The effect of raloxifene, a SERM, on extracellular matrix protein expression of pelvic fibroblasts.

INTRODUCTION AND HYPOTHESIS: We hypothesize that the abnormal extracellular matrix (ECM) turnover in pelvic tissues of women with prolapse may be attenuated by raloxifene. We examine the effect of raloxifene on ECM protein expression in pelvic fibroblasts. METHODS: Pelvic fibroblasts were isolated from cases (N = 6) and controls (N = 3). Cells were treated with raloxifene. Dose-response analyses were performed by ANOVA. mRNA and protein expression of collagen I, III, MMPs, and TIMPs were determined by RT-PCR and Western blot. MMP activity was analyzed by zymography. RESULTS: The mRNA expression of TIMP-3 and protein expression of TIMP-1 and TIMP-3 were significantly increased by raloxifene in fibroblasts from both cases and controls (P < 0.05). Collagen I, III, and MMP mRNA and protein expressions were not affected. CONCLUSIONS: Raloxifene selectively attenuates abnormal matrix degradation by increasing inhibitors of proteases, TIMPs, in pelvic fibroblasts. This opens the possibility for SERMs to be used as preventive therapy for pelvic floor disorders.

Identification of protein marker in vaginal wall tissues of women with stress urinary incontinence by protein chip array.

AIM: We sought to investigate protein biomarkers for stress urinary incontinence (SUI) in vaginal tissues using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and examine if this is a reliable methodology to examine proteins in small tissue specimens. MATERIAL AND METHODS: We compared protein expression profile of vaginal tissue from women with SUI and continent controls. A 22.6kDa peak was identified by subsequent weak cation-exchange, reverse-phase fractionation, gel electrophoresis, and trypsin digestion, then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS-MS. Biomarker identity and expression level were confirmed by Western-blotting and immunohistochemistry. RESULTS: Expression of the 22.6kDa protein, identified as SM-22α, was significantly higher in women with SUI versus controls. A 3×3-mm tissue sample was sufficient for identification. Western-blot/immunohistochemistry confirmed the SELDI-TOS MS findings. CONCLUSION: SM-22α, a marker for myofibroblasts, was identified as a biomarker of SUI. Differential protein profiling by SELDI-TOF MS is a powerful and reliable tool for urogynecological research as it allows us to study an array of proteins simultaneously using small tissue samples.

Expression of apoptotic factors in vaginal tissues from women with urogenital prolapse.

AIMS: Increased apoptotic activity in pelvic tissues may contribute to development of pelvic floor disorders. We evaluated expression of apoptotic factors (Bcl-2 family) in vaginal tissues from women with pelvic organ prolapse (POP) and how these factors correlate with severity of prolapse. METHODS: mRNA and protein expression of anti-apoptotic and pro-apoptotic factors in vaginal tissues from subjects and controls were determined by real-time PCR and Western blot. Severity of prolapse was staged using POP-Q criteria. RESULTS: Differential expression of Bcl-2 family factors was observed in protein rather than in gene expression. During the secretory phase, the anti-apoptotic (Bcl-2, Bcl-xl) and pro-apoptotic protein (Bax) were upregulated in controls compared to cases (P < 0.05). The ratios of Bcl-2/Bax and Bcl-2/Bad, which determine cellular sensitivity to induction of apoptosis, were higher in controls versus cases. Higher ratios indicate reduced cellular sensitivity to apoptosis. Protein expression of Bax and Bad was higher in women with severe compared to mild prolapse (P < 0.05). CONCLUSION: Increased expression of Bad, Bax, and decreased ratios of Bcl-2/Bax, Bcl-2/Bad suggest increased apoptotic activity or sensitivity to induction of apoptosis in vaginal tissues of women with POP.

Evaluation of a community intervention for promotion of safe motherhood in Eritrea.

INTRODUCTION: We evaluated a community-based intervention to promote safe motherhood, focusing on knowledge and behaviors that may reduce maternal mortality and birth complications. The intervention aimed to increase women's birth preparedness, knowledge of birth danger signs, use of antenatal care services, and birth at a health care facility. METHODS: Volunteers from a remote rural community in Northern Eritrea were trained to lead participatory educational sessions on safe motherhood with women and men. The evaluation used a quasiexperimental design (nonequivalent group pretest-posttest) including cross-sectional surveys with postpartum women (pretest n = 466, posttest n = 378) in the intervention area and in a similar remote rural comparison area. RESULTS: Women's knowledge of birth danger signs increased significantly in the intervention area but not in the comparison area. There was a significant increase in the proportion of women who had the recommended 4 or more antenatal care visits during pregnancy in the intervention area (from 18% to 80%, P < .001), although this proportion did not change significantly in the comparison area (from 53% to 47%, P = .194). There was a greater increase in birth in a health care facility in the intervention area. DISCUSSION: Participatory sessions led by community volunteers can increase safe motherhood knowledge and encourage use of essential maternity services.

Transforming growth interacting factor expression in leiomyoma compared with myometrium.

OBJECTIVE: To investigate the expression of transforming growth interacting factor (TGIF), a Smad transcriptional corepressor, in leiomyoma and matched myometrial tissue samples and the effect of TGIF overexpression in myometrial cells. DESIGN: Experimental study. SETTING: Tertiary university hospital. PATIENT(S): Uterine leiomyoma and myometrial tissues from 16 patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The distribution of TGIF in leiomyoma and myometrial tissues by immunohistochemistry stain, mRNA, and protein expression levels by real-time quantitative polymerase chain-reaction (QPCR) and Western blot. Transcriptional regulation of TGIF in myometrial cells with overexpressed TGIF. RESULT(S): Although TGIF is present in the smooth muscle cells of the leiomyoma and the myometrium, it is not found in the extracellular matrix. The TGIF mRNA and protein expressions were statistically significantly higher in the leiomyoma compared with the matched, unaffected myometrial tissues in both phases of the menstrual cycle. There were no differences in mRNA or protein expression throughout the menstrual cycle. Overexpression of TGIF protein in myometrial cells statistically significantly suppressed up-regulation of plasminogen activator inhibitor (PAI-1) induced by TGF-beta1 treatment. CONCLUSION(S): Expression of TGIF is increased in leiomyoma compared with myometrium. This increase in TGIF expression is not affected by endogenous ovarian hormones. Thus, TGIF is a potential repressor of TGF-beta pathways in myometrial cells.

The role of counseling for obstetric fistula patients: lessons learned from Eritrea.

OBJECTIVE: The goal of this study was to evaluate the first formal counseling program for obstetric fistula patients in Eritrea. METHODS: To evaluate the impact of the counseling program, clients were interviewed both before pre-operative counseling and again after post-operative counseling. A questionnaire was used in the interviews to assess women's knowledge about fistula, self-esteem, and their behavioral intentions for health maintenance and social reintegration following surgical repair. In addition, two focus groups were conducted with a total of 19 clients assessing their experiences with the surgical care and counseling. RESULTS: Data from the questionnaires revealed significant improvements in women's knowledge about fistula, self-esteem, and behavioral intentions following counseling. Focus group data also supported increased knowledge and self-esteem. CONCLUSION: Evaluation of the short-term impact of an initial formal counseling program for fistula patients in sub-Saharan Africa affirmed the positive effects that such a program has for fistula patients, with increased knowledge about the causes of fistula, fistula prevention and enhanced self-esteem. PRACTICAL IMPLICATIONS: Culturally appropriate counseling can be incorporated into services for surgical repair of obstetric fistula in low-resource settings and has the potential to improve the physical and mental well-being of women undergoing fistula repair.

Experience with a low-pressure colonic pouch (Mainz II) urinary diversion for irreparable vesicovaginal fistula and bladder extrophy in East Africa.

INTRODUCTION AND HYPOTHESIS: We report our experience with a low-pressure colonic pouch for urinary diversion in women with irreparable vesicovaginal fistulas and bladder extrophy. METHODS: This is a case series of 35 women with irreparable vesicovaginal fistula who underwent urinary diversion and two cases performed for bladder extrophy. RESULTS: Partial or complete loss of the urethra was present in over 90% of fistula cases. Fifty-five percent had prior vaginal repairs. The median length of stay was 21 days. Median follow-up for 29 (78%) patients was 18 months. Nighttime urinary incontinence occurred in 31%. Twenty-one (91%) of 23 patients had a serum creatinine <1.5 although all patients had evidence of acidosis. Two patients died 4 years after surgery from sepsis and renal failure. CONCLUSIONS: Urinary diversion using the Mainz pouch II can be performed in the developing world with low perioperative morbidity and mortality. Acidosis and nighttime incontinence are the most common complications.

Relaxin increases elastase activity and protease inhibitors in smooth muscle cells from the myometrium compared with cells from leiomyomas.

We investigated the effect of relaxin on extracellular matrix remodeling in cultured myometrial compared with leiomyoma smooth muscle cells. Relaxin increases elastase activity and protease inhibitor expressions in myometrial smooth muscle cells, but no effect was observed in leiomyoma cells.

Effect of relaxin on TGF-beta1 expression in cultured vaginal fibroblasts from women with stress urinary incontinence.

The objective of this study is to compare relaxin's effect on transforming growth factor (TGF)- beta1 and latent TGF-beta1-binding protein (LTBP-1) in vaginal fibroblasts from women with stress urinary incontinence (SUI) to continent women (controls) in both phases of the menstrual cycle. Fibroblasts were treated with relaxin. TGF-beta1 levels were measured by enzyme-linked immunosorbent assay. LTBP-1 expression was evaluated by Western blot. In the proliferative phase, total TGF-beta1 level in the supernatant, cells, and extracellular matrix (ECM) of SUI fibroblasts decreased with increasing relaxin concentration (P < .05). Active TGF-beta1 levels increased at a low concentration of relaxin (P < .05) in the supernatant but decreased in the ECM of SUI fibroblasts at high concentration (P < .05). In the secretory phase, total TGF-beta1 levels decreased with relaxin treatment (P < .05) in the supernatant, cells and ECM of both women with SUI and controls. Relaxin decreased the levels of total and active TGF-beta1 in the ECM isolated from SUI vaginal fibroblasts.

Increased expression of latent TGF-beta binding protein-1 and fibrillin-1 in human uterine leiomyomata.

We compared latent TGF-ss binding protein-1 (LTBP-1) and fibrillin-1 (FBN-1) expression in leiomyomata and myometrium, correlated with leiomyomata size. We studied in vivo and in vitro effects of ovarian steroids using matched leiomyomata and myometrium samples from both phases of the menstrual cycle. Leiomyomata were divided into small (or=6 cm) groups. We validated LTBP-1 and FBN-1 expression using QPCR, western blot and immunohistochemistry. LTBP-1 and FBN-1 mRNA and protein expressions were higher in the medium-sized group compared with myometrium in the proliferative phase (P = 0.01; P = 0.01). FBN-1 mRNA expression was higher in the secretory phase (P = 0.01). LTBP-1 mRNA and protein expression was higher in the medium group compared with the small and large groups in the proliferative phase (P = 0.04; P = 0.04). No differences between groups were seen in FBN-1 expression in either phase. 17Beta-estradiol (E2) increased mRNA and protein expression of LTBP-1 and FBN-1 in cultured leiomyoma smooth muscle cells (LSMC) (P < 0.05). No change in FBN-1 and LTBP-1 expression was observed when cells were treated with E2 plus progesterone. Estrogen may be involved in LTBP-1 and FBN-1 expression in leiomyomata. Extracellular matrix metabolism may be different in medium-sized leiomyoma.

Expression of CD9 in frozen-thawed mouse oocytes: preliminary experience.

CD9 mRNA and protein expression levels in mouse slow frozen-rapid thawed oocytes were compared with those in fresh oocytes by using comparative quantitative real time reverse transcription-PCR and semiquantitative Western blot, respectively. The expression levels of both CD9 mRNA and protein in the frozen oocytes were significantly lower than those found in the fresh oocytes.

The role of neutrophil elastase in elastin metabolism of pelvic tissues from women with stress urinary incontinence.

OBJECTIVE: Altered elastin metabolism is implicated in pelvic floor disorders. We studied neutrophil elastase (NE) and matrix metalloproteinase (MMP) activities in vaginal tissues from premenopausal women with stress urinary incontinence (SUI). METHODS: Elastase and NE activities in vaginal tissues were assessed. Protein and mRNA expressions were determined by RT-PCR and Western blot. Total elastin and collagen contents were evaluated. To compare the relative elastolytic effect of NE and MMP-2, we used their respective antibodies to immunoprecipitate these proteins from vaginal fibroblast extracts prior to assessing elastase activity. RESULTS: Elastase activity in vaginal wall tissues was significantly higher in the secretory compared to the proliferative phase. NE mRNA and protein expressions were similar between control and SUI tissues from the secretory phase. However, NE activity in the SUI tissues was higher compared to control tissues. The mRNA expression of alpha-1 antitrypsin (ATT) was higher in control tissues from the proliferative phase compared to those from the secretory phase, while no difference was observed in SUI tissues between either phase. Protein expression of the active form of ATT was decreased in SUI tissues compared to controls during the secretory phase. Anti-NE antibody reduced total elastase activity by 60-70%, compared to less than 20% reduction with anti-MMP-2 antibody. CONCLUSION: During the secretory phase, elastolytic activity is increased in pelvic tissues from women with SUI, through an increase in NE activity and a concurrent decrease in ATT expression. The serine protease, NE, appears to be a more significant modulator of elastase activity compared to MMP-2.

Experiences of women seeking medical care for obstetric fistula in Eritrea: implications for prevention, treatment, and social reintegration.

This article presents findings from qualitative interviews with women seeking medical care for obstetric fistula in Eritrea. The interviews were designed to inform programme design for the prevention and treatment of obstetric fistula. Interviews were conducted with 11 new fistula repair patients, 15 women returning for follow-up for their fistula repairs, and five accompanying family members at Massawa Hospital in the Northern Red Sea Zone of Eritrea during November-December 2004. The women described long delays in accessing emergency obstetric care due to delayed recognition of the seriousness of the problem and lack of transportation from remote villages. Follow-up patients described improvements in their conditions, but many continued to have problems with incontinence and sexual health. Both new and returning patients lacked specific information about their condition, what to expect in terms of treatment and recovery, and how to care for themselves. The findings point to a need for community mobilization and education on safe motherhood for prevention of fistula, as well as for improved information, counselling, follow-up, and social services for women who develop obstetric fistulas.

The enhancement of female sexual function with ArginMax, a nutritional supplement, among women differing in menopausal status.

We conducted a double-blind, placebo-controlled study to determine the role of dietary supplementation on sexual function in women of differing menopausal status. One hundred eight (108) women, age 22-73 years, who reported a lack of sexual desire, enrolled as participants. Of these, 55 received ArginMax for women and 53 received placebo. ArginMax for women contains L-arginine, ginseng, ginkgo, damiana, multivitamins, and minerals. The 108 women, given definitions, self-reported as 59 premenopausal (PRE); 20 perimenopausal (PERI), and 29 postmenopausal (POST). After 4 weeks, PRE women on ArginMax primarily reported significant improvement in level of sexual desire (72%; p = 0.03) and satisfaction with overall sex life (68%; p = 0.007), compared with placebo group, according to the Female Sexual Function Index (FSFI; Kaplan et al., 1999) scales. Frequency of sexual desire (60%; p = 0.05) and frequency of intercourse (56% p = 0.01) also increased among the PRE women. In contrast, among PERI women, primary improvements were reported for frequency of intercourse (86%; p = 0.002), satisfaction with sexual relationship (79%; p = 0.03), and vaginal dryness (64%; p = 0.03) compared with placebo group. POST women primarily showed an increased in level of sexual desire, with 51% showing improvement, compared with only 8% in the placebo group (p = 0.008). Nutritional intervention plays an important role in women's sexual health, but issues and areas of greatest improvement differ among women of different menopausal states. The largest number of attribute improvements were seen in PRE and PERI women, although attribute types vary among these groups. Level of desire was shown to increase significantly in POST women. Since ArginMax for women has been shown to exhibit no estrogen activity, it may be desirable alternative to hormone therapy for sexual concerns.

Regulation of cyclooxygenase activity in cultured endometrial stromal cells by granulocyte-macrophage colony-stimulating factor.

OBJECTIVE: To assess the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to regulate cyclooxygenase (COX) enzyme activity and prostaglandins (PGs) synthesis, specifically PGE2 production in stromal cells, neither of which have been addressed in the literature. DESIGN: Prospective study. SETTING: Department of obstetrics and gynecology at a university hospital. PATIENT(S): Human luteal phase endometrium was obtained from surgical specimens (n = 6) for clinical indications. INTERVENTION(S): Confluent stromal cells were stimulated with GM-CSF. MAIN OUTCOME MEASURE(S): Expression of COX mRNA, COX enzyme activity, and PGE2 level in cultured stromal cells. RESULT(S): Confluent stromal cell cultures treated with P and E2 for 9 days were stimulated with GM-CSF. After treatment of 12 hours, low-dose GM-CSF (0.001-0.1 ng/mL) increased COX-2 mRNA levels in stromal cell, whereas high dose GM-CSF (1-100 ng/mL) decreased COX-1 and COX-2 mRNA levels. After treatment of 48 hours, low concentrations of GM-CSF (0.001-0.1 ng/mL) increased total COX and COX-2 enzyme activity, whereas high concentrations of GM-CSF (1-100 ng/mL) inhibited COX and COX-2 activity; The PGE2 levels decreased by 31% to 393.3 pg/mL (P < .05) with concentrations of GM-CSF increasing from 1 ng/mL to 100 ng/mL. CONCLUSION(S): There appeared to be a biphasic pattern of COX-2 enzyme response to GM-CSF with low concentrations increasing activity and high concentrations inhibiting activity. It is possible that GM-CSF may provide critical regulation of PG production in the preimplantation period.

Do extracellular matrix protein expressions change with cyclic reproductive hormones in pelvic connective tissue from women with stress urinary incontinence?

BACKGROUND: To evaluate differential expression of transforming growth factor (TGF-beta1), latent transforming factor-binding proteins (LTBP-1, LTBP-2) and elastin microfibril components (fibrillin-1 and fibrillin-2) in vaginal tissue from women with stress urinary incontinence (SUI). METHODS: In this case-control study, vaginal tissue from women in both phases of the menstrual cycle was obtained. Messenger RNA (mRNA) expressions of LTBP-1, LTBP-2, fibrillin-1, fibrillin-2 and TGF-beta1 were determined by relative real-time quantification PCR. Tissue localization was analysed by immunohistochemistry, and semiquantitative protein expression was evaluated by Western blot analysis. RESULTS: Vaginal wall fibroblasts synthesized all proteins tested. LTBP-1, LTBP-2 and TGF-beta1 co-localized with elastin microfibrils, fibrillin-1 and fibrillin-2 in the extracellular matrix. LTBP-1 mRNA and protein expressions were higher in control versus women affected with SUI in the proliferative phase (P = 0.04), while in the secretory phase, mRNA expression in cases was higher (P = 0.04). Fibrillin-1 mRNA was higher in women affected by SUI versus controls in both phases, but no statistical differences in fibrillin-1 protein expression were observed between the two groups in either phase. LTBP-2 and TGF-beta1 mRNA expressions showed the same trends as LTBP-1. CONCLUSION: LTBP-1, LTBP-2, TGF-beta1, fibrillin-1, and fibrillin-2 expressions are hormonally regulated in vaginal wall fibroblasts and differ in women affected by SUI when compared to controls. These data suggest a mechanism to regulate TGF-beta1 activity in pelvic connective tissue.

Microarray analysis of differentially expressed genes in vaginal tissues from women with stress urinary incontinence compared with asymptomatic women.

BACKGROUND: The pathophysiology of pelvic floor dysfunction resulting in stress urinary incontinence (SUI) in women is complex. Evidence suggests that there is also a genetic predisposition towards SUI. We sought to identify differentially expressed genes involved in extracellular matrix (ECM) metabolism in vaginal tissues from women with SUI in the secretory phase of menses compared with asymptomatic women. METHODS: Tissue samples were taken from the periurethral vaginal wall of five pairs of premenopausal, age-matched SUI and continent women and subjected to microarray analysis using the GeneChip Human Genome U133 oligonucleotide chip set. RESULTS: Extensive statistical analyses generated a list of 79 differentially expressed genes. Elafin, keratin 16, collagen type XVII and plakophilin 1 were consistently identified as up-regulated ECM genes. Elafin, a serine protease inhibitor involved in the elastin degradation pathway and wound healing, was expressed in pelvic fibroblasts and confirmed by Western blot, quantitative competitive PCR and immunofluorescence cell staining. CONCLUSIONS: Genes involved in elastin metabolism were differentially expressed in vaginal tissue from women with SUI, suggesting that elastin remodelling may be important in the molecular aetiology of SUI.

Surgical management of complex obstetric fistula in Eritrea.

OBJECTIVES: To evaluate the incidence of and demographic characteristics associated with obstetric fistula in Eritrea. To determine the outcomes of surgical repair of complex fistula in Eritrea by a visiting surgical team. METHODS: A surgical team comprising expert gynecologic surgeons traveled to Eritrea in September 2004. We evaluated 50 patients with genitourinary fistula and performed surgical repairs of these fistulas on 37 women via both vaginal and abdominal approaches. Demographic and basic medical data were obtained at the time of evaluation, and follow-up questionnaires were completed at 4 weeks postoperative. RESULTS: The majority of the women had fistulas related to obstructed labor at their first pregnancy unattended by any healthcare professional. The average duration of labor was 3 days, and more than half had resulted in stillbirths. The rate of successful repair in women with primary vesicovaginal fistulas (VVF) was 63%, and that in women with recurrent vesicovaginal fistulas was 61%. Two women required urinary diversion procedures because of the severity of the damage to the genital tract. Urethral reconstruction in women with urethrovaginal fistulas (UVFs) was successfully accomplished in 77% of patients. The rate of successful repair of rectovaginal fistulas (RVFs) was 87%. CONCLUSIONS: We have demonstrated that a team of specialized surgeons can successfully accomplish surgical procedures and repairs of very complex urinary tract fistulas in a very short mission to a resource-poor nation.

Exogenous granulocyte-macrophage colony-stimulating factor promotes follicular development in the newborn rat in vivo.

BACKGROUND: Expression and selective cellular localization of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor in ovarian tissue imply an autocrine/paracrine role in ovarian function. Evidence indicating a functional role for GM-CSF in ovarian follicular cell function has been provided by studies with GM-CSF knockout (GM-/-) mice, which suggest that GM-CSF influences events associated with murine follicular maturation. METHODS: Immature female rats were treated with GM-CSF, FSH or saline for 5 or 10 days. Ovaries were collected for histologic examination and immunostaining determination of CYP17, a theca cell marker. In addition, ovarian section slides were evaluated by immunofluorescence for CD45, an ovarian leukocyte marker. To investigate the possible mechanism of GM-CSF action on follicular development, theca-interstitial cells (T-I) were separated and cultured. Cells were treated with increasing concentrations of GM-CSF, then evaluated for CYP17 mRNA and protein expression assays. RESULTS: After 10 days of treatment with GM-CSF, the number of small preantral and large preantral follicles was significantly increased compared with the control group (P < 0.05). Similarly, treatment with FSH increased the number of small preantral and large preantral follicles (P < 0.05). CD45 expression measured by immunofluorescence was not different in the three groups, indicating that the distribution of leukocytes was unchanged. In addition, CYP17 was increased in the T-I cells both in vivo and in vitro after GM-CSF treatment. CONCLUSION: The present results suggest that GM-CSF may play a significant role in follicular development.