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Original Articles: Hormones and Reproductive Health |
Departments of Obstetrics and Gynecology (M.I., R.N.) and Pathology (Y.M., A.O.), Wakayama Medical College, Wakayama 641-0012, Japan
Address all correspondence and requests for reprints to: Masaaki Iwahashi, M.D., Department of Obstetrics and Gynecology, Wakayama Medical College, 811-1 Kimiidera, Wakayama 641-0012, Japan. E-mail: masaaki{at}wakayama-med.ac.jp
Abstract
To investigate the characteristic structure of hydatidiform mole, type
IV collagen expression was determined in human villous tissues obtained
from normal pregnancies (n = 17) and complete hydatidiform moles
(n = 10). Indirect immunofluorescent staining was performed to
detect type IV collagen with specific monoclonal antibody, and Northern
blot analysis was performed to assess expression of messenger
ribonucleic acid for the 
1(IV) chain. In addition, serum levels of
type I, III, and IV collagen were measured by RIA. Immunohistochemical
staining for type IV collagen revealed stronger staining of the
trophoblastic basement membrane in hydatidiform mole than in
normal pregnancy. Northern blot analysis revealed that the villous
expression of messenger ribonucleic acid for the 1(IV) chain was
significantly increased in hydatidiform moles compared with normal
pregnancy (P < 0.01). Although there were no
differences in the serum type I and III collagen levels between
hydatidiform mole and normal pregnancy, the type IV collagen level was
significantly higher in patients with hydatidiform mole than in normal
pregnancy (P < 0.05). These results suggest that
type IV collagen might play an important role in determining the
pathophysiology and structure of hydatidiform mole.
THE EXTRACELLULAR matrix (ECM) is considered to play an important role in the stability of tissues and in regulating the growth and differentiation of cells (1, 2). Synthesis, accumulation, and catabolism of the ECM occur during wound healing and during the initiation and progression of numerous diseases (3). The ECM of normal villous tissues has been studied (4, 5), but changes in the ECM in villous tissues of hydatidiform mole are not fully understood.
The hydatidiform mole has such a characteristic macroscopic appearance that it may be diagnosed by the ultrasonographer, attendant at delivery, or the pathologist. Microscopic examination discloses diffuse, marked villous enlargement due to massive stromal edema. The edema displaces the mesenchymal stroma centrally, creating an acellular clear space called a central cistern. There is marked proliferation of cyto- and syncytiotrophoblasts (6).
In the present study we investigated the expression of type IV collagen, the major component of basement membrane (BM), in hydatidiform moles by immunofluorescent staining and Northern blot analysis and determined by RIA serum levels of various types of collagen in patients with hydatidiform mole. The results were compared with those obtained in normal pregnancy.
Materials and Methods
This project was approved by the committee on investigations involving human subjects of Wakayama Medical College. Informed consent was obtained from each subject after the purpose and nature of the study had been fully explained.
Tissues
Normal villous tissues were obtained from 17 women, aged 20–38 yr, during early pregnancy (7–12 weeks) by dilatation and curettage (D&C) for termination of pregnancy and were immediately frozen in liquid nitrogen. Molar tissues were obtained from 10 women, aged 22–41 yr, by D&C at 7–13 weeks gestation. The pathological diagnosis of all 10 cases was complete hydatidiform mole. We excluded necrotic villous tissue from analysis by histological examination. Gestational age was determined from the date of the last menstrual period and by ultrasonographic measurements performed in early pregnancy.
No statistical difference was recognized in gestational age between the normal and molar pregnancy groups (mean ± SD, 9.7 ± 2.4 and 9.1 ±1.9 weeks) at serum sampling and D&C.
Primary antibodies
Specific monoclonal antibody (mAb) against the 1-chain of
human type IV collagen was used. The preparation of the antibodies has
been described previously (7). In brief, BALB/c mice were
immunized with the 1-chain of human type IV collagen after it had
been extracted from human placentas. The spleen cells of these mice
were then hybridized with myeloma cells. After
hypoxanthine-aminopterine-thymidine selection, positive hybrids were
identified by enzyme-linked immunosorbent assay. The specificity of the
mAb was determined by immunoblotting or by inhibition in an
enzyme-linked immunosorbent assay.
Immunohistochemistry
Immunohistochemical analysis was performed by the standard indirect immunofluorescence method. In brief, 3-µm frozen sections were rehydrated in PBS at room temperature and then incubated with the primary antibody (diluted 1:100 in PBS) for 12 h at 4 C in a humidified chamber. After incubation, the sections were washed twice in PBS for 3 min each time. Each section was then incubated for 1 h at room temperature with human plasma-preabsorbed, fluorescein isothiocyanate-conjugated goat antibodies against mouse Igs diluted 1:100 in PBS (Organon Teknik, West Chester, PA). Subsequently, the sections were washed again in PBS, mounted in buffered glycerol, and examined under a fluorescence microscope (Olympus Corp., Tokyo, Japan).
Northern blot analysis
A 916-bp BamHI-HindIII fragment from the
plasmid HT 21, encoding for the NC domain of 1(IV) collagen
and part of the 3'-untranslated region of the corresponding gene
(8), was cloned into the M13 polylinker site of the pSP64
and pSP65 vectors (Promega Corp., Madison, WI). A
riboprobe transcription kit (Promega Corp.) was used for
transcription, and the transcripts were labeled with
[32P]CTP for Northern blot analysis. Total
ribonucleic acid (RNA) was isolated from villous tissues as described
previously (9, 10), followed by size fractionation on 1%
denaturing agarose-formaldehyde gels and transfer to nitrocellulose
membranes (Schleicher & Schuell, Inc., Keene, NH) by
overnight capillary blotting in 20 x SSC (sodium chloride-sodium
citrate) solution. For normalization of 1(IV) messenger RNA (mRNA)
levels, duplicate membranes were prepared from the same RNA samples for
separate hybridization to the 1(IV) probe and a probe for ß-actin
mRNA. Before Northern blot analysis, the transferred RNA was covalently
cross-linked to the nitrocellulose membranes with a UV cross-linker
(Stratagene, La Jolla, CA). Northern blots were
prehybridized for 3 h at 65 C [1(IV)] or 42 C (ß-actin) in
the presence of 50% (vol/vol) formamide under standard conditions,
followed by hybridization with the appropriate radiolabeled probe at
the same temperature for 16 h. Membranes were washed in 2 x
SSC-0.1% SDS for 10 min at room temperature and then washed twice in
0.1 x SSC-0.1% SDS at 65 C [1(IV)] or at 55 C (ß-actin).
Then the membranes were exposed to Kodak X-Omat film
(Eastman Kodak Co., Rochester, NY) for 18 h at -70
C. Autoradiographs were analyzed by densitometry to quantitate
differences in transcript levels between normal pregnancy and
hydatidiform mole.
RIA of serum collagen
Serum samples from the patients were obtained before D&C and were stored at -20 C until analysis. Serum levels of type I and III collagen were measured with RIA kits for the carboxyl-terminal end of type I procollagen propeptide and the amino-terminal end of the type III collagen procollagen propeptide, as described previously (11, 12). Serum levels of type IV collagen were measured with RIA kits for 7S collagen (Hoechst AG, Frankfurt, Germany) The control subjects were 32 nonpregnant female volunteers, aged 19–45 yr, without liver dysfunction or any other diseases. All serum samples were tested in duplicate.
Statistical analysis
Densitometric analysis of the expression of type IV collagen mRNA was conducted after normalization for the level of ß-actin mRNA in each sample. Densitometric data were expressed as the mean ± SEM. Serum collagen levels were expressed as the mean ± SD. Mean values were compared by Student’s t test or ANOVA using a StatView software program on a Macintosh computer. Two-tailed P < 0.05 was considered statistically significant.
Results
Immunohistochemical analysis of the villous tissues
Control sections were stained with goat antibodies against mouse
IgG without prior application of the appropriate primary antibody (Fig. 1A
). When the mAb against type IV
collagen was first allowed to react with an excess of type IV collagen,
no immunostaining was observed.
|
). Enhanced
immunostaining for type IV collagen was seen on the BMs beneath the
trophoblastic epithelium in all 10 hydatidiform moles investigated in
this study (Fig. 1C). In addition, immunostaining was present in the
cytoplasm of both cyto- and syncytiotrophoblasts in all cases of
hydatidiform moles (Fig. 1C).
Northern blot analysis of 1(IV) mRNA
Northern blot analysis was performed to determine the expression
of the 1(IV) gene in villous tissues from normal pregnancy and
hydatidiform moles (Fig. 2, lanes 1 and
2). The results of densitometric analysis of 1(IV) mRNA expression
are shown in Fig. 3. In villous tissues
from hydatidiform mole, 1(IV) mRNA expression was significantly
higher than in tissues from normal pregnancy (0.42 ± 0.20
vs. 3.72 ± 1.25 densitometry units; P
< 0.01). In contrast, ß-actin mRNA levels were similar in villous
tissues from normal pregnancy and hydatidiform moles (Fig. 3).
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Serum concentrations of the various types of collagen were
determined in the pregnant women and in a control group. The results
were presented as the mean ± SD (Table 1). No difference was recognized in the
serum concentration of the carboxyl-terminal end of type I procollagen
propeptide and the amino-terminal end of the type III collagen
procollagen propeptide between normal pregnancy and molar pregnancy.
However, the concentration of 7S collagen in the patients with
hydatidiform mole was significantly elevated compared with that in the
normal pregnant women (P < 0.05) or the control
subjects (P < 0.01).
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In the present study we investigated the expression of type IV collagen in human villous tissues obtained from normal pregnancy and hydatidiform mole. Our findings suggested that increased production of type IV collagen might induce significant elevation of the serum 7S collagen level in patients with hydatidiform mole. It is well known from previous immunohistochemical studies that laminin and type IV collagen are permanent constituents of the BM of late and early chorionic villi and placental blood vessels (4, 5). It has also been reported that BM components, such as type IV collagen and laminin, can be synthesized by trophoblastic cells (13). It has been reported that type IV collagen promotes the attachment of human trophoblast cells to the substratum in vitro (14), and that the mouse trophoblast cells bind to ECM molecules such as collagens (15), laminin, and fibronectin (16). The BM may be involved in the mechanisms controlling selective permeability to macromolecules as well as providing structural tissue support (17). Therefore, type IV collagen might be an essential constituent of placental BMs and might play an important role in the function and structure of the villi.
In addition, increased type IV collagen expression in the BM beneath the trophoblastic epithelium might modulate the characteristics of trophoblastic cells in hydatidiform moles in an autocrine and/or paracrine fashion. Thus, a reduction of type V collagen in the villi might result in reduced attachment or increased metastatic ability of trophoblast cells in hydatidiform moles.
Serum 7S collagen levels are reported to be high in patients with fibrosis of the liver (18, 19, 20) (e.g. chronic hepatitis, liver cirrhosis, and alcoholic liver disease), various malignant tumors (21), and diabetes mellitus (22, 23). In the case of liver fibrosis, increased deposition of BM components, such as type IV collagen and laminin, in the perisinusoidal walls of the liver has been reported (24, 25). In the case of malignancy, Brocks et al. (26) reported that serum BM component levels were increased in about 50% of cancer patients. This increase may be due to the destruction of the BM by tumor invasion and increased synthesis of fibrous components surrounding the tumor. In addition, serum type IV collagen levels were significantly higher in diabetic patients than in healthy subjects and were increased with the prevalence or incidence of diabetic complications. Especially in the patients with diabetic microangiopathy or diabetic nephropathy, serum type IV collagen levels became higher (22, 23). These observations indicated that enhanced synthesis of type IV collagen in BM of capillaries and small vessels might induce elevation of the circulating type IV collagen in the patients with diabetic microangiopathy. Our data suggest that the increased serum 7S collagen level in patients with hydatidiform mole might be caused by excessive trophoblastic type IV collagen production rather than by destruction of type IV collagen in the BM. We have also previously shown that the expression of laminin-1, one of the ECM components in the BM, was higher increased in villi from hydatidiform moles at the gene and protein level than in villi from normal pregnancies (27). We have had no case of invasive mole or choriocarcinoma; therefore, type IV collagen expression in these diseases remains to be elucidated. In conclusion, enhanced type IV collagen accumulation in the BM beneath the trophoblastic epithelium and in the trophoblastic cytoplasm was observed in hydatidiform moles. Therefore, overexpression of type IV collagen might result in the morphological and functional characteristics of hydatidiform mole.
This study provides some clues to understanding the pathophysiology of hydatidiform mole in terms of the ECM metabolism. Further work is needed to elucidate the mechanisms regulating the trophoblastic expression of genes for other types of collagen in normal pregnancy and hydatidiform mole.
Received May 23, 2000.
Revised October 24, 2000.
Revised December 4, 2000.
Accepted December 10, 2000.
References
1(IV) chain of human type IV
procollagen reveal an unusual homology of amino acid sequences in two
halves of the carboxyterminal domain. J Biol Chem. 260:7681–7687.1(III) chain of type III procollagen exposes an arginine and causes
Ehlers-Danlos syndrome IV. J Biol Chem. 264:1349–1352.1(IV)
chain genes by cells of early human placenta and gestational
endometrium. Lab Invest. 67:191–200.[Medline]
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