Citrate synthase, sarcoplasmic reticular calcium ATPase, and choline acetyltransferase activities of specific pelvic floor muscles of the rabbit

There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic–endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity.     

Electromyography of pelvic floor muscles

Pelvic floor muscles (PFM) are intimately involved in function of lower urinary tract, the anorectum and sexual functions, therefore their neural control transcends the primarily important somatic innervation of striated muscle, as they are directly involved in “visceral activity”. Neural control of pelvic organs is affected by a unique co-ordination of somatic and autonomic motor nervous systems. Visceral and somatic sensory fibres supply sensory information from pelvic organs; their input influences through central integrative mechanisms also pelvic floor muscle activity. Anatomically, somatic afferent and efferent nerves of the sacral cord segments, reflexly integrated at the spinal cord and brainstem level, conduct neural control of PFM. The inputs from several higher centres influence the complex reflex control and are decisive for voluntary control, and for socially adapted behaviour related to excretory functions.     

Measuring morphological parameters of the pelvic floor for finite element modelling purposes

The goal of this study was to obtain a complete data set needed for studying the complex biomechanical behaviour of the pelvic floor muscles using a computer model based on the finite element (FE) theory. The model should be able to predict the effect of surgical interventions and give insight into the function of pelvic floor muscles. Because there was a lack of any information concerning morphological parameters of the pelvic floor muscle structures, we performed an experimental measurement to uncover those morphological parameters. Geometric parameters as well as muscle parameters of the pelvic floor muscles were measured on an embalmed female cadaver. A three-dimensional (3D) geometric data set of the pelvic floor including muscle fibre directions was obtained using a palpator device. A 3D surface model based on the experimental data, needed for mathematical modelling of the pelvic floor, was created. For all parts of the diaphragma pelvis, the optimal muscle fibre length was determined by laser diffraction measurements of the sarcomere length. In addition, other muscle parameters such as physiological cross-sectional area and total muscle fibre length were determined. Apart from these measurements we obtained a data set of the pelvic floor structures based on nuclear magnetic resonance imaging (MRI) on the same cadaver specimen. The purpose of this experiment was to discover the relationship between the MRI morphology and geometrical parameters obtained from the previous measurements. The produced data set is not only important for biomechanical modelling of the pelvic floor muscles, but it also describes the geometry of muscle fibres and is useful for functional analysis of the pelvic floor in general. By the use of many reference landmarks all these morphologic data concerning fibre directions and optimal fibre length can be morphed to the geometrical data based on segmentation from MRI scans.These data can be directly used as an input for building a mathematical model based on FE theory.     

Effects of pelvic floor muscle contraction on anal canal pressure

The role of pelvic floor muscle contraction in the genesis of anal canal pressure is not clear. Recent studies have suggested that vaginal distension increases pelvic floor muscle contraction. We studied the effects of vaginal distension on anal canal pressure in 15 nullipara asymptomatic women. Anal pressure, rest, and squeeze were measured using station pull-through manometry techniques with no vaginal probe, a 10-mm vaginal probe, and a 25-mm vaginal probe in place. Rest and squeeze vaginal pressures were significantly higher when measured with the 25-mm probe compared with the 10-mm probe, suggesting that vaginal distension enhances pelvic floor contraction. In the presence of the 25-mm vaginal probe, rest and squeeze anal pressures in the proximal part of the anal canal were significantly higher compared with no vaginal probe or the 10-mm vaginal probe. On the other hand, distal anal pressures were not affected by any of the vaginal probes. Ultrasound imaging of the pelvic floor revealed that vaginal distension increased the anterior-posterior length of the puborectalis muscle. Atropine at 15 micro g/kg had no influence on the rest and squeeze anal pressures with or without vaginal distension. Our data suggest that pelvic floor contractions increase pressures in the proximal part of the anal canal, which is anatomically surrounded by the puborectalis muscle. We propose that pelvic floor contraction plays an important role in the fecal continence mechanism by increasing anal canal pressure.     

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P <0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.     

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.     

A shell finite element model of the pelvic floor muscles

The pelvic floor gives support to the organs in the abdominal cavity. Using the dataset made public in (Janda et al. J. Biomech. (2003) 36(6), pp. 749-757), we have reconstructed the geometry of one of the most important parts of the pelvic floor, the levator ani, using NURB surfaces. Once the surface is triangulated, the corresponding mesh is used in a finite element analysis with shell elements.Based on the 3D behavior of the muscle we have constructed a shell that takes into account the direction of the muscle fibers and the incompressibility of the tissue. The constitutive model for the isotropic strain energy and the passive strain energy stored in the fibers is adapted from Humphrey's model for cardiac muscles. To this the active behavior of the skeletal muscle is added.We present preliminary results of a simulation of the levator ani muscle under pressure and with active contraction. This research aims at helping simulate the damages to the pelvic floor that can occur after childbirth.     

Effects of nonlinear muscle elasticity on pelvic floor mechanics during vaginal childbirth

The role of the pelvic floor soft tissues during the second stage of labor, particularly the levator ani muscle, has attracted much interest recently. It has been postulated that the passage of the fetal head through the pelvis may cause excessive stretching of the levator ani muscle, which may lead to pelvic floor dysfunction and pelvic organ prolapse later in life. In order to study the complex biomechanical interactions between the levator ani muscle and the fetal head during the second stage of labor, finite element models have been developed for quantitative analysis of this process. In this study we have simulated vaginal delivery using individual-specific anatomical computer models of the pelvic floor interacting with a fetal head model with minimal restrictions placed upon its motion. Two constitutive relations were considered for the levator ani muscle (of exponential and neo-Hookean forms). For comparison purposes, the exponential relation was chosen to exhibit much greater stiffening at higher strains beyond the range of the experimental data. We demonstrated that increased nonlinearity in the elastic response of the tissues leads to considerably higher (56%) estimated force required for delivery, accompanied by a more homogeneous spatial distribution of maximum principal stretch ratio across the muscle. These results indicate that the form of constitutive relation beyond the presently available experimental data markedly affects the estimated function of the levator ani muscle during vaginal delivery, due to the large strains that occur. Further experimental data at higher strains are necessary in order to more reliably characterize the constitutive behavior required for modeling vaginal childbirth.     

Subject specific finite elasticity simulations of the pelvic floor

An anatomically realistic computational model of the pelvic floor and anal canal regions was used in this study to examine the mechanics of normal defecatory function within the female pelvic floor. This subject specific, MRI-based model enabled mechanical simulations to be performed and quantitatively assessed against experimental data retrieved from the same volunteer. The levator ani muscle group mesh was used as the domain over which the governing equations of finite elasticity were solved using the finite element method with a Mooney-Rivlin material law. Deformation of the levator ani was simulated during a 'bear down' maneuver in order to visualize the way this muscle group functions in an asymptomatic subject. A pressure of 4 kPa was imposed on the mesh and the computed mesh displacements were compared to those obtained from dynamic MR images with an average, experimentally consistent, downwards displacement of 27.2 mm being achieved. The RMS error for this movement was 0.7 mm equating to a percentage error of 2.6% in the supero-inferior direction and 13.7 mm or 74.5% in the antero-posterior direction.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Analysis of pituitary gonadotropin concentration in blood serum and immunolocalization and immunoexpression of follicle stimulating hormone and luteinising hormone receptors in ovaries of postmenopausal women

The participation of gonadotropins in ovarian carcinogenesis is well known and is supported by studies with inhibition of pituitary gonadotropin secretion, which results in a diminished risk of cancer. However, there are few data on localization and expression of Follicle Stimulating Hormone and Luteinising Hormone Receptors (FSHR and LHR) in ovaries of healthy postmenopausal women, and their correlation with FSH and LH concentration in blood serum is unknown. The aim of our study was to analyze gonadotropin concentration in blood serum and the expression of FSHR and LHR in ovaries of 207 postmenopausal women. Patients included in the study were divided into three groups depending on the number of years since menopause. We analyzed the concentration of FSH and LH in blood serum and the expression of FSHR and LHR in ovaries. Ovaries of postmenopausal women showed numerous morphological changes in the cortex and medulla when compared to the structure of ovaries of women at reproductive age. In all groups of patients clefts in the surface epithelium and epithelial inclusion cysts were found. The concentration of FSH and LH in the blood serum of women studied increased significantly with time from menopause. Significant differences between analyzed menopausal groups were found. The highest FSH and LH concentration in blood serum were found in women with the longest period of time from menopause. Quantitatively similar expression of FSHR and LHR was found in ovarian surface epithelial cells, in epithelial inclusion cysts and in the connective tissue cells of ovarian stroma. The intensity of the immunohistochemical reaction decreased with time from menopause and with age.     

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.     

Subcellular distribution of aromatase in human placenta and ovary.

The aromatization of androstenedione in human ovarian microsomes is inhibited by an antibody to porcine hepatic microsomal NADPH-cytochrome c reductase. Likewise, the antibody inhibits aromatization in mitochondria isolated from human ovaries and placentae. A given quantity of the antibody produces the same percent inhibition of aromatization in microsomes and mitochondria of both ovaries and placentae. These data, in addition to the low specific activity observed for the mitochondrial aromatase, indicate that aromatization in mitochondria probably results from microsomal contamination.     

FSHR、LHR基因突变与多态对女性生殖的影响

卵泡刺激素(FSH)与黄体生成素(LH)促进性腺的发育和成熟、调节人类配子的产生,在生殖方面起着关键的作用。两受体(FSHR与LHR)的突变会导致生殖能力的改变,出现一系列临床症状,引起组织学,分子水平等多方面的变化,同时FSHR的多态性与卵巢反应性、女性的月经周期等许多方面相关。本文从多方面阐述FSHR与LHR对女性生殖的影响。     

Modeling the contraction of the pelvic floor muscles

We performed numerical simulation of voluntary contraction of the pelvic floor muscles to evaluate the resulting displacements of the organs and muscles. Structures were segmented in Magnetic Resonance (MR) images. Different material properties and constitutive models were attributed. The Finite Element Method was applied, and displacements were compared with dynamic MRI findings. Numerical simulation showed muscle magnitude displacement ranging from 0 to 7.9 mm, more evident in the posterior area. Accordingly, the anorectum moved more than the uterus and bladder. Dynamic MRI showed less 0.2 mm and 4.1 mm muscle dislocation in the anterior and cranial directions, respectively. Applications of this model include evaluating muscle impairment, subject-specific mesh implant planning, or effectiveness of rehabilitation.     

Management of disorders of the posterior pelvic floor

INTRODUCTION: Constipation is a relatively common problem affecting 15 percent of adults in the Western world, and over half of these cases are related to pelvic floor disorders. This article reviews the clinical presentation and diagnostic approach to posterior pelvic floor disorders, including how to image and treat them. METHODS: A Pubmed search using keywords "rectal prolapse," "rectocele," "perineal hernia," and "anismus" was performed, and bibliographies of the revealed articles were cross-referenced to obtain a representative cross-section of the literature, both investigational studies and reviews, that are currently available on posterior pelvic floor disorders. DISCUSSION: Pelvic floor disorders can occur with or without concomitant physical anatomical defects, and there are a number of imaging modalities available to detect such abnormalities in order to decide on the appropriate course of treatment. Depending on the nature of the disorder, operative or non-operative therapy may be indicated. CONCLUSION: Correctly diagnosing pelvic floor disorders can be complex and challenging, and the various imaging modalities as well as clinical history and exam must be considered together in order to arrive at a diagnosis.     

The management of episiotomy technique and its effect on pelvic floor muscles during a malposition childbirth

Vaginal childbirth is the leading cause of pelvic floor muscles injury, which contributes to pelvic floor dysfunction, being enhanced by fetal malposition. Therefore, the aim of the present study is to verify the influence of mediolateral episiotomies in the mechanics of the pelvic floor with the fetus in occiput posterior position when compared to the occiput anterior position. Numerical simulations of vaginal deliveries, with and without episiotomy, are performed based on the Finite Element Method. The biomechanical model includes the pelvic floor muscles, a surface to delimit the anterior region of the birth canal and a fetus. Fetal malposition induces greater extension of the muscle compared to the normal position, leading to increases of stretch. The faster enlargement may be responsible for a prolonged second stage of labor. Regarding the force required to achieve delivery, the difference between the analyzed cases are 35 N, which might justify the increased need of surgical interventions. Furthermore, episiotomy is essential in reducing the damage to values near the ones obtained with normal position, making the fetal position irrelevant. These biomechanical models have become extremely useful tools to provide some understanding of pelvic floor function during delivery helping in the development of preventative strategies.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.