additional specific tube number. This combination forms a unique registration number. All following steps in processing, storing and data management of the samples are linked to this unique number.
All blood samples from the mother and partner were taken by research nurses and temporally stored at our research centre or one of the obstetric departments at room temperature for a maximum of 3 h. At least every 3 h, these blood samples were transported to a dedicated laboratory facility of the regional laboratory in Rotterdam, the Netherlands (STAR-MDC) for further processing and storage. Participants delivered their child either at home or at one of the hospitals in Rotterdam. The midwife or obstetrician collected cord blood samples. Subsequently, courier services with a 7-days and 24-h availability were responsible for transportation of these cord blood samples to our laboratory within 2 h.
After collection and transportation, all blood and urine samples have been centrally processed and stored at the STAR-MDC laboratory. Samples for DNA extraction have initially been stored as EDTA whole blood samples and subsequently transported to the Erasmus Medical Center for further processing.
All blood and urine samples are registered in Labosys (Philips) [3]. Both the anonymous person unique study numbers and all sample numbers are registered which enables matching of each sample to a study subject. Also, picking and defrosting of samples in registered in Labosys. A back-up of this registration is available at the Erasmus Medical Center.
Response rates
Response rates for collection of biological specimens are presented in Table 4. Overall response rates for blood collection in mothers are high. To get these high response rates, we have used the venous punctures that were already planned in routine care for additional blood sample collection when this was possible. Blood for DNA extraction is available in 91, 83 and 67% of the eligible mothers, partners and children. The larger number of missings in partners and children were mainly due to lack of consent and logistical constraints during delivery, respectively. As Table 4 shows, response rates for the various blood samples at one occasion were similar. Absolute numbers of urine samples are lower than the absolute number of blood samples because the urine sample collection was performed during a limited period in the prenatal phase of the study. Tables 5 and 6 show characteristics of children and partners with and without available DNA. In children, the number of missing blood samples in children with perinatal complications including low birth weight and preterm birth is higher because of the limited possibilities and priorities during these deliveries. At this moment, efforts are carried out for completing DNA collection in the children by buccal samples.
Table 4Response rates biological specimensEligibleAvailable % (n)Mother    DNA8,88091 (8,055)    Early pregnancy    Plasma (EDTA)6,74895 (6,398)    Serum6,74894 (6,337)    Urine2,79785 (2,375)    Mid-pregnancy    Plasma (EDTA)8,24193 (7,682)    Serum8,24193 (7,631)    Urine3,38097 (3,279)    Late-pregnancy    Urine3,91896 (3,762)Partner    DNA6,34783 (5,289)Child    DNA8,82167 (5,908)    Plasma (EDTA)8,82166 (5,857)    Serum8,82166 (5,821)Number of eligible subjects reflects those participating in the study during pregnancy (DNA mother and partner) and visit our research centre in early, mid- or late pregnancy (plasma, serum and urine). The number of eligible subjects for urine sample collection is lower since this data-collection was added after starting the study. Number of eligible children reflects the number of live born children of mothers who were enrolled in the prenatal phase of the study with a known date of birthTable 5Characteristics of partners with and without DNA availablePartners (n = 6,347)DNA available (n = 5,289)No DNA available (n = 1,058)Age (years)32.8 (5.7)32.2 (5.7)Ethnicity (%)    Dutch, other-European 68.266.1    Surinamese6.86.6    Moroccan 3.86.2    Turkish 6.87.2    Dutch Antilles 2.93.4    Cape Verdian 2.71.9    Others 8.88.6Values are percentages or means (standard deviation)Table 6Characteristics of children with and without DNA availableChildren (n = 8,821)DNA available (n = 5,908)No DNA available (n = 2,913)Male (%)50.650.1Birth weight (grams)3,444 (520)3,280 (671)Birth weight <2,500 g (%)3.310.8Gestational age (weeks)40.1 (37.1–42.0)39.9 (34.7–42.1)Gestational age <37 weeks (%)3.911.2Ethnicity (%)    Dutch, other-European 64.754.6    Surinamese7.49.0    Moroccan 6.57.9    Turkish 7.59.5    Dutch Antilles 3.84.8    Cape Verdian 2.43.5    Others 7.710.7Values are percentages, means (standard deviation) or median (95% range)
DNA
For DNA extraction, one 10 ml EDTA tube from mothers, partners and children at birth have been collected and stored directly as whole blood sample at −80°C. After completion of the blood sample collection, DNA extraction was started from white blood cells. First, DNA extraction from all children has been conducted manually using the Qiagen FlexiGene Kit (Qiagen Hilden, Germany). Currently, DNA extraction from 5 ml whole blood samples from the mothers and partners is performed by a Hamilton STAR multi-channel robot using AGOWA magnetic bead technology (at 72 samples per run) and is expected to be finished in spring 2008.
Extracted DNA is automatically collected in stock tubes (2D Matrix, Micronic) that are organized in 96-wells format, but that can be individually addressed. These stock samples are split into two tubes that are stored at different locations. One of the stocks will then be used for normalising the DNA concentrations in 96 deep well (DW) plates. This protocol, as well as other manipulations of the DNA samples are performed on a Caliper ALH3000 pipetting robot (8/96/384 channels) with a Twister module and a 96/384 wells Tecan GENios plus UV reader. In total, 90 DNA samples are normalised per 96 DW plate with 6 water blancs per plate. Additionally, a random selection of 5% of the total number of samples are put into separate 96 DW plates, again with 6 blancs per plate, for control purposes. For normalisation, sample and diluent volumes are automatically calculated and produced to obtain equal DNA concentrations for all samples (25–50 ng/μl). From these stock 96-DW plates, 384 DW plates are created, which comprise 24 blancs each. Subsequently, replica PCR plates (384 wells) with a concentration of 1 ng/μl are created for genotype studies.
Stock 96-well plates (DW and 2D Matrix, Micronic) are stored at −20°C at two different locations within the Erasmus Medical Center (Genetic Epidemiology Laboratory, Genetic Laboratory Internal Medicine). The 384 DW stock and PCR plates are stored at the Genetic Laboratory Internal Medicine, Erasmus Medical Center. All genotyping studies with Generation R DNA material are performed in-house at the high throughput genotyping facility in this Laboratory.
Genome Wide Association database
A new approach to identify most important common genetic variants that contribute to explain variance in phenotypes by a systematic analysis of our genome has only recently become available as the Genome Wide Association (GWA) analysis [4]. The GWA analysis is now generally considered a critical tool to identify genes for complex diseases and traits. The GWA analysis is based on genotyping large numbers (>500,000–2,000,000) of Single Nucleotide Polymorphisms (SNPs), and subsequently performing association analyses of the SNP genotypes (or haplotypes thereof) with the phenotype of interest. The power of GWA analyses has been recently demonstrated with the identification of susceptibility genes involved in a wide range of disorders varying from common and rare diseases including coronary heart disease, diabetes and multiple sclerosis [5–7]. These developments have led to a stream of novel disease genes, highlighting new etiological pathways and are expected to improve the understanding of the molecular basis of these diseases. Currently, we are preparing a GWA analysis in all children participating in the Generation R Study of whom DNA is available. For this, the infrastructure, used for the GWA analysis in Rotterdam Study, a population-based cohort study in about 15,000 adults in Rotterdam, the Netherlands is used [8].
Blood for phenotypes
Results of analyses performed in blood samples (5 ml EDTA plasma) for routine care for pregnant women (Hb, Ht, HIV, HBsAg, Lues, Rhesus factor and irregular antibodies) are obtained from midwife and obstetric registries.
All other EDTA plasma and serum samples from mothers and children have been processed and stored for future studies. Processing was aimed to be finished within a maximum of 3 h after venous puncture. Total processing time took 15 min. The samples were spun and the plasma and serum volume was distributed into 250 μl aliquots and transferred to 0.65 ml polypropylene tubes (Micronic) by a Tecan automatic liquid handler. From the mother, a set of on average 16 aliquots tubes (with a total volume of 4 ml) each from one plasma and serum sample were divided over four different microtiter trays. From the plasma and serum samples from the children, on average 11 tubes were filled with 250 μl aliquots (in total 2.75 ml) divided over three microtiter trays. The trays were immediately stored at −80°C. Each Micronic tube is uniquely coded on the bottom with the Traxis® 2D code.
The aliquots were divided over 4 trays of 96 tubes for storage in the freezers. Figure 2 shows the distribution of the samples in the 96 trays. All samples are stored at −80°C at one location (STAR-MDC laboratory) where they are distributed over four different freezers with different power supplies.
Fig. 2Distribution of blood samples in the 96 trays (see text)
Urine
Urine samples (65 ml) were added to the data collection between February 2004 and November 2005. The urine samples were kept at room temperature for a maximum of 3 h and transported to the STAR-MDC laboratory for further processing. At the laboratory, the urine was filled out manually in one 5 ml and three 20 ml tubes. Before the urine was filled out, a gravity measurement was performed. The 5 ml urine tubes were send to the Department of Medical Microbiology of the Erasmus Medical Centre for Chlamydia analysis [9]. The remaining urine tubes (20 ml) were each stored at −20°C at the STAR-MDC laboratory.
Other population-based birth cohort studies
The Generation R Study is not the first birth-cohort study with an extensive collection of biological specimens. Compared to other birth-cohort studies, the size of the Generation R Study cohort is not larger but the measurements are more detailed and the study group has a large ethnic variety [10–14]. Studies having a similar design and biological specimens collection include the Avon Longitudinal Study of Parents and Children (ALSPAC) in Bristol, UK, the Danish National Birth Cohort Study, the Norwegian mother and child Cohort Study, the Southampton Women’s Survey (SWS) in Southampton, UK and The National Children’s Study in the USA.
Collaboration with these studies for funding, standardization, replication and quality management is necessary. Currently, we are exploring possibilities for formal collaboration.
Requests for biological samples use
The study has an open policy with regard to collaboration with other research groups. Request for collaboration and use of biological specimens should primarily be pointed to Albert Hofman (a.hofman@erasmusmc.nl). These requests are discussed in the Generation R Study Management Team regarding their scientific merits, study aims, overlap with ongoing studies, logistic consequences and financial contributions.
General policy is that collaborating researchers and groups are responsible themselves for the finances and that all laboratory tests are conducted in the Eramus Medical Center, Rotterdam. After approval of the project by the Generation R Study Management Team and the Medical Ethical Committee of the Erasmus Medical Center, the collaborative research project is embedded in one of the four research areas supervised by the specific principal investigator.