Pelvic floor muscle training with and without supplementary KAATSU for women with stress urinary incontinence - a randomized controlled pilot study.

AIMS: To explore if adding occlusion training of a thigh (KAATSU) to low-intensity pelvic floor muscle training (PFMT) could increase effect of PFMT in women with stress urinary incontinence (SUI). METHODS: Single-blinded randomized controlled pilot study. Women with SUI and an ICIQ-UI-SF (International Consultation on Incontinence Questionnaire-Short form) score of ≥12 were randomized to a low-intensity PFMT program followed by KAATSU (KAATSU + PFMT) or to a low-intensity PFMT program without KAATSU (PFMT group), both performed four times a week for 12 weeks. PRIMARY OUTCOME: Change in the ICIQ-UI-SF score at a 12-week follow-up. SECONDARY OUTCOMES: a 3-day leakage diary, the PGI-I (Patient Global Index of Improvement scale), bother with KAATSU in a numeric rank scale and change in urethral opening pressure (UOP) measured with urethral pressure reflectometry (UPR) at rest, contraction and straining at the 12-week follow-up. RESULTS: Forty-one women with SUI and an ICIQ-UI-SF of 13 (range 12-16) were included. Fourteen in the KAATSU + PFMT and 17 in the PFMT group completed the study. Both groups had a significant and clinically relevant improvement of the ICIQ-UI-SF score and decrease in number of incontinence episodes with no significant between group differences. UOP did not increase significantly in either group. Bother with KAATSU was low but seven of 14 women expressed dislike with KAATSU. CONCLUSIONS: The added KAATSU protocol did not increase the effect of low-intensity PFMT and it was not well tolerated. While subjective effect was significant in both intervention groups this was not reflected in the UPR measures.

Regenerative medicine provides alternative strategies for the treatment of anal incontinence.

INTRODUCTION AND HYPOTHESIS: Anal incontinence is a common disorder but current treatment modalities are not ideal and the development of new treatments is needed. The aim of this review was to identify the existing knowledge of regenerative medicine strategies in the form of cellular therapies or bioengineering as a treatment for anal incontinence caused by anal sphincter defects. METHODS: PubMed was searched for preclinical and clinical studies in English published from January 2005 to January 2016. RESULTS: Animal studies have demonstrated that cellular therapy in the form of local injections of culture-expanded skeletal myogenic cells stimulates repair of both acute and 2 - 4-week-old anal sphincter injuries. The results from a small clinical trial with ten patients and a case report support the preclinical findings. Animal studies have also demonstrated that local injections of mesenchymal stem cells stimulate repair of sphincter injuries, and a complex bioengineering strategy for creation and implantation of an intrinsically innervated internal anal sphincter construct has been successfully developed in a series of animal studies. CONCLUSION: Cellular therapies with myogenic cells and mesenchymal stem cells and the use of bioengineering technology to create an anal sphincter are new potential strategies to treat anal incontinence caused by anal sphincter defects, but the clinical evidence is extremely limited. The use of culture-expanded autologous skeletal myogenic cells has been most intensively investigated and several clinical trials were ongoing at the time of this report. The cost-effectiveness of such a therapy is an issue and muscle fragmentation is suggested as a simple alternative.

Tissue-engineering with muscle fiber fragments improves the strength of a weak abdominal wall in rats.

INTRODUCTION AND HYPOTHESIS: Alternative approaches to reinforce the native tissue in patients with pelvic organ prolapse (POP) are needed to improve surgical outcome. Our aims were to develop a weakened abdominal wall in a rat model to mimic the weakened vaginal wall in women with POP and then evaluate the regenerative potential of a quickly biodegradable synthetic scaffold, methoxypolyethylene glycol polylactic-co-glycolic acid (MPEG-PLGA), seeded with autologous muscle fiber fragments (MFFs) using this model. METHODS: In an initial pilot study with 15 animals, significant weakening of the abdominal wall and a feasible technique was established by creating a partial defect with removal of one abdominal muscle layer. Subsequently, 18 rats were evenly divided into three groups: (1) unrepaired partial defect; (2) partial defect repaired with MPEG-PLGA; (3) partial defect repaired with MPEG-PLGA and MFFs labeled with PKH26-fluorescence dye. After 8 weeks, we performed histopathological and immunohistochemical testing, fluorescence analysis, and uniaxial biomechanical testing. RESULTS: Both macroscopically and microscopically, the MPEG-PLGA scaffold was fully degraded, with no signs of an inflammatory or foreign-body response. PKH26-positive cells were found in all animals from the group with added MFFs. Analysis of variance (ANOVA) showed a significant difference between groups with respect to load at failure (p = 0.028), and post hoc testing revealed that the group with MPEG-PLGA and MFFs showed a significantly higher strength than the group with MPEG-PLGA alone (p = 0.034). CONCLUSION: Tissue-engineering with MFFs seeded on a scaffold of biodegradable MPEG-PLGA might be an interesting adjunct to future POP repair.

Muscle fragments on a scaffold in rats: a potential regenerative strategy in urogynecology.

INTRODUCTION AND HYPOTHESIS: The use of permanent synthetic meshes to improve the outcome of pelvic organ prolapse (POP) repair causes frequent and serious complications. The use of the synthetic, biodegradable scaffold methoxypolyethyleneglycol-polylactic-co-glycolic acid (MPEG-PLGA) seeded with autologous muscle fiber fragments (MFF), as an adjunct to native tissue POP repair, is a potential new alternative. METHODS: A rat abdominal wall model of native repair was used with six animals in each of three groups: native repair, native repair + MPEG-PLGA, and native repair + MPEG-PLGA + MFF. MFF were labeled with PKH26-fluorescence dye. After 8 weeks labeled cells were identified in tissue samples and histopathological and immunohistochemical analyses of connective tissue organization and desmin reactivity of muscle cells were performed. Fresh tissue samples were subjected to uniaxial biomechanical testing. Statistical analyses were performed using one-way analysis of variance (ANOVA). RESULTS: MPEG-PLGA was fully degraded after 8 weeks. Desmin-immunopositive (6/6) and PKH26-positive cells (6/6) were found only after native repair + MPEG-PLGA + MFF, indicating survival, proliferation, and integration of cells originating from the MFF. This group also showed significantly increased stiffness in the high stiffness zone compared with native repair + MPEG-PLGA (p = 0.032) and borderline significantly higher stiffness compared to native repair (p = 0.054). CONCLUSIONS: In this pilot study, MPEG-PLGA scaffolds seeded with autologous MFF affected some histological and biomechanical properties of native tissue repair in an abdominal wall defect model in rats. The method thus appears to be a simple tissue engineering concept with potential relevance for native tissue repair of POP.

Intraurethral injection of autologous minced skeletal muscle: a simple surgical treatment for stress urinary incontinence.

PURPOSE: Intraurethral injection of in vitro expanded autologous skeletal muscle derived cells is a new regenerative therapy for stress urinary incontinence. We examined the efficacy and safety of a simpler alternative strategy using freshly harvested, minced autologous skeletal muscle tissue with its inherent content of regenerative cells. MATERIALS AND METHODS: A total of 20 and 15 women with uncomplicated and complicated stress urinary incontinence, respectively, received intraurethral injections of minced autologous skeletal muscle tissue and were followed for 1 year. Efficacy was assessed by the number of leakages in a 3-day diary and by ICIQ-SF scores. We calculated the rates of cure, defined as zero leaks in 3 days plus an ICIQ-SF score of 5 or less, and improvement, defined as simultaneous decreases in each outcome measure. RESULTS: Significant reductions were observed in each group in the mean number of leakages (p <0.01) and in ICIQ-SF scores (p <0.001). In the uncomplicated group cure and improvement were observed in 25% and 63% of patients, and in the complicated group they were noted in 7% and 57%, respectively. No voiding dysfunction developed and only minor adverse events were noted. CONCLUSIONS: Intraurethral injection of minced autologous muscle tissue is a simple surgical procedure that appears safe and moderately effective in women with uncomplicated stress urinary incontinence. It compares well to a more complicated regenerative strategy using in vitro expanded muscle derived cells.

The Reliability of 3-Dimensional Endoanal Ultrasonography Early and Late Postpartum.

IMPORTANCE: There is no consensus on how to define obstetric anal sphincter defects detected by 3-dimensional endoanal ultrasonography (3D-EAUS), and the reported rates vary significantly in the postpartum period. OBJECTIVE: The objective of this study was to establish a diagnostic strategy with a high and clinically relevant interrater reliability both early and late postpartum. STUDY DESIGN: The study was prospective and observational, and 3D-EAUS was performed 10-14 days and 9-12 months postpartum in an unselected cohort of primiparous women with vacuum-assisted deliveries. Two experienced examiners evaluated the ultrasonographic results, which were divided into the categories intact, inconclusive, small, moderate, and large defects based on Starck scores. Three different diagnostic strategies were validated, and the prevalence- and bias-adjusted kappa (PABAK) values calculated. RESULTS: Of 334 eligible women, 184 (55.1%) completed both examinations. Disagreements involving small defects were predominant and observed in 34 and 39 cases, respectively, at the 2 time points. The highest overall agreement rates (91.3% and 92.4%, respectively) and PABAK values (0.83 and 0.85, respectively) were reached when the disagreements were minimized by dichotomizing the results into Starck scores >4 (designated a significant defect) versus Starck scores 0-4 (all others). CONCLUSIONS: The interrater reliability of detecting small anal sphincter defects by 3D-EAUS was low at both time points for the 2 experienced raters. In contrast, the interrater reliability of detecting a significant defect was classified as almost perfect agreement at both time points.

Expression of the clock genes Per1 and Bmal1 during follicle development in the rat ovary. Effects of gonadotropin stimulation and hypophysectomy.

Daily oscillations of clock genes have recently been demonstrated in the ovaries of several species. Clock gene knockout or mutant mice demonstrate a variety of reproductive defects. Accumulating evidence suggests that these rhythms act to synchronise the expression of specific ovarian genes to hypothalamo-pituitary signals and that they are regulated by one or both of the gonadotropins. The aim of this study has been to examine the spatio-temporal expression of the clock genes Per1 and Bmal1 during gonadotropin-independent and gonadotropin-dependent follicle development in the rat ovary. We have examined the ovaries of prepubertal rats, of prepubertal rats stimulated with equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and of hypophysectomised adult animals. Using quantitative reverse transcription with the polymerase chain reaction, in situ hybridisation histochemistry and immunohistochemistry, we have demonstrated that the expression of the two clock genes is low and arrhythmic in ovarian cells during early gonadotropin-independent follicle development in prepubertal animals and in hypophysectomised animals. We have also demonstrated that the expression of the clock genes becomes rhythmic following eCG stimulation in the theca interna cells and the secondary interstitial cells and that, following additional hCG stimulation, the expression of the clock genes also becomes rhythmic in the granulosa cells of preovulatory follicles. These findings link the initiation of clock gene rhythms in the rat ovary to the luteinising hormone receptor and suggest a functional link to androgen and progesterone production. In hypophysectomised animals, rhythmic clock gene expression is also observed in the corpora lutea and in secondary interstitial cells demonstrating that, in these compartments, entrainment of clock gene rhythms is gonadotropin-independent.

Fresh muscle fiber fragments on a scaffold in rats-a new concept in urogynecology?

OBJECTIVE: To investigate if a synthetic, biodegradable scaffold with either autologous in vitro cultured muscle-derived cells or autologous fresh muscle fiber fragments could be used for tissue repair. STUDY DESIGN: Twenty scaffolds with muscle-derived cells and 20 scaffolds with muscle fiber fragments were implanted subcutaneously on the abdomen of rats, 2 in each rat, and examined after 3 weeks (10 of each preparation) and 8 weeks (10 of each preparation). Immonohistochemistry and histopathology was undertaken for assessment of growth pattern and biocompatibility, respectively. RESULTS: At 3 weeks, both muscle-derived cells and muscle fiber fragments could be identified. At 8 weeks, the muscle fiber fragments generated fragmented, striated muscle tissue in 6 of 10 explants, whereas the muscle-derived cells and all scaffolds had vanished. CONCLUSION: Autologous fresh muscle fiber fragments on a biodegradable scaffold seem useful for tissue repair. This study introduces a promising new concept with possible implications for the surgical reconstruction of pelvic organ prolapse.

The clinical relevance of cell-based therapy for the treatment of stress urinary incontinence.

Stress urinary incontinence is a common disorder affecting the quality of life for millions of women worldwide. Effective surgical procedures involving synthetic permanent meshes exist, but significant short- and long-term complications occur. Cell-based therapy using autologous stem cells or progenitor cells presents an alternative approach, which aims at repairing the anatomical components of the urethral continence mechanism. In vitro expanded progenitor cells isolated from muscle biopsies have been most intensely investigated, and both preclinical trials and a few clinical trials have provided proof of concept for the idea. An initial enthusiasm caused by positive results from early clinical trials has been dampened by the recognition of scientific irregularities. At the same time, the safety issue for cell-based therapy has been highlighted by the appearance of new and comprehensive regulatory demands. The influence on the cost effectiveness, the clinical relevance and the future perspectives of the present clinical approach are discussed.

Examinations of a new long-term degradable electrospun polycaprolactone scaffold in three rat abdominal wall models.

Alternative approaches to reinforce native tissue in reconstructive surgery for pelvic organ prolapse are warranted. Tissue engineering combines the use of a scaffold with the regenerative potential of stem cells and is a promising new concept in urogynecology. Our objective was to evaluate whether a newly developed long-term degradable polycaprolactone scaffold could provide biomechanical reinforcement and function as a scaffold for autologous muscle fiber fragments. We performed a study with three different rat abdominal wall models where the scaffold with or without muscle fiber fragments was placed (1) subcutaneously (minimal load), (2) in a partial defect (partial load), and (3) in a full-thickness defect (heavy load). After 8 weeks, no animals had developed hernia, and the scaffold provided biomechanical reinforcement, even in the models where it was subjected to heavy load. The scaffold was not yet degraded but showed increased thickness in all groups. Histologically, we found a massive foreign body response with numerous large giant cells intermingled with the fibers of the scaffold. Cells from added muscle fiber fragments could not be traced by PKH26 fluorescence or desmin staining. Taken together, the long-term degradable polycaprolactone scaffold provided biomechanical reinforcement by inducing a marked foreign-body response and attracting numerous inflammatory cells to form a strong neo-tissue construct. However, cells from the muscle fiber fragments did not survive in this milieu. Properties of the new neo-tissue construct must be evaluated at the time of full degradation of the scaffold before its possible clinical value in pelvic organ prolapse surgery can be evaluated.

Diurnal rhythmicity of the clock genes Per1 and Per2 in the rat ovary.

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0-2 and peaked at Zeitgeber time 12-14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.

Role of pituitary adenylate cyclase-activating peptide (PACAP) in the cyclic recruitment of immature follicles in the rat ovary.

Following the midcyclic gonadotropin surge, PACAP is transiently expressed for approximately 12 h in the cyclic adult rat ovary. PACAP is observed in granulosa/lutein cells of the large mature follicles destined to ovulate and is believed to be a regulator of acute progesterone production and luteinization in these follicles. PACAP is also observed in solitary theca cells of immature follicles and in interstitial glandular cells intimately surrounding immature follicles. To examine if PACAP could be involved in the process of cyclic recruitment of such immature follicles, we primed immature granulosa cells from prepubertal ovaries with PACAP (1 nM and 100 nM) for 12 h. The treatment significantly stimulated the subsequent 24 h FSH-induced estradiol production (2.2 and 2.4 fold, respectively). The response seemed to be caused by a stimulation of aromatase activity. Estradiol production induced by testosterone was increased 2.4 and 2.6 fold, respectively, whereas functional FSH-receptors (cAMP production following FSH stimulation) or spontaneous apoptosis (immunohistochemical detection of DNA fragments) was unaffected. We conclude that PACAP priming of immature rat granulosa cells for 12 h increases subsequent FSH induced estradiol production and that PACAP could be involved in the cyclic recruitment of immature follicles in the adult rat ovary.