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Reproductive Endocrinology |
Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics-Gynecology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051
Address all correspondence and requests for reprints to: M. Linette Casey, Ph.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: casey{at}grnctr.swmed.edu
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Introduction |
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Despite the breadth and depth of the world-wide health problem created by the sequelae of PT-PROM, there have been few studies of the ontogenesis of the formation of interstitial collagens in amnion tissue during fetal development. Even the cellular site of origin of these interstitial collagens was established only recently (9).
The human fetal membranes, viz. the amnion and chorion
laeve, are avascular viscoelastic structures (10). Resistance to
tearing and rupture (i.e. tensile strength) of this tissue
is provided primarily by the innermost membrane, the amnion (11), a
tissue in which there are no demonstrable vascular, nerve, lymphatic,
or smooth muscle components (11). The inner surface of the amnion,
which is contiguous with the amniotic fluid, is comprised of an
uninterrupted single layer of cuboidal epithelial cells, believed to be
derived from embryonic ectoderm (12). The amnion epithelium is attached
firmly to a clearly delineated basement membrane, which is adjacent to
the zona compacta, an acellular layer that is composed primarily of
interstitial collagens types I and III and lesser amounts of types V
and VI (13, 14, 15, 16, 17). On the outer aspect of the zona compacta of the
near-term amnion, there are widely dispersed fibroblast-like
mesenchymal cells. In early embryogenesis (
8 weeks gestation),
however, the mesenchymal cells are contiguous with the epithelial
cells, forming a double layer of amnion cells. The compactness of the
mesenchymal cells begins to diminish at about the same time that
interstitial collagens are laid down between these two layers of cells
(18). As the compact layer of interstitial collagens forms, the
epithelium becomes separated from the mesenchymal cells, which
ultimately become widely dispersed.
Bourne (11), who described the layers of the amnion, pointed to the
collagenous compact layer of this tissue as the source of tensile
strength. We have demonstrated that the interstitial collagens (type I
and III) are produced in the mesenchymal cells of this tissue (9), and
very little of these collagens is produced in the epithelial cells.
Moreover, the apparent capacity for interstitial procollagen
synthesis/processing is much greater in amnion tissue early in
pregnancy (12–14 weeks gestation) than during the third trimester of
pregnancy. Namely, the levels of procollagen I and III [
1(I) and
2(I) as well as 1(III)] messenger ribonucleic acids (mRNAs) are
much greater in amnion early in pregnancy (9). The same is true of the
specific activities of amnion tissue lysyl and prolyl hydroxylase,
enzymes crucial to the intracellular processing of the procollagens
(9).
The cross-links of interstitial collagens provide tensile strength and resistance to hydrolysis by collagenases. Pinnell and Martin discovered that the initial reaction in the covalent cross-linking of collagen is effected by a Cu2+-dependent enzyme they termed lysyl oxidase, which is secreted from the cell and acts in the extracellular space (19). Lysyl oxidase is encoded by the ras recision gene (20); increased expression of this gene prevents/reverses ras oncogene-induced cell transformation (21). The relationship between lysyl oxidase activity and the recision of ras action(s), if any, however, has not been defined.
Decreased tensile strength can be caused by inadequate cross-linking of interstitial collagen. Noncross-linked, or soluble, collagens are better substrates for mammalian interstitial collagenases than are cross-linked, insoluble, collagens (22). Deficient collagen cross-linking is found in animals and humans with Cu2+ deficiency and in mice and persons with heritable abnormalities of intracellular Cu2+ transport (e.g. Menkes disease) (23). In this investigation, we evaluated lysyl oxidase activity and the levels of lysyl oxidase mRNA and immunoreactive protein in human amnion tissues obtained from pregnancies of 12–43 weeks gestation. The levels of lysyl oxidase mRNA also were assessed in separated amnion mesenchymal and epithelial cells and in these cells maintained in monolayer culture.
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Materials and Methods |
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Amnion tissues, at or near term, were obtained from normal pregnancies at the time of cesarean section conducted before or after the onset of labor or at the time of spontaneous vaginal delivery at term. Amnion tissues also were obtained from pregnancies between 25–36 weeks gestation. In all such cases, the fetal membranes were intact until the time of delivery or after the onset of labor. In the 25–36 week gestation pregnancies, preterm delivery was elected because of severe pregnancy-induced hypertension (n = 3) or the spontaneous onset of preterm labor (n = 10). None of these pregnancies was complicated by chorioamnionitis or was from a pregnancy known to be associated with the use of cocaine. We cannot conclude, however, that the pregnancy-induced hypertension or the spontaneous onset of preterm labor did not affect the activity of lysyl oxidase in the amnions of these pregnancies. In addition, amnion tissues were collected from presumably normal (by history, sonographic criteria, and direct examination of the products of conception after delivery) midtrimester pregnancies after elective termination of pregnancy (12–24 weeks). These tissues were used for this research after health care professionals unrelated to the research team obtained consent in writing from the pregnant woman. The consent forms and protocols used were approved by the institutional review board of the University of Texas Southwestern Medical Center (Dallas, TX). In all, amnion tissues from 111 amnionic sacs (including 9 twin and 1 triplet pregnancies) were evaluated, and for most amnionic sacs, both placental and reflected (and fused, if present) amnion tissues were studied.
Amnion tissue was used for the assay of lysyl oxidase activity, immunoreactive lysyl oxidase protein estimation, or assessment of the levels of mRNA. In some studies, amnion epithelial and mesenchymal cells were separated and evaluated. The amnion was separated from chorion laeve by delamination with blunt dissection. For some studies, placental and reflected amnion tissues were assayed separately for lysyl oxidase activity. Amnion tissue also was obtained from the fused portion of fetal membranes in diamnionic-dichorionic and diamnionic-monochorionic twin placentas of twin and triplet pregnancies. Within 15 min of delivery or surgery, the tissues were placed at 4 C; dissection was conducted within 30 min to 2 h thereafter. Before 20 weeks of pregnancy, gestational age (given in weeks since the first day of the last menstrual period) was estimated from fetal foot length according to the criteria of Moore (24) and Streeter (25).
Amnion cells in culture
Amnion epithelial cells were isolated by incubation of amnion tissue fragments (5–15 g) in Eagle’s MEM-trypsin (150 mL) [2 g of 1:250 diluted trypsin (Life Technologies, Grand Island, NY)/L culture medium] at 37 C for 30 min with stirring as previously described (26). The medium from the first incubation was discarded, the tissue was minced again, and the incubation with trypsin was repeated three times. After each 30-min incubation, the medium was poured over gauze to separate the dispersed amnion epithelial cells; viability was assessed by trypan blue exclusion. In some cases, the acutely separated cells were used for study. In other cases, the cells were plated in plastic culture plates at a density of about 300,000 cells/cm2. The yield of amnion epithelial cells was approximately 8–12 x 106/g amnion tissue; viability was about 98%. The cells were placed in culture dishes (60- or 100-mm diameter or 24-well plates) and allowed to replicate to confluence (7–10 days) in Ham’s F-12-DMEM (1:1, vol/vol) that contained heat-inactivated FBS (10%, vol/vol), penicillin (200 U/mL), streptomycin (200 µg/mL), fungizone (0.5 µg/mL), kanamycin (200 µg/mL), and gentamicin (200 µg/mL). The cells were maintained in monolayer culture in a humidified atmosphere of air and CO2 (5%). Mesenchymal cells were isolated from the amnion tissue pieces after removal of epithelial cells (evaluated microscopically after hematoxylin-eosin staining) as previously described (9). The tissue pieces were incubated at 37 C for 60 min in Eagle’s MEM with collagenase (0.75 mg/mL) and deoxyribonuclease (0.075 mg/mL). The dispersed mesenchymal cells were collected by filtration of the mixture through gauze and centrifugation and plated in plastic dishes at a density of about 400,000 cells/cm2. The yield of mesenchymal cells was approximately 1 x 106 cells/g tissue.
Lysyl oxidase enzyme activity
Lysyl oxidase activity was assayed in urea (6 mol/L) extracts of amnion tissue according to the method of Harris et al. (27) using radiolabeled insoluble substrate (principally elastin) synthesized by incubation of [4,5-3H]lysine (102 Ci/mmol) with aorta tissue minces from 1- or 2-day-old chicks. For preparation of the substrate, minces of aorta tissues were incubated with [4,5-3H]lysine (0.1 mCi/aorta) in otherwise lysine-free RPMI culture medium that contained ß-aminoproprionitrile (50 µg/mL) for 16–24 h. At the end of the incubation, the minces were homogenized in a solution of NaCl (0.15 mol/L). The homogenate was centrifuged at 11,000 x g for 20 min at 4 C; the pellet was suspended in NaCl (0.15 mol/L solution, and homogenized and centrifuged twice more. The insoluble elastin substrate in water was placed in a boiling water bath for 9 min, chilled on ice, and centrifuged and suspended twice more. Finally, the substrate was suspended in assay buffer that consisted of potassium phosphate buffer (0.15 mol/L, pH 7.6) and NaCl (0.12 mol/L). The yield of radiolabeled insoluble elastin substrate prepared in this manner was about 350 x 106 dpm.
In preparation for assay of lysyl oxidase activity, amnion tissues (stored frozen at -80) were pulverized with a mortar and pestle at liquid nitrogen temperature. Portions of the pulverized tissues were homogenized with a Polytron-like (Tissue-Tearer) homogenizer in potassium phosphate buffer (0.16 mol/L; pH 7.8) that contained NaCl (0.12 mol/L), ethylenediamine tetraacetate (EDTA; 1 mmol/L), phenylmethylsulfonylfluoride (1 mmol/L), pepstatin A (1 µmol/L), and leupeptin (6 µmol/L). The homogenates were centrifuged at 10,000 x g for 20 min at 4 C. The pellets were suspended in the same buffer, homogenized in a glass-Teflon homogenizer, and centrifuged again. The pellets were suspended in potassium phosphate buffer (0.16 mol/L; pH 7.8) that contained NaCl (0.12 mol/L), EDTA (1 mmol/L), phenylmethylsulfonylfluoride (1 mmol/L), pepstatin A (1 µmol/L), leupeptin (6 µmol/L), and urea (6 mol/L). These mixtures were rotated at 4 C for 2 or 18 h (routinely for 18 h). Thereafter, the mixtures were centrifuged at 10,000 x g for 30 min at 4 C, and the supernatants (urea extracts) were used for assay of lysyl oxidase activity. The urea extracts were dialyzed at 4 C for 20 h against potassium phosphate buffer (0.15 mol/L; pH 7.6) and NaCl (0.12 mol/L).
Lysyl oxidase reaction mixtures (1 mL total volume) consisted of dialyzed urea extracts in various amounts (routinely, 50 µg protein), 3H-insoluble elastin substrate (2.5 x 106 cpm), and assay buffer. After incubation at 40 C for various times (routinely, 4 h), the reaction was stopped by freezing. Water was distilled from the reaction mixtures, and an aliquot (0.7 mL) of the water was assayed for radioactivity by liquid scintillation spectrometry. All assays were conducted in duplicate.
The specific activity of the substrate generated in chick aorta incubated with [3H]lysine is not known. Therefore, to compare lysyl oxidase enzyme activities in this large number of amnion tissue samples, several precautions and safeguards were used. First, large numbers of tissues were studied in each separate assay. Second, the substrate was prepared in an identical manner for each assay. Third, the data were corrected for interassay variation, which was evaluated by use of a urea extract of a pool of amnion tissue and a urea extract of a pool of chick aorta.
Western analysis of immunoreactive lysyl oxidase protein
SDS-PAGE was conducted by the method of Laemelli (28) on 7.5% slab gels. After separation, the proteins were transferred electrophoretically (4 C, 20 V, 15–16 h) from the polyacrylamide gel to nitrocellulose paper in buffer that contained Tris (0.02 mol/L), glycine (0.15 mol/L), and methanol (20%, vol/vol). The blot was blocked by incubation at 37 C for 45 min in a solution of Tris-Cl (10 mmol/L; pH 7.5), NaCl (0.15 mol/L), BSA (5%, wt/vol), and Nonidet P-40 (0.2%, vol/vol). Thereafter, the blot was incubated for 3 h in the same buffer solution that contained rabbit antiserum (diluted 1:500) raised against bovine aorta lysyl oxidase, provided by Dr. Herbert M. Kagan, Boston University (Boston, MA). The blot was incubated at 25 C for 2 h in a solution of the blocking buffer that contained horseradish peroxidase-conjugated goat antirabbit IgG (diluted 1:1000; Bio-Rad Laboratories, Richmond, CA). The proteins that bound the first and then the second antibodies were visualized by color development with the horseradish peroxidase color reagent (Bio-Rad). Specificity of the antibody for lysyl oxidase was demonstrated; the approximately 31-kDa protein was not detected by Western analysis using rabbit nonimmune serum or irrelevant rabbit antiserum (against renin). The relative molecular mass of the proteins was estimated from the migration distances of molecular mass (Mr) markers purchased from Bio-Rad. The proteins used were phosphorylase b (Mr = 92,500), BSA (Mr = 66,200), ovalbumin (Mr = 45,000), carbonic anhydrase (Mr = 31,000), soybean trypsin inhibitor (Mr = 21,500), and lysozyme (Mr = 14,400).
Northern analysis of lysyl oxidase mRNA
Total RNA was prepared from tissues or cells in culture by the guanidine thiocyanate-ultracentrifugation method of Chirgwin et al. (29). Briefly, tissues were pulverized with a mortar-pestle cooled with liquid N2. The tissue powder was solubilized in guanidine thiocyanate (4 mol/L); alternatively, amnion epithelial or mesenchymal cells in monolayer culture were solubilized in guanidine thiocyanate (4 mol/L). The mixture was layered over a CsCl (5.7 mol/L) cushion and centrifuged at 238,000 x g for 18 h at 25 C. The pellet was suspended in sodium acetate (0.3 mol/L; pH 6.0) and precipitated at -80 C after the addition of ethanol. Total RNA was suspended in Tris (10 mmol/L; pH 7.4) and EDTA (1 mmol/L) and quantified by spectrophotometry.
For Northern analyses, total RNA (10–20 µg per lane) was
size-fractionated by electrophoresis on 1% formaldehyde-agarose gels
and transferred electrophoretically to a Hybond-N+ membrane
(Amersham, Arlington Heights, IL). The membranes were heated at 80 C
in vacuo for 60 min. Prehybridization was conducted for
16 h at 65 C in buffer [formamide (50%, vol/vol), NaCl (0.25
mol/L), Na2HPO4 (0.25 mol/L; pH 7.2), EDTA (1
mmol/L), and SDS (7%, wt/vol)]. Hybridization was conducted for
16 h at 43 C in this buffer with a human lysyl oxidase
complementary DNA probe (1609 bp; provided by Dr. Herbert M. Kagan,
Boston University School of Medicine, Boston, MA) radiolabeled with
[-32P]deoxy-CTP by random hexamer priming. Blots were
washed with 2 x SSC (standard saline citrate) and SDS (0.1%,
wt/vol) for 15 min at 25 C, twice with 0.1 x SSC and SDS (0.1%,
wt/vol) for 10 min at 25 C, once for 15 min at 43 C, and once or twice
at 52 C. Autoradiography of the membranes was performed at -80 C using
Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). The presence of
equal amounts of total RNA in each lane was verified by visualization
of 28S and 18S ribosomal RNA subunits and analysis of
glyceraldehyde-3-phosphate dehydrogenase mRNA, using a specific 24-mer
oligonucleotide probe (5'-TCT AGA CGG CAG GTC AGG TCC ACC-3') end
labeled with [
-32P]ATP.
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Results |
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In amnion tissue preparations assayed for lysyl oxidase activity, the formation of product with extracts of amnion tissue was linear with time of incubation up to 6 h. Linearity with protein was maintained when the amount of 6 mol/L urea-extracted protein in the incubation mixture was less than 200 µg. The activity of lysyl oxidase was similar in preparations extracted with urea for 2 or 18 h. Based on these findings, we conducted assays routinely with preparations extracted with urea (6 mol/L) for 18 h, using an incubation time of 4 h and amnion urea-extracted protein levels of 50 or 100 µg/assay tube.
Lysyl oxidase activity, in urea-extracted amnion tissue
preparations (n = 111) ranged from undetectable (<250 cpm/h·mg
protein) to 107,134 cpm/h·mg protein, but varied inversely with
gestational age in a highly significant manner (Fig. 1
).
Lysyl oxidase activity in amnion (expressed per mg tissue protein) at
12 (menstrual) weeks gestational age was similar to that in newborn
chick aorta. The activity of lysyl oxidase in amnion tissue of
singleton term pregnancies with intact membranes (
38 weeks; n =
18) was not affected by labor status (data not shown). The data for
lysyl oxidase in reflected amnion tissue from singleton pregnancies
with intact membranes were evaluated as a function of gestational age
and fitted to a third order polynomial for regression analysis (Fig. 1). For all data (n = 70), including pregnancies complicated by
preterm labor or pregnancy-induced hypertension, the relationship was
significant (P < 0.001; power 1.0; = 0.5). The
same was true when only pregnancies with no known complication were
evaluated.
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). In 9 of 12 cases, the activity of lysyl oxidase in
the fused portion of the membranes, whether dichorionic (in the twin
pregnancy) or monochorionic (in the twins of the triplet pregnancy),
was considerably greater than that in placental or reflected
amnion.
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By Western analysis, an immunoreactive protein of approximately 31
kDa was detected in urea-extracted amnion tissue preparations (Fig. 2). The amount of immunoreactive lysyl oxidase protein
in early amnion tissue (per mg tissue protein) was greater than that in
amnion tissues from near-term pregnancies.
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Lysyl oxidase mRNA was detected by Northern analysis of total RNA
from amnion tissues at various gestational ages (Fig. 3). The major mRNA species was about 4.3 kilobases in
length. Although lysyl oxidase mRNA was detected in all amnion tissues
evaluated, the levels of lysyl oxidase mRNA in amnion tissue at
midtrimester of pregnancy were greater (in four of five pregnancies)
than those in amnion tissue from pregnancies delivered during the third
trimester (n = 6). The level of lysyl oxidase mRNA in amnion
mesenchymal cells was much greater than that in epithelial cells
isolated from the same tissue (Fig. 4). The preferential
expression of lysyl oxidase mRNA in amnion mesenchymal cells was
maintained in these cells in culture; namely, the level of lysyl
oxidase mRNA was much greater in mesenchymal cells than in amnion
epithelial cells isolated from amnion of the same pregnancy maintained
under identical conditions (Fig. 5).
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Discussion |
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1(I), 2(I), and 1(III),
are greatest in amnion tissue early in pregnancy, as are the specific
activities of lysyl and prolyl hydroxylases. In addition, it was
established that the mesenchymal cells of the amnion are the cellular
site of interstitial procollagen formation (9). There also is a
decrease in the amnion content of DNA per unit dry weight as a function
of gestational age, which may correspond to a decline in the density of
mesenchymal cells as pregnancy progresses (9). The procollagen I and
III subunit mRNAs are found almost exclusively in freshly isolated
amnion mesenchymal cells (compared with epithelial cells), and this
difference as well as the capacity for interstitial collagen synthesis
are maintained in mesenchymal cells in monolayer culture (9). This study was conducted to evaluate the capacity for cross-linking of collagen fibrils in amnion tissue (with gestational age) and to define the amnion cell (mesenchymal or epithelial) in which this enzyme activity is expressed. Specifically, lysyl oxidase enzyme activity and the levels of lysyl oxidase immunoreactive protein and mRNA in amnion with fetal development were evaluated. Lysyl oxidase was chosen for study for two reasons: 1) the cross-linking of collagen is essential for the provision of optimum tensile strength to a tissue and for greatest resistance to proteinases (22); and 2) deficiencies in lysyl oxidase activity may result from Cu2+ deficiency or abnormal transport/metabolism of Cu2+. Thus, the activity of lysyl oxidase can be a reflection of both the synthesis of this enzyme and the availability of copper to lysyl oxidase in a given tissue or specific cell.
Lysyl oxidase enzyme activity is very high in presumably normal midtrimester amnion tissues (delivered by elective abortion by dilatation and extraction). At 12–14 (menstrual) weeks gestation, amnion lysyl oxidase activity (expressed as counts per min/mg tissue protein) was comparable with that in chick aorta (27). Thereafter, the lysyl oxidase activity in amnion declined. By Western analysis, immunoreactive lysyl oxidase protein was found in high levels in midtrimester amnion. By Northern analysis of total RNA, the same was true of the levels of lysyl oxidase mRNA in these tissues. Thereafter, the levels of lysyl oxidase protein declined, and at term were appreciably lower than those before 16 weeks gestation. The decrease in the level of lysyl oxidase protein between 16 weeks and term, however, was not as great as expected from the steep decline in lysyl oxidase activity between these two stages of gestation. The meaning of this finding is not entirely clear. A relatively greater lysyl oxidase protein level compared with lysyl oxidase enzyme activity is observed with Cu2+ deficiency (30, 31). The possibility of Cu2+ deficiency at the level of lysyl oxidase in the amnion mesenchymal cells of these pregnancies, therefore, should be considered.
The reason for the much higher level of lysyl oxidase activity in placental amnion compared with that of reflected amnion in some pairs of tissues is not resolved. Several potential explanations can be proposed. Placental amnion is that portion of this tissue that covers the fetal surface of the placenta. Reflected amnion is the portion of the tissue that is contiguous with the chorion laeve. There is no chorion laeve beneath the placental amnion; rather, the placental amnion is directly contiguous with the fetal chorionic vessels that traverse the placenta in the chorionic plate. This anatomical arrangement may favor greater lysyl oxidase in placental amnion because of 1) a greater number of mesenchymal cells in placental amnion compared with that in reflected amnion, and 2) a potential source of Cu2+ from fetal vessels, the adventitial surfaces of which are contiguous with amnion mesenchymal cells. In addition, however, the lysyl oxidase activity in fused amnion tissues of diamnionic-dichorionic twin placentas were greater than those in the reflected amnions of the same pregnancies. Therefore, the role of decidua parietalis tissue in lysyl oxidase in reflected amnion tissues also must be considered; namely, the lysyl oxidase in amnion removed from the decidual-chorion laeve interface (i.e. placental and fused amnions) was greater than that in amnions contiguous with chorion laeve-decidua.
The level of lysyl oxidase mRNA in separated amnion mesenchymal cells was much greater than that in epithelial cells. The higher level of lysyl oxidase mRNA in mesenchymal cells compared with that in epithelial cells was maintained during monolayer culture. These findings together with those relative to collagen I and III synthesis (9) indicate that the interstitial collagens in the compact layer of amnion arise by synthesis/processing of procollagens in the mesenchymal cells of this tissue and by cross-linking of the collagen fibrils by lysyl oxidase secreted by mesenchymal cells.
Some evidence has been presented previously that the collagen content and tensile strength of amnion are greater in early than in late gestational age amnions. Polishuk and colleagues (32), for example, discovered that there was a decrease in tensile strength as fetal weight (gestational age) increased from 250 to 3250 g. Skinner et al. (33) found that the hydroxyproline content of amnion declined as a function of gestational age from 26–43 weeks gestation. These findings, which suggest a greater amnion content of interstitial collagen in early pregnancy, may mean that the maintenance of amnion tensile strength during the final 20 weeks of gestation is highly dependent upon a long t1/2 of interstitial collagen in avascular amnion together with low rates of collagen replacement. Such a naturally occurring diapause in the capacity for amnion collagen formation could be devastating in pregnancies in which there is either an intrinsic disorder in the formation of adequate amounts of structurally stable amnion collagen or a nutrition/xenobiotic-induced compromise in the stability of interstitial collagen, giving rise to an increased risk for PT-PROM.
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Acknowledgments |
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Footnotes |
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Received June 24, 1996.
Revised August 7, 1996.
Accepted September 3, 1996.
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References |
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-aminoadipic-
-semialdehyde (allysine) by an extract from bone. Proc Natl Acad Sci USA. 61:708–716.This article has been cited by other articles:
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  D. A. Kirschmann, E. A. Seftor, S. F. T. Fong, D. R. C. Nieva, C. M. Sullivan, E. M. Edwards, P. Sommer, K. Csiszar, and M. J. C. Hendrix A Molecular Role for Lysyl Oxidase in Breast Cancer Invasion Cancer Res., August 1, 2002; 62(15): 4478 - 4483. [Abstract] [Full Text] [PDF]
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 P.C. McParland, S.C. Bell, J.H. Pringle, and D.J. Taylor Regional and cellular localization of osteonectin/SPARC expression in connective tissue and cytotrophoblastic layers of human fetal membranes at term Mol. Hum. Reprod., May 1, 2001; 7(5): 463 - 474. [Abstract] [Full Text] [PDF]
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H. Lei, R. Kalluri, E. E. Furth, A. H. Baker, and J. F. Strauss III Rat Amnion Type IV Collagen Composition and Metabolism: Implications for Membrane Breakdown Biol Reprod, January 1, 1999; 60(1): 176 - 182. [Abstract] [Full Text]
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H. Lei, E. E. Furth, R. Kalluri, P. Wakenell, C. B. Kallen, J. J. Jeffrey, P. S. Leboy, and J. F. Strauss III Induction of Matrix Metalloproteinases and Collagenolysis in Chick Embryonic Membranes before Hatching Biol Reprod, January 1, 1999; 60(1): 183 - 189. [Abstract] [Full Text]
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M. L. Casey and P. C. MacDonald Keratinocyte Growth Factor Expression in the Mesenchymal Cells of Human Amnion J. Clin. Endocrinol. Metab., October 1, 1997; 82(10): 3319 - 3323. [Abstract] [Full Text] [PDF]
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, severe pregnancy-induced hypertension (not in labor); 
,
spontaneous preterm labor.
