Human Reproduction Vol.23, No.2 pp. 387–393, 2008 doi:10.

1093/humrep/dem370
Advance Access publication on December 11, 2007

Is a2-macroglobulin important in female stress

urinary incontinence?

Y. Wen1, W.C. Man, E.R. Sokol, M.L. Polan and B.H. Chen
Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive,
H333 Stanford, CA 94305, USA
1
Correspondence address. Tel: þ1-650-723-9536; Fax: þ1-650-723-7737; E-mail: yanwen@stanford.edu

BACKGROUND: Loss of mechanical stability of the urethra and bladder is thought to be important in the
development of stress urinary incontinence (SUI). The vaginal wall is the main supporting tissue for pelvic organs
and changes in components of supporting tissues are known to be involved in the pathophysiology of SUI.
METHODS: We evaluated changes in expression of a2-macroglobulin (a2-M), a protease inhibitor, in vaginal wall

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tissues from premenopausal women (aged 42–45 years) with SUI (n 5 28) compared with menstrual cycle-matched
continent women (controls, n 5 29). The distribution of a2-M in vaginal wall tissues and fibroblasts was analysed
by immunohistochemistry and immunofluorescence. Expression levels of a2-M mRNA and protein was determined
by relative real-time quantitative PCR and enzyme-linked immunosorbent assay, respectively. Protease inhibition
was measured to assess bioactivity. RESULTS: Vaginal wall tissues do express a2-M. Expression of a2-M mRNA
and protein was significantly higher in tissues from controls compared to women with SUI in both proliferative
and secretory phases (P < 0.05). Protease inhibitory activity of a2-M was significantly higher in tissues from controls
compared to women with SUI in the secretory phase (P < 0.05), but we found no difference in the proliferative phase
between groups. a2-M protein level was lower in the proliferative phase than the secretory phase in both controls and
SUI patients, while for a2-M mRNA this was found only in controls. CONCLUSIONS: Decreased expression of a2-M
mRNA and protein and protease inhibitory activity in the vaginal wall tissues of women with SUI may contribute to
the development of SUI.

Keywords: extracellular matrix; stress urinary incontinence; a2-macroglobulin; vaginal wall; proteases

Introduction fibrous elements (collagen and elastic fibers) and a viscoelastic

Stress urinary incontinence (SUI) is a common, costly and
distressing condition for women (Nygaard and Heit, 2004;
Anger et al., 2006; Subak et al., 2006). Frequency of SUI
increases with age, and peaks in prevalence first around meno-
pause and then after age 65 (Hannestad et al., 2000). SUI is
defined as the involuntary loss of urine which occurs when
intra-abdominal pressure exceeds urethral pressure. Factors
affecting urethral pressure include bladder neck mobility; ure-
thral sphincter muscle and nerve integrity; urethral smooth
muscle and vascular plexuses; and surrounding tissue
support. The vaginal wall is the main supporting tissue and pro-
vides a stable base for the urethra and bladder neck, preventing
urethral and bladder neck descent (Nygaard and Heit, 2004).
The loss of mechanical stability of the urethra and bladder is
thought to be an important factor in the development of SUI
(Weber et al., 2004).
The vaginal wall comprises sequanous epithelium, sub-
epithelium, smooth muscle and adventia. All elements are
embedded in an extracellular matrix (ECM), composed of
matrix (proteoglycans, glycoproteins). The collagens and
elastin fibers confer tissue strength and elasticity, respectively,
whereas structural glycoproteins create tissue cohesiveness.
The ECM of the vaginal tissue largely determines its tissue
tensile strength and its mechanical stability. Therefore, the
relationship between production and degradation of the ECM
components is crucial for maintaining mechanical integrity of
pelvic supporting tissues. Abnormal quantity and quality of
ECM components in pelvic supporting tissues have been
reported to be involved in the pathophysiology of SUI and/
or pelvic organ prolapse (Liapis et al., 2001; Ewies et al.,
2003; Goepel et al., 2003; Liu et al., 2004; Karam et al.,
2007; Wen et al., 2007).
ECM undergoes continuous tissue remodeling in response to
different environmental stimuli (Alperin and Moalli, 2006).
Proteolysis regulates ECM assembly, degradation of excess
ECM components, remodeling of ECM structures and the
release of growth factors and bioactive fragments (Weber
et al., 2004). Matrix metalloproteinases (MMPs) and serine

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Wen et al.
protease (neutrophil elastase, NE) are capable of degrading
ECM components (collagen, elastin, proteoglycans and glyco-
proteins). In previous studies, we documented that the relative
activity of MMPs and NE are increased whereas their respect-
ive inhibitors [tissue inhibitor of metalloproteinase-1 (TIMP-1)
and a1-antitrypsin (ATT)] is decreased in the vaginal wall
tissues of women with SUI compared with controls (Chen
et al., 2002; Chen et al., 2004; Chen et al., 2007). Other inves-
tigators have also observed accelerated remodeling of ECM in
the pelvic supporting tissues in women with SUI and/or pelvic
organ prolapse compared with controls (Moalli et al., 2005;
Gabriel et al., 2006; Phillips et al., 2006). a-2macroglobulin
(a2-M) has been characterized as an extracellular panprotei-
nase inhibitor with the unique ability to inhibit almost all
known proteases, regardless of their mechanistic classes
(serine, metallo, cysteine and aspartate). Information from a
knock-out mice study suggests that a2-M is involved in
tissue remodeling during both pregnancy and wound repair
(Umans et al., 1995). However, a2-M’s role in the develop-
ment of SUI has not been examined.
In this study, we sought to investigate possible changes in
the expression of a2-M by measuring its expression level
and inhibitory capacity in vaginal wall tissues from premeno-
pausal women with SUI compared with controls in both the
proliferative and secretory phases of the menstrual cycle.
Material and Methods
Patient selection and tissue collection
We selected women with SUI (n ¼ 28) and controls (n ¼ 29) from
both the proliferative and secretory phases of the menstrual cycle
for this study. Phase of cycle was confirmed by endometrial histology.
Women with a history of endometriosis, gynecologic malignancies,
pelvic inflammatory conditions, connective tissue disorders, emphy-
sema, prior pelvic surgery and advanced pelvic organ prolapse
(greater than stage II by pelvic organ prolapse-quantification) were
excluded. The Institutional Review Board of Stanford University
School of Medicine approved this study. Informed consents were
obtained from all participants.
In women undergoing surgery for SUI, 1 cm2 of full-thickness,
peri-urethral vaginal mucosa was excised 1 cm lateral to the urethro-
vesical junction identified by a Foley balloon. Smaller, 0.5 cm2 biop-
sies of vaginal mucosa from a similar area were excised from controls
undergoing benign gynecologic surgeries for fibroids, dysfunctional
bleeding or ovarian cysts. At the time of tissue collection, the epi-
thelial layers were removed with a razor blade. A representative cross-
section was fixed in 10% buffered formalin for 16 h, processed with
paraffin embedding and used for immunohistochemistry. Tissue
from a patient was collected into a tube containing Dulbecco’s modi-
fied Eagle’s medium for the isolation of fibroblasts from vaginal wall
tissue for immunofluorescence staining as described previously (Chen
et al., 2004; Wen et al., 2006). The remainder of the tissue was frozen
immediately in liquid nitrogen and then stored at 2808C for further
processing.
Immunofluorescence staining for a2-M
Immunofluorescence staining of fibroblasts from the vaginal wall was
performed as previously described (Chen et al., 2004). Briefly, fibro-
blast cells from the vaginal cuff were cultured in a 4-well chamber
slide. The cells were fixed with 4% paraformaldehyde and treated
388
with 5% Triton. After washing with Tris –HCl-Tween buffer
(TBS-T) and blocking with 5% normal secondary serum, the slides
were incubated overnight with rabbit anti-a2-M (1/200, Sigma, St
Louis, MO, USA) primary antibody at 4ºC. Non-immune serum of
the primary antibody was used as a negative control. Then, after
washing, the slides were stained with goat anti-rabbit-immunoglobulin
(Ig)G- fluorescein isothiocyanate (1/50, Sigma) at room temperature
for 1 h. 40 ,6-Diamidino-2-phenylindole staining was used to observe
nuclei. The slides were washed three times and mounted with Vecta-
shield (Vector Laboratory, Burlingame, CA, USA).
Immunohistochemistry for a2-M
Immunohistochemical staining for a2-M was performed on fixed
embedded tissue as described previously (Wen et al., 2006) to localize
the expression of a2-M in vaginal wall specimens. Briefly,
paraffin-embedded specimens were cut into 5 mm sections,
de-waxed in Xylene and rehydrated through graded ethanol solutions.
After washing with TBS-T, endogenous peroxidases were blocked
with 3% H2O2 in TBS-T, and non-specific binding was blocked
with 1% bovine serum albumin, 5% normal secondary antibody host
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serum in TBS-T at room temperature for 1 h. After rinsing with
TBS-T, the slides were incubated with rabbit anti-a2-M (1/20,
Sigma) primary antibody overnight at 48C. Omission of the primary
antibody was used as a negative control. After rinsing with TBS-T,
slides were incubated with a secondary antibody, goat anti-rabbit
biotin conjugate (1/50, Vector Laboratory). The slides were incubated
with Vectastatin ABC Kit (Vector Laboratory) reagent for 30 min at
room temperature. Immunoreactive products were visualized by
incubating slides with the substrate solution in TBS-T buffer with
levamisole to block alkaline phosphatase activity. Slides were coun-
terstained with 25% of hematoxylin (Fisher, Fair Lawn, NJ, USA).
They were visualized and photographed with AxioCam (Zeiss,
Oberkochen, Germany).
Relative real-time quantitative PCR
Expression of a2-M mRNA was analysed by real-time quantitative
PCR (QPCR) in SUI and control patients. The extraction of RNA
from the tissue sample was carried out with the RNA-STAT-60
reagent (Tel-Test, Inc., Friendswood, TX, USA). The complementary
DNA (cDNA) was generated from total RNA, as described previously
(Chen et al., 2004). PCR primers used to amplify a2-M cDNA were
forward: 50 -TTCGTAAACCCAAAA TGTGTCCA-30 and reverse:
50 -GTGAGGCTCTTCAACAT GCAC-30 . Real-time QPCR was
carried on the Mx3005P Multiplex Quantificative PCR System with
MxPro QPCR software (Stratagene, La Jolla, CA, USA). Brilliant
SYBR Green QPCR Master Mix (Stratagene) was used to perform
PCR. QPCR was performed as described previously (Wen et al.,
2006). The amplifications were carried out following a 10-min hot
start at 958C in a three-step protocol with 30 s denaturation (948C),
1 min annealing (558C) and extension at 728C for 30 s. Forty cycles
were performed. Hypoxanthine phosphoribosyl-transferase 1
(HPRT1) was used as an endogenous reference (Wen et al., 2006)
against which the different template values were normalized. All
PCR reactions were performed in duplicate. Cycle of threshold (Ct)
methods was used for quantification. Relative quantification of the
gene of a2-M, corrected for the quantity of the normalizer gene
(HPRT1), was divided by one normalized control sample value (cali-
brator sample) to generate the relative quantification to calibrator (Rel.
Quant. to Cal.). The PCR products were sequenced to ensure that the
correct gene sequence was amplified, and were also loaded on 2%
agarose gel to confirm their size (134 bp).
We were only able to run 24 samples (two groups of 24, in dupli-
cate) at a time for RT–PCR because the Mx3005P Multiplex

Quantificative PCR System only has a 96-well blot. Samples from
controls and women with SUI from the same phase of the menstrual
cycle were run together. Then, samples from the controls or women
with SUI from the different phases of the menstrual cycle were run
together.
Western blot analysis
Protein extraction was performed as described previously (Chen et al.,
2004). Total protein concentrations were determined using the
Bradford method (Bio-Rad, Hercules, CA, USA). The samples were
reduced with a sodium dodecyl sulfate (SDS) sample buffer contain-
ing 5% of 2-mercaptoethanol and boiled for 5 min. The samples
were subjected to 8% SDS– polyacrylamide gel electrophoresis
(SDS –PAGE). The gels were blotted onto nitrocellulose membranes
(Pierce, Rockford, IL, USA) in an electrophoretic transfer cell
(Bio-Rad). Blots were blocked with 5% non-fat milk at 48C overnight,
then probed with goat anti-a2-M (1:1000, Affinity Biologicals, Inc.,
Ontario, Canada) at room temperature for 1 h. After washing three
times with phosphate buffered saline with 0.1% Triton, pH 7.4
(PBS-T), the membrane was then incubated in 1:10 000 dilution of
mouse anti-goat IgG conjugated to horseradish peroxidase (HRP)
(GE Healthcare, Pittsburgh, PA, USA) for 1 h at room temperature,
followed by three washes in PBS-T. Blots were developed by chemi-
luminescence. The blots were re-probed with rabbit anti-
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal
antibody (1/2500, Abcam, Inc., Cambridge, MA, USA), then
1/10 000 dilution of donkey anti-rabbit IgG conjugated to HRP (GE
Healthcare, Sunnyvale, CA, USA). The band density was determined
by Bio-Rad Quality One Software (Bio-Rad).
Measurement of total a2-M
We used an enzyme-linked immunosorbent assay (ELISA) kit (Assay-
pro, St Charles, Mo, USA) to measure total a2-M in the vaginal wall
tissue extracts prepared as described above for the Western blot.
A 96-well plate was pre-coated with a polyclonal anti-a2-M antibody.
Fifty microliters of standard or samples were added into the wells in
duplicates for 2 h at room temperature. After washing, anti-a2-M bio-
tinylated polyclonal antibody was added to the plates for 1 h at room
temperature. Plates were then incubated with 50 ml of streptavidin-
peroxidase conjugate for 30 min at room temperature. Substrate was
added to the wells for 15 min for color development, and then termi-
nated by addition of 0.5 N HCl. The absorbance at 450 nm was
measured on a plate reader (Molecular Devices, Sunnyvale, CA,
USA). According to the manufacturer, intra-assay and inter-assay
coefficients of variation are 5.1% and 7.8%, respectively. The level
of total a2-M in the samples was normalized by protein concentration.
The a2-M protease (thermolysin) inhibitory activity assay
The ability of a2-M in vaginal wall tissue extracts to inhibit proteases
was assessed using QuantiCleave Protease Assay Kit (Pierce) (Wu and
Pizzo, 2001). Briefly, 50 ml of the tissue extracts or a2-M standard
were added in duplicate to 96-well plates containing 50 ml of thermo-
lysin (0.02 mg/ml, Sigma) per well, and pre-incubated at 378C
for 30 min. After pre-incubation, 100 ml of Tris –HCl buffer
(0.01 M Tris –HCl, 0.002 M CaCl2, pH 8.2, control blank) or 100 ml
of succinylated casein (2 mg/ml) was added into each well and
incubated at 378C for 1 h. Finally, 50 ml of trinitrobenzene sulfonate
solution was added and incubated at room temperature for 20 min.
The plates were read on a microplate reader at 450 nm. According
to the manufacturer’s protocol, a2-M attributable thermolysin
inhibitory activity was calculated for the change in absorbance at
450 nm by subtracting the absorbance at 450 of the control blank
a2-Macroglobulin expression in vaginal wall tissues
from that of the corresponding succinylated casein well. The a2-M
protease inhibitory activity was then normalized by total protein
concentration.
Statistical analysis
Statistical analysis was performed using unpaired t-tests. The level of
significance was set at P , 0.05. JMPIN software version 5.1 was
used (SAS Institute, Inc., Cary, NC, USA).
Results
We recruited 57 participants: 14 (mean age, 43.2 + 1.6 years)
with SUI, and 14 (mean age, 45.6 + 1.4 years) controls in the
proliferative phase, as well as 14 (mean age, 45.6 + 1.2 years)
with SUI and 15 (mean age, 42.7 + 1.0 years) controls in the
secretory phase of the menstrual cycle. There were no signifi-
cant differences in mean age and in number of vaginal deliv-
eries between SUI and control groups in both phases of the
cycle. The small tissue sample size limited our ability to
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perform all the assays on each specimen. Where tissue
samples were large enough, we performed all assays. Where
tissue samples were too small for the entire panel of assays,
we included more participants from each group. This accounts
for the discrepancy in the number of tissue samples included in
each experiment.
Identification and localization of a2-M expression
in vaginal wall tissue
The presence of a2-M in cultured human embryonic lung fibro-
blasts has been reported previously (Ma et al., 2004). Our
immunoflouresence study shows that a2-M is also expressed
in the fibroblasts isolated from vaginal wall tissue (Fig. 1).
The immunohistochemistry results further indicate that a2-M
is expressed in both cells and ECM of the vaginal wall
tissue, but the expression is primarily localized in the cells
(Fig. 2).
Expression levels of a2-M mRNA in vaginal wall tissue
from controls and women with SUI
The expression levels of a2-M in tissues from women with SUI
and control groups from the proliferative and secretory phases
of menstrual cycle were evaluated using relative real-time
QPCR. The RT – PCR fragments of a2-M run on 2% agarose
gels confirmed their size (134 bp, Fig. 3A). The expression
level of a2-M mRNA in the control group (n ¼ 8) was 11
times higher than that in the SUI group (n ¼ 8) during the pro-
liferative phase (P ¼ 0.03) and the level of a2-M mRNA in the
continent control group (n ¼ 9) was 8 times higher
(P ¼ 0.01) than that in the SUI group (n ¼ 9) during the
secretory phase (Fig. 3B).
To examine the in vivo ability of reproductive hormones to
alter the mRNA expression levels of a2-M in vaginal wall
tissue from both SUI and control groups, we compared the
expression levels of a2-M mRNA between the proliferative
and the secretory phases of the menstrual cycle in both
groups. The expression level of a2-M mRNA in the control
group was 3 times higher (P ¼ 0.01) in the secretory phase
(n ¼ 12) compared to the level in the proliferative phase
389
Wen et al.

Figure 1: Fibroblasts from the vaginal wall of a control patient were cultured
The cells were fixed and subjected to immunofluorescence staining analysis using anti-a2-M antibody (green). 40 ,6-Diamidino-2-phenylindole
was used to stain nuclei (blue)

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Figure 2: Immunohistochemical localization of a2-M, which was detected in the cells and throughout the ECM of vaginal wall tissue

(n ¼ 9) (Fig. 4). However, the expression level of a2-M
mRNA in the SUI group was similar during the proliferative
(n ¼ 10) and the secretory phases (n ¼ 10) (Fig. 4).
Characterization of proteolytic a2-M fragments
in the vaginal wall tissues from continent
controls and women with SUI
a2-M may be proteolytically degraded during severe inflam-
matory processes, resulting in decreased inhibitory function
and increased proteolysis. This proteolytic process is character-
ized in reducing SDS – PAGE and Western blot as the appear-
ance of additional a2-M fragment bands (smaller than 90 kDa)
rather than 180 and 90 kDa bands (Wu and Pizzo, 2001).
Figure 5 shows Western blot analysis of the proteolytic state
of a2-M in the tissue extracts of both control and SUI groups
in the secretory phase. In controls, a2-M shows a similar
protein pattern as the SUI group, consisting of the 180 kDa sub-
units and the 90 kDa fragments resulting from a2-M bait
region proteolysis, together with the 50 kDa fragments from
390
a2-M secondary proteolysis. Densitometric analysis shows
that the 180/90 kDa and 180/50 kDa ratios for a2-M were
similar for control and SUI groups. Both groups also had equi-
valent a2-M (180 kDa) /GAPDH (40 kDa) ratios. Western blot
analysis of the proteolytic state of a2-M in the tissue extracts of
both controls and SUI groups in the proliferative phase shows
similar results (data not shown).
Total concentrations of a2-M protein in vaginal
wall tissues determined by ELISA
We measured a2-M concentrations in the tissue extracts using
ELISA (Table I). For controls (n ¼ 7) in the proliferative
phase, the mean concentration of total a2-M was 2-fold
higher than for women with SUI (n ¼ 8, P ¼ 0.048, Table I).
For controls (n ¼ 8) in the secretory phase, the mean concen-
tration of total a2-M was roughly 3-fold higher than for
women with SUI (n ¼ 7, P ¼ 0.044, Table I). Within the SUI
group, the mean concentration of total a2-M was also 2-fold
higher in the secretory phase compared with the mean
 a2-Macroglobulin expression in vaginal wall tissues

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Figure 3: Relative real-time QPCR analyses were performed to determine and compare the mRNA expression levels of a2-M in vaginal wall tissue
from both the controls and SUI patients in the proliferative and the secretory phases
Relative quantification of the gene of a2-M, normalized to the internal control (hypoxanthine phosphoribosyl-transferase 1), was divided by one
normalized control sample value (calibrator sample) to generate the relative quantification to calibrator (Rel.Quant. to Cal.). (A) The RT–PCR frag-
ments of a2-M shown on a 2% agarose gel confirm their size (134 bp). (B) The bars represent the mean + SEM. a2-M mRNA expression level was
higher in both control groups than in the SUI groups during the proliferative phase (11-fold increase, P ¼ 0.03) and secretory phase (8-fold increase,
P ¼ 0.01). Relative Quantity to Calibrator Average (baseline-corrected, reference dye—normalized fluorescence) [Rel. Quant. to Cal. Avg (dRn)]

concentration in the proliferative phase (P ¼ 0.049, Table I).
Within controls, the mean concentration of total a2-M
was 2-fold higher in the secretory phase compared with the
mean concentration in the proliferative phase (P ¼ 0.015,
Table I).
Protease inhibitory activity of a2-M in vaginal wall tissues
Having shown that a2-M protein levels are increased in controls
compared with women with SUI in both proliferative and
secretory phases, we sought to correlate these differences with
the ability of a2-M to inhibit proteases (Table II). We specifi-
cally measured the a2-M-attributable thermolysin inhibitory
activity and found a significantly higher mean protease

Figure 4: Comparative relative real-time QPCR analyses were per-

formed to determine the ability of reproductive hormones to modulate
mRNA expression of a2-M in vaginal wall tissue from women with
SUI and the continent controls in both the proliferative and the
secretory phases
The bars represent the mean + SEM. The expression level of a2-M
mRNA in SUI groups shows no difference between the proliferative
(n ¼ 10) and secretory phases (n ¼ 10).The expression level of
a2-M mRNA in the continent control group was 3 times higher
(P ¼ 0.01) in the secretory phase (n ¼ 12) compared with the level
in the proliferative phase (n ¼ 9)
Figure 5: Western blotting of a2-M in vaginal wall tissues from 9
SUI and 10 continent control patients in the secretory phase
a2-M shows three main bands on the blot; the 180 kDa subunit and the
90 kDa fragments resulting from a2-M bait region proteolysis,
together with the 50 kDa fragments from a2-M secondary proteolysis.
‘M’ represents protein marker. ‘C’ represents controls. ‘S’ represents
women with SUI. Densitometric analysis showed that the 180/90 kDa
or 180/50 kDa and a2-M (180 kDa) / glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (40kDa) ratios were similar between the
two groups

391
Wen et al.
Table I. Total a2-M concentrations in vaginal wall tissues (mg/mg/ml).
Menstrual Phase SUI (mean + SE) Controls (mean + SE) P-values
Proliferative phase 3.45 + 0.63** 7.58 + 1.65*** 0.048*
Secretory phase 6.20 + 2.19** 17.73 + 4.5*** 0.044*
Total a2-M concentrations in the tissue extracts were determined by
enzyme-linked immunosorbent assay. Values are mean + SEM for the group.
*Significant difference between SUI and control groups.
**P ¼ 0.049 [difference between the proliferative (n ¼ 8) and the secretory
phases (n ¼ 7) in SUI group].
***P ¼ 0.015 [difference between the proliferative (n ¼ 7) and secretory
phases (n ¼ 8) in the continent control group].
Table II. a2-M attributable-thermolysin inhibitory activity was measured by
QuantiCleave Protease Assay Kit.
Menstrual Phase SUI (mean + SE) Controls (mean + SE) P-values
Proliferative phase 0.125 + 0.016** 0.139 + 0.03*** 0.64*
Secretory phase 0.096 + 0.008** 0.166 + 0.019*** 0.02*
The mean + SEM for the group is presented in the table
*Significant difference between SUI and control groups.
**P ¼ 0.3 [comparison between the proliferative (n ¼ 9) and secretory
phases (n ¼ 8) in SUI group].
***P ¼ 0.34 [comparison between the proliferative (n ¼ 8) and secretory
phases (n ¼ 8) in continent control group].
inhibitory level in the controls (n ¼ 8) compared to women with
SUI (n ¼ 8) during the secretory phase (P ¼ 0.02, Table II). We
saw no significant difference between the mean protease inhibi-
tory level in controls (n ¼ 8) compared to women with SUI
(n ¼ 9) during the proliferative phase (P ¼ 0.64, Table II).
Within each group, there was no significant difference
between the proliferative and secretory phases.
Discussion
In the present study, we investigated the expression and pro-
tease inhibitory capacity of a2-M in the vaginal wall tissues
of women with and without SUI during the proliferative and
secretory phases of the menstrual cycle. We found that the
expression levels of a2-M mRNA and protein in vaginal wall
tissues are significantly higher in controls than in women
with SUI during both proliferative and secretory phases. The
capacity of a2-M to inhibit protease activity in vaginal wall
tissues is also higher in controls than in women with SUI
during the secretory phase. In the proliferative phase, we did
not observe a significant difference between the SUI and
control groups in a2-M protease inhibitory capacity, which
differs from our PCR and ELISA data. This may be due to
the small number of patients in each group or because the
assay was not sensitive enough to pick up small differences.
This could also be due to cyclic variation in other factors
which affect enzyme activity.
Collagen and elastin play a major role in maintaining tissue
tensile strength and the mechanical stability of pelvic support-
ing tissues. Their functions are complementary, with collagen
being involved with tensile strength and elastin with defor-
mation and recoil. The quantity and quality of collagen,
elastin and other components in the pelvic supporting tissues
392
are maintained through a precise balance between proteolytic
and a2-M inhibitory activity in the ECM. a2-M has a broad-
specificity proteinase inhibitory ability in the ECM (Jensen
and Sottrup-Jensen, 1986). Decreased a2-M expression and
protease inhibitory activity could result in higher proteolytic
activity in the ECM. Excessive degradation of collagen,
elastin and other components in the ECM then weakens the
pelvic supporting tissues.
a2-M is not only a broad-specificity proteinase inhibitor, but
also a carrier of tissue repair growth factors, such as TGF-b
(Ma et al., 2004). TGF-b1, a profibrotic factor, enhances col-
lagen and elastin production (McGowan and McNamer,
1990; Gleizes et al., 1997). It also suppresses ECM degradation
by down-regulating the expression of proteinases, such as col-
lagenase and elastase, and up-regulating proteinase inhibitors,
such as the TIMPs (Overall et al., 1991). Low level of a2-M
in SUI vaginal tissue may also cause decreased synthesis of
ECM components through a decrease in TGF-b1. As a result,
the weakened connective tissues lose their tensile strength
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and mechanical stability, accelerating the development of SUI.
The bands of 180 and 85– 90 kDa of a2-M can normally be
observed by reducing SDS– PAGE. The enhanced degradation
of a2-M by secondary proteolysis could produce the smaller
fragments of a2-M (lower than 85 –90 kDa), as has been
observed in rheumatoid arthritis synovial fluids and the
plasma of patients with severe and lethal acute pancreatitis
(Wu and Pizzo, 2001; Bı́saro de Lorenc et al., 2005). Our
Western blot data show that the protein pattern of a2-M in
the SUI group is similar to that in the control group with
three forms of a2-M fragments. The ratios of 180/90 kDa
and 180/50 kDa bands are not significantly different between
the SUI and control groups, indicating that higher secondary
protease activity involved in a2-M cleavage is not observed
in vaginal wall tissues from women with SUI or controls.
This suggests that SUI is a chronic, as opposed to an acute,
inflammatory process that gradually develops years after an
injury resulting from vaginal childbirth and/or pregnancy.
a2-M is synthesized by granulosa cells of follicles in the rat
ovary after the LH surge (Gaddy-Kurten and Richards, 1991).
In our present study, we observed a significant increase in
a2-M mRNA expression in vaginal wall tissues during the
secretory phase compared with the proliferative phase in the
control group. However, a2-M mRNA expression did not
change in the SUI group. This difference between groups
suggests that a2-M expression may be regulated by reproduc-
tive hormones, and that pregnancy may favor the development
of SUI in women with a genetic predisposition to SUI, through
a relative decrease in a2-M expression compared with controls.
Given the scope of this study, we are unable to differentiate
whether the observed difference in a2-M expression is the
cause or the result of SUI. This requires further study.
Our study suggests that the imbalance of ECM synthesis and
degradation in vaginal wall tissues of women with SUI could
be caused in part by decreased levels of a2-M expression
and a corresponding decrease in protease inhibitory activity.
These data, combined with our previous observation of low
expression of the protease inhibitors, ATT and TIMP-1,
strongly suggest that decreased expression of protease

inhibitors in ECM may contribute to the development of SUI.
This information may lead to the discoveries of new methods
to prevent and treat SUI and/or pelvic organ prolapse.
Acknowledgement
The authors thank Lorna Groundwater for her excellent editorial help.
Funding
National Institute of Health (AG 17907); Mary Lake Polan
Transition Fund from Stanford University.
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Submitted on August 17, 2007; resubmitted on October 15, 2007; accepted on
October 19, 2007
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