Effect of gelatin methacryloyl hydrogel on healing of the guinea pig vaginal wall with or without mesh augmentation.

INTRODUCTION AND HYPOTHESIS: The aims of this study were to evaluate the effectiveness of gelatin methacryloyl as an adjunct to anterior vaginal wall injury with or without vaginal mesh compared with traditional repair with suture. METHODS: Virginal cycling Hartley strain guinea pigs (n = 60) were randomized to undergo surgical injury and repair using either polyglactin 910 suture or gelatin methacryloyl for epithelium re-approximation or anterior colporrhaphy with mesh augmentation using either polyglactin 910 suture or gelatin methacryloyl for mesh fixation and epithelium re-approximation. Noninjured controls (n = 5) were also evaluated. After 4 days, 4 weeks, or 3 months, tissues were analyzed by hematoxylin & eosin in addition to immunolabeling for macrophages, leukocytes, smooth muscle, and fibroblasts. RESULTS: Surgical injury repaired with suture was associated with increased inflammation and vessel density compared with gelatin methacryloyl. Vimentin and α-smooth muscle actin expression were increased with gelatin methacryloyl at 4 days (p = 0.0026, p = 0.0272). There were no differences in changes in smooth muscle or overall histomorphology after 3 months between the two closure techniques. Mesh repair with suture was also associated with increased inflammation and vessel density relative to gelatin methacryloyl. Quantification of collagen content by picrosirius red staining revealed increased thick collagen fibers throughout the implanted mesh with gelatin methacryloyl compared with suture at 4 weeks (0.62 ± 0.01 µm2 vs 0.55 ± 0.01, p = 0.018). Even at the long-term time point of 3 months, mesh repair with suture resulted in a profibrotic encapsulation of the mesh fibers, which was minimal with gelatin methacryloyl. Smooth muscle density was suppressed after mesh implantation returning to baseline levels at 3 months regardless of fixation with suture or gelatin methacryloyl. CONCLUSIONS: These results suggest that gelatin methacryloyl might be a safe alternative to suture for epithelium re-approximation and anchoring of prolapse meshes to the vagina and may improve chronic inflammation in the vaginal wall associated with mesh complications.

Endometrial stromal cell attachment and matrix homeostasis in abdominal wall endometriomas.

STUDY QUESTION: How does progesterone alter matrix remodeling in abdominal wall endometriomas compared with normal endometrium? SUMMARY ANSWER: Progesterone may prevent attachment of endometrial cells to the abdominal wall, but does not ameliorate abnormal stromal cell responses of abdominal wall endometriomas. WHAT IS KNOWN ALREADY: Menstruation is a tightly orchestrated physiologic event in which steroid hormones and inflammatory cells cooperatively initiate shedding of the endometrium. Abdominal wall endometriomas represent a unique form of endometriosis in which endometrial cells inoculate fascia or dermis at the time of obstetrical or gynecologic surgery. Invasion of endometrium into ectopic sites requires matrix metalloproteinases (MMPs) for tissue remodeling but endometrium is not shed externally. STUDY DESIGN SIZE, DURATION: Observational study in 14 cases and 19 controls. PARTICIPANTS /MATERIALS, SETTING, METHODS: Tissues and stromal cells isolated from 14 abdominal wall endometriomas were compared with 19 normal cycling endometrium using immunohistochemistry, quantitative PCR, gelatin zymography and cell attachment assays. P values < 0.05 were considered significant and experiments were repeated in at least three different cell preps to provide scientific rigor to the conclusions. MAIN RESULTS AND THE ROLE OF CHANCE: The results indicate that MMP2 and MMP9 are not increased by TGFß1 in endometrioma stromal cells. Although progesterone prevents attachment of endometrioma cells to matrix components of the abdominal wall, it does not ameliorate these abnormal stromal cell responses to TGFß1. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Endometriomas were collected from women identified pre-operatively. Not all endometriomas were collected. Stromal cells from normal endometrium were from different patients, not women undergoing endometrioma resection. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insight into the mechanisms by which progesterone may prevent abdominal wall endometriomas but, once established, are refractory to progesterone treatment. STUDY FUNDING/COMPETING INTEREST(S): Tissue acquisition was supported by NIH P01HD087150. Authors have no competing interests.

Estrogen-regulated miRNA-27b is altered by bisphenol A in human endometrial stromal cells.

MicroRNAs (miRs) are small molecules important for regulation of transcription and translation. The objective was to identify hormonally regulated miRs in human endometrial stromal cells and to determine the impact of the endocrine disruptor, bisphenol A (BPA), on those miRs. miR microarray analysis and multiple confirmatory cell preparations treated with 17ß-estradiol (E2) and BPA altered miR-27b, let-7c, let-7e and miR-181b. Further, decidualization downregulated miR-27b. VEGFB and VEGFC were validated as targets of miR-27b. Identification of miR-27b target genes suggests that BPA and E2 downregulate miR-27b thereby leading to upregulation of genes important for vascularization and angiogenesis of the endometrium during the menstrual cycle and decidualization.

Effects of vaginal conjugated equine estrogens and ospemifene on the rat vaginal wall and lower urinary tract.

Although the positive effects of vaginal estrogens and the selective estrogen receptor modulator, ospemifene (OS), on the vaginal epithelium are well recognized, less is known regarding the effects of these therapies on the lower urinary tract or vaginal muscularis. Clinical evidence suggests that vaginally administered estrogen may improve overactive bladder-related symptoms. The objective of this study was to compare the effects of OS, vaginal conjugated equine estrogens (CEE), or both on the vaginal wall and lower urinary tract in a rat model of menopause. Contractile force of the bladder neck, dome, and external urethral sphincter at optimal field stimulation did not differ significantly among treatment groups. Pharmacologic responses to atropine, carbachol, and potassium chloride were similar among groups. Vaginal epithelial thickness and differentiation were differentially regulated by CEE or OS. Ospemifene altered epithelial differentiation pathways in vaginal epithelium in a unique way, and these effects were additive with local CEE. Unless contraindicated, the beneficial effects of vaginal CEE on the vaginal wall outweigh those of OS.

Urinary Biomarkers in Women with Refractory Urgency Urinary Incontinence Randomized to Sacral Neuromodulation versus OnabotulinumtoxinA Compared to Controls.

PURPOSE: We measured urinary biomarker levels in women with refractory urgency urinary incontinence and controls at baseline and 6 months after treatment with sacral neuromodulation or intradetrusor injection of onabotulinumtoxinA. We also assessed the association of baseline biomarkers with posttreatment urgency urinary incontinence episodes and overactive bladder symptom bother outcomes. MATERIALS AND METHODS: First morning urine samples were collected from consented trial participants and age matched women without urgency urinary incontinence. Biomarkers reflecting general inflammation, neuroinflammation, afferent neurotransmitters and tissue remodeling were measured using standardized enzyme-linked immunosorbent assay and activity assays as appropriate. Symptom bother was assessed by the overactive bladder questionnaire and urgency urinary incontinence episodes were determined by bladder diary. Linear models were used to examine differences in mean biomarker levels and the change in urgency urinary incontinence episodes and symptom bother between baseline and 6 months. Modest evidence of a potential association was represented by p ≤0.01 and p ≤0.004 represented moderate evidence of an association with outcomes. RESULTS: Baseline biomarker levels differed little between cases and controls except tropoelastin (p = 0.001) and N-terminal telopeptide collagen type 1 (p <0.001). Changes in biomarker levels 6 months after intervention included decreases in collagenase (p <0.001) in both treatment groups and increases in interleukin-8 (p = 0.002) and matrix metalloprotease-9 (p <0.001) in the onabotulinumtoxinA group. Higher baseline calcitonin gene-related peptide across both treatments (p = 0.007) and nerve growth factor in the onabotulinumtoxinA arm (p = 0.007) were associated with less reduction in overactive bladder symptom bother. CONCLUSIONS: Refractory urgency urinary incontinence is a complex condition. These data suggest that matrix remodeling and neuropeptide mediation may be involved in its pathophysiological mechanisms and response to treatment.

Sex-Linked Skeletal Phenotype of Lysyl Oxidase Like-1 Mutant Mice.

Lysyl oxidases are required for collagen and elastin cross-linking and extracellular matrix maturation including in bone. The lysyl oxidase family consists of lysyl oxidase (LOX) and 4 isoforms (LOXL1-4). Here we investigate whether deletion of LOXL1, which has been linked primarily to elastin maturation, leads to skeletal abnormalities. Left femurs (n = 8), L5 vertebrae (n = 8), and tibiae (n = 8) were analyzed by micro-computed tomography in 13-week-old wild-type (WT) and LOXL1-/- male and female mice. Right femurs (n = 8) were subjected to immunohistochemistry for LOXL1, and histochemical/histology analyses of osteoclasts and growth plates. Sera from all mice were analyzed for bone turnover markers. Results indicate strong expression of LOXL1 in wild-type growth plates in femurs. Significant deterioration of trabecular bone structure in long bones and vertebrae from female was observed but not from male, mutant mice compared with WT. Decreases in BV/TV, Conn.D, trabecular thickness, and number in the femoral distal metaphysis were observed in female, but not in male, mutant mice. Trabecular spacing was increased significantly in femurs of female mutant mice. Findings were similar in trabeculae of L5 vertebrae from female mutant mice. The number of TRAP positive osteoclasts at the trabecular bone surface was increased in female mutant mice compared with WT females, consistent with increased serum RANKL and decreased OPG levels. Analysis of bone turnover markers confirmed increased bone resorption as indicated by significantly elevated CTX-1 in the serum of female LOXL1-/- mice compared to their wild-type counterparts, as well as decreased bone formation as measured by decreased serum levels of PINP. Picrosirius red staining revealed a loss of heterogeneity in collagen organization in female LOXL1-/- mice only, with little to no yellow and orange birefringence. Organization was also impaired in chondrocyte columns in both female and male LOXL1-/- mice, but to a greater extent in females. Data indicate that LOXL1-/- mutant mice develop appendicular and axial skeletal phenotypes characterized by decreased bone volume fraction and compromised trabecular microstructure, predominantly in females.

Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.

Effect of vaginal or systemic estrogen on dynamics of collagen assembly in the rat vaginal wall.

The objective of this study was to compare the effects of systemic and local estrogen treatment on collagen assembly and biomechanical properties of the vaginal wall. Ovariectomized nulliparous rats were treated with estradiol or conjugated equine estrogens (CEEs) either systemically, vaginal CEE, or vaginal placebo cream for 4 wk. Low-dose local CEE treatment resulted in increased vaginal epithelial thickness and significant vaginal growth without uterine hyperplasia. Furthermore, vaginal wall distensibility increased without compromise of maximal force at failure. Systemic estradiol resulted in modest increases in collagen type I with no change in collagen type III mRNA. Low-dose vaginal treatment, however, resulted in dramatic increases in both collagen subtypes whereas moderate and high dose local therapies were less effective. Consistent with the mRNA results, low-dose vaginal estrogen resulted in increased total and cross-linked collagen content. The inverse relationship between vaginal dose and collagen expression may be explained in part by progressive downregulation of estrogen receptor-alpha mRNA with increasing estrogen dose. We conclude that, in this menopausal rat model, local estrogen treatment increased total and cross-linked collagen content and markedly stimulated collagen mRNA expression in an inverse dose-effect relationship. High-dose vaginal estrogen resulted in downregulation of estrogen receptor-alpha and loss of estrogen-induced increases in vaginal collagen. These results may have important clinical implications regarding the use of local vaginal estrogen therapy and its role as an adjunctive treatment in women with loss of vaginal support.

Effect of myogenic stem cells on the integrity and histomorphology of repaired transected external anal sphincter.

INTRODUCTION AND HYPOTHESIS: The objective was to evaluate the effect of myogenic stem cells on histological properties and the volume of striated muscle of the external anal sphincter after transection and repair. METHODS: Histological analysis was performed on the external anal sphincters of 40 young female rats euthanized at 7 or 90 days after transection and repair and randomization to injection of either phosphate buffered solution (PBS) or myogenic stem cells (SC) at the transection site. Sphincter complexes, previously evaluated for neurophysiological function, were processed for histology and analyzed for possible disruption, amount of inflammation, and volume of striated muscle. The relationship between the muscular disruption and contractile force of sphincters was evaluated. RESULTS: Disruption was seen in 100 % of sphincters 7 days after repair for both SC and control animals. Eighty-nine percent of controls and 78% of SC-administered animals had intact sphincters at 90 days. Significant inflammatory infiltrate was seen in repaired anal sphincters for both the PBS and the SC groups at 7 days, and persisted at 90 days, with no difference between treatment groups. Striated muscle volume increased from 7 to 90 days for both control and SC-administered animals. Although there was no difference in volume between treatments, there was substantial temporal improvement in contractile force generation of the sphincters receiving SC compared with those receiving PBS. CONCLUSION: In this animal model, administration of myogenic stem cells to transected/repaired anal sphincters did not alter the amount of inflammation nor the volume of striated muscle, suggesting that stem cells might improve contractile function through other cellular processes.

Myogenic stem cell-laden hydrogel scaffold in wound healing of the disrupted external anal sphincter.

OBJECTIVE: To evaluate the effect of myogenic stem cell-laden hydrogel scaffold on contractile function and histomorphology of the external anal sphincter (EAS) after transection without repair. METHODS: Eighty female rats underwent anal sphincter transection without repair. After 2 weeks, animals were injected at the transection site with: nothing (non-repaired control, NRC group); a polyethylene glycol-based hydrogel matrix scaffold combined with phosphate-buffered saline (PBS/hydrogel group); a hydrogel matrix scaffold combined with myogenic stem cells (stem cell/hydrogel group): or type I collagen (collagen) group. 4 (n = 40) or 12 (n = 40) weeks later, the anal sphincter complexes were dissected out and analyzed for contractile function, disruption, and striated muscle volume. Time-matched unoperated controls (UOC) were utilized for each of the two time points (n = 20). RESULTS: After 4 weeks, maximal electrical field-stimulated (EFS) contractions were significantly decreased in all four non-repaired treatment groups compared with UOC. However, EFS-stimulated contractions, tetanic force generation, and twitch tension were improved in non-repaired EAS injected with stem cell/hydrogel group relative to the NRC, PBS/hydrogel, or collagen groups. NRC and sphincters injected with PBS/hydrogel deteriorated further by 12 weeks, while those receiving stem cell/hydrogel maintained improved contractile function at varying frequencies and voltages. Striated muscle volume increased from 4 to 12 weeks for PBS/hydrogel and stem cell/hydrogel animals. At 12 weeks, stem cell/hydrogel animals had greater sphincter striated muscle volumes compared with all other treatment groups. CONCLUSION: In this animal model, sustained improvement of contractile responses in non-repaired EAS treated with biogel scaffold and myogenic stem cells suggests that a biologically compatible matrix may facilitate stem cell survival, differentiation, or function leading to recovery of contractile function even after persistent disruption.

Fetal fibronectin signaling induces matrix metalloproteases and cyclooxygenase-2 (COX-2) in amnion cells and preterm birth in mice.

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E(2) synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.

Two distinct subtypes of hepatitis B virus-related acute liver failure are separable by quantitative serum immunoglobulin M anti-hepatitis B core antibody and hepatitis B virus DNA levels.

UNLABELLED: Hepatitis B virus (HBV)-related acute liver failure (HBV-ALF) may occur after acute HBV infection (AHBV-ALF) or during an exacerbation of chronic HBV infection (CHBV-ALF). Clinical differentiation of the two is often difficult if a previous history of HBV is not available. Quantitative measurements of immunoglobulin M (IgM) anti-hepatitis B core antibody (anti-HBc) titers and of HBV viral loads (VLs) might allow the separation of AHBV-ALF from CHBV-ALF. Of 1,602 patients with ALF, 60 met clinical criteria for AHBV-ALF and 27 for CHBV-ALF. Sera were available on 47 and 23 patients, respectively. A quantitative immunoassay was used to determine IgM anti-HBc levels, and real-time polymerase chain reaction (rtPCR) was used to determine HBV VLs. AHBV-ALFs had much higher IgM anti-HBc titers than CHBV-ALFs (signal-to-noise [S/N] ratio median: 88.5; range, 0-1,120 versus 1.3, 0-750; P < 0.001); a cut point for a S/N ratio of 5.0 correctly identified 44 of 46 (96%) AHBV-ALFs and 16 of 23 (70%) CHBV-ALFs; the area under the receiver operator characteristic curve was 0.86 (P < 0.001). AHBV-ALF median admission VL was 3.9 (0-8.1) log10 IU/mL versus 5.2 (2.0-8.7) log10 IU/mL for CHBV-ALF (P < 0.025). Twenty percent (12 of 60) of the AHBV-ALF group had no hepatitis B surface antigen (HBsAg) detectable on admission to study, wheras no CHBV-ALF patients experienced HBsAg clearance. Rates of transplant-free survival were 33% (20 of 60) for AHBV-ALF versus 11% (3 of 27) for CHBV-ALF (P = 0.030). CONCLUSIONS: AHBV-ALF and CHBV-ALF differ markedly in IgM anti-HBc titers, in HBV VLs, and in prognosis, suggesting that the two forms are, indeed, different entities that might each have a unique pathogenesis.

Effect of repeat acute injury on contractile function of the external anal sphincter in an animal model.

INTRODUCTION AND HYPOTHESIS: Our aim was to estimate the physiologic effects of early repeat transection and repair on the contractile properties of the external anal sphincter (EAS) in a rat model. METHODS: Eighty young female rats underwent anal sphincter transection and repair. After 7 days, they were randomized to repeat sphincter transection (injury-injury, n = 40) or sham operation (injury-sham, n = 40). Thereafter, the anal sphincter complex was dissected, mounted, and analyzed for contractile function 7 days, 21 days, 3 months, or 6 months after the second operation. Contractile function was also determined in 40 age-matched unoperated controls (n = 10 for each time point). Statistical analysis was performed using analysis of variance (ANOVA) with Tukey-Kramer adjustment for multiple testing. P ≤ 0.05 was considered significant. RESULTS: Although single injury (injury-sham) resulted in modest compromise of sphincter function, repeat injury (injury-injury) resulted in profound impairment of twitch tension, maximal tetanic responses, and maximal electrical-field stimulation (EFS) induced-force generation at 7 days. After single injury, parameters of contractile function returned to baseline uninjured levels by 21 days. In contrast, sphincter function remained reduced 21 days after repeat injury. Contractile function of sphincters from both injury-sham and injury-injury animals were no longer impaired at 3 and 6 months. CONCLUSION: In this animal model, repeat injury and repair of the EAS 7 days after the initial injury resulted in prolonged compromise of EAS function compared with single injury. Nevertheless, contractile function of the double-injured sphincter fully recovered with time, resulting in no long-term impairment.

Fibulin-4 deficiency results in ascending aortic aneurysms: a potential link between abnormal smooth muscle cell phenotype and aneurysm progression.

RATIONALE: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. OBJECTIVE: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. METHODS AND RESULTS: We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4(GKO) and Fbln4(SMKO), respectively). Fbln4(GKO) and Fbln4(SMKO) aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4(GKO) and Fbln4(SMKO) mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4(GKO) SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. CONCLUSIONS: The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.

Impact of vaginal distention on cell senescence in an animal model of pelvic organ prolapse.

OBJECTIVE: Cellular senescence, associated with aging, leads to impaired tissue regeneration. We hypothesize that vaginal injury initiates cell senescence, further propagated during aging resulting in pelvic organ prolapse (POP). Our objective was to employ a mouse model of POP (Fibulin-5 knockout mice, Fbln5-/-) to determine if vaginal distention leads to cellular senescence and POP. METHODS: 6wk old females [wild-type (WT), n = 81; Fbln5-/-, n = 47)] were assigned to control vs vaginal distention, which approximated vaginal delivery. Serial POP measurements were obtained until vagina were harvested from euthanized mice at 24, 48, 72 h and 1wk. Markers of cell senescence were quantified by immunofluorescence. DNA damage was assessed with γ-H2Ax. RESULTS: WT distended mice showed decreased p53 (p = 0.0230) and γ-H2Ax (p = 0.0008) in vaginal stromal cells at 1wk compared to controls. In WT mice, SA-ß-Gal activity increased 1wk after distention (p = 0.05). In Fbln5-/- mice, p53 and γ-H2Ax did not decrease, but p16 decreased 72 h after distention (p = 0.0150). SA-ß-Gal activity also increased in Fbln5-/-, but at earlier time points and 1wk after distention (p < 0.0001). Fbln5-/- mice developed POP after distention earlier than non distended animals (p = 0.0135). CONCLUSIONS: Vaginal distention downregulates p53 and γ-H2Ax in WT mice, thereby promoting cell proliferation 1wk after injury. This was absent among Fbln5-/- distention mice suggesting they do not escape senescence. These findings indicate a failure of cellular protection from senescence in animals predisposed to POP.

Failure of pelvic organ support in mice deficient in fibulin-3.

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3(-[supi]/-)), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3(-[supi]/-) mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3(-[supi]/-) animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3(-[supi]/-) mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3(-[supi]/-) animals with prolapse compared with Fbln3(-[supi]/-) mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse.

Healing Mechanism of Ruptured Fetal Membrane.

Preterm premature rupture of membranes (pPROM) typically leads to spontaneous preterm birth within several days. In a few rare cases, however, amniotic fluid leakage ceases, amniotic fluid volume is restored, and pregnancy continues until term. Amnion, the collagen-rich layer that forms the load-bearing structure of the fetal membrane, has regenerative capacity and has been used clinically to aid in the healing of various wounds including burns, diabetic ulcers, and corneal injuries. In the healing process of ruptured fetal membranes, amnion epithelial cells seem to play a major role with assistance from innate immunity. In a mouse model of sterile pPROM, macrophages are recruited to the injured site. Well-organized and localized inflammatory responses cause epithelial mesenchymal transition of amnion epithelial cells which accelerates cell migration and healing of the amnion. Research on amnion regeneration is expected to provide insight into potential treatment strategies for pPROM.

Protease Inhibition Improves Healing of The Vaginal Wall after Obstetrical Injury: Results from a Preclinical Animal Model.

Vaginal delivery with obstetrical trauma is a risk factor for pelvic organ prolapse later in life. Loss of fibulin-5 (FBLN5), an elastogenesis-promoting cellular matrix protein, results in prolapse in mice. Here, we evaluated effects of pregnancy, parturition, and obstetrical injury on FBLN5 content, elastic fibers, biomechanics, and histomorphology of the vaginal wall in rats. Further, we analyzed the effects of actinonin, a protease inhibitor, on obstetrical injury of the vaginal wall. Vaginal FBLN5 decreased significantly in pregnancy, and injury resulted in further downregulation. Stiffness of the vaginal wall decreased 82% in pregnant rats and 74% (p = 0.019) with injury relative to uninjured vaginal delivery controls at 3d. Actinonin ameliorated loss of FBLN5, rescued injury-induced loss of elastic fibers and biomechanical properties after parturition, and reduced the area of injury 10-fold. We conclude that pregnancy and parturition have a profound impact on vaginal FBLN5 and biomechanics of the vaginal wall. Further, obstetrical injury has significant deleterious impact on recovery of the vaginal wall from pregnancy. Actinonin, a non-specific matrix metalloprotease inhibitor, improved recovery of the parturient vaginal wall after obstetrical injury.

Controlled Ovarian Stimulation Protocols Alter Endometrial Histomorphology and Gene Expression Profiles.

Although it is well appreciated that ovarian stimulation protocols for in vitro fertilization (IVF) alter endometrial receptivity, the precise cellular mechanisms are not known. To gain insights into potential mechanisms by which different ovarian stimulation protocols alter the endometrium, we compared histologic and gene expression profiles of endometrium from women undergoing conventional ovarian stimulation for IVF (C-IVF) with those undergoing minimal stimulation with clomiphene citrate (MS-IVF). Sixteen women undergoing MS-IVF (n = 8) or C-IVF (n = 8) were recruited for endometrial biopsy at the time of oocyte retrieval. Endometrial glands were large, tortuous, and secretory with C-IVF but small and undifferentiated with MS-IVF. Whereas RNA sequencing did not reveal changes in estrogen receptor or its co-regulators or classic proliferation associated genes in MS-IVF, together with immunohistochemistry, Wnt signaling was disrupted in endometrium from MS-IVF cycles with significant upregulation of Wnt inhibitors. Secreted frizzled-related protein 1 (sFRP1) was increased fourfold (p < 0.01), and sFRP4 was upregulated sixfold (p < 0.01) relative to C-IVF. Further these proteins were localized to subepithelial endometrial stroma. These data indicate that MS-IVF protocols with CC do not seem to impact endometrial estrogen signaling as much as would be expected from the reported antiestrogenic properties of CC. Rather, the findings of this study highlight Wnt signaling as a major factor for endometrial development during IVF cycles.

Melatonin synergizes with oxytocin to enhance contractility of human myometrial smooth muscle cells.

CONTEXT: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. OBJECTIVE: The purpose of this study was to determine the effects of MEL on contractility and the contractile machinery in telomerase-immortalized human myometrial cells. DESIGN: To ascertain the effect of MEL on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodomelatonin +/- oxytocin (OT). The effects of iodomelatonin on gap junctions were also investigated. Additionally, expression levels of the type 2 MEL receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor. RESULTS: MEL was found to synergistically enhance OT-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched nonlaboring pregnant women. MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner. CONCLUSIONS: MEL synergizes with OT to promote myometrial cell contractions and to facilitate gap junction activity in vitro. Such a synergy in vivo would promote coordinated and forceful contractions of the late term pregnant uterus necessary for parturition.