Bovine herpesviruses induce different cell death forms in neuronal and glial-derived tumor cell cultures.

Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40 % of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40 % in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80 % of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

Modified pubovaginal sling technique in the surgical management of female stress urinary incontinence / Técnica modificada do sling pubovaginal no tratamento cirúrgico da incontinência urinária de esforço feminina

Objective: To assess the application of aponeurotic sling by a modified technique with direct visualization of needles in patients with stress urinary incontinence. Methods: we applied the Kings Health Questionnaire (KHQ) for quality of life, gynecological examination, urinalysis I and urine culture approximately seven days prior to the urodynamic study (UDS) and the one-hour PAD test in patients undergoing making aponeurotic sling with its passing through the retropubic route with direct visualization of the needle, PAD test and King's Helth Questionnaire before and after surgery. Results: The mean age was 50.6 years, BMI of 28 and Leak Pressure (LP) 58,5cm H2O; 89% were Caucasian. Forty-six of them were monitored for three and six months, 43 for 12 months. The objective cure rate at 12 months postoperatively was approximately 93.5%. In evaluating quality of life, we observed a significant improvement in 12 months postoperatively compared with the preoperative period. There was no no urethral/bladder injury. As adverse results, we had one persistent urinary retention (2.3%), who was submitted to urethrolysis, currently without incontinence. Conclusion: The proposed procedure is safe as for the risk of bladder or urethral injuries, promoting significant improvement in quality of life and objective cure.

Objetivo: avaliar a aplicação de faixa aponeurótica por técnica modificada com visibilização direta das agulhas em pacientes com incontinência urinária de esforço. Métodos: foi aplicado o questionário Kings Health Questionaire (KHQ) de qualidade de vida, exame ginecológico, exame de urina I e urocultura aproximadamente sete dias antes da realização do estudo urodinâmico (EUD) e PAD test de uma hora submetidas à confecção de faixa aponeurótica com passagem de faixa pela via retropúbica, com agulha sob visibilização direta, PAD test e King´s Helth Questionaire, no pré e pós operatórios. Resultados: a média de idade foi 50,6 anos, 89% da cor branca, IMC de 28 e PPE de 58,5cm de H2O. Quarenta e seis delas tiveram acompanhamento de três e seis meses, 43 de 12 meses. A taxa de cura objetiva, em 12 meses de pós-operatório foi aproximadamente 93,5%. Ao avaliarmos a qualidade de vida das pacientes, observamos a melhora significante em 12 meses de pós-operatório, quando comparada ao pré-operatório. Não foi observada nenhuma lesão uretral/vesical. Como resultados adversos tivemos uma retenção urinária persistente (2,3%), sendo submetida à uretrolíse, estando atualmente sem perda. Conclusão: a operação proposta é segura quanto ao risco de lesões vesicais ou uretrais, promovendo melhora acentuada na qualidade de vida e cura objetiva.

Modified pubovaginal sling technique in the surgical management of female stress urinary incontinence.

OBJECTIVE: To assess the application of aponeurotic sling by a modified technique with direct visualization of needles in patients with stress urinary incontinence. METHODS: we applied the Kings Health Questionnaire (KHQ) for quality of life, gynecological examination, urinalysis I and urine culture approximately seven days prior to the urodynamic study (UDS) and the one-hour PAD test in patients undergoing making aponeurotic sling with its passing through the retropubic route with direct visualization of the needle, PAD test and King's Health Questionnaire before and after surgery. RESULTS: The mean age was 50.6 years, BMI of 28 and Leak Pressure (LP) 58,5 cm H2O; 89% were Caucasian. Forty-six of them were monitored for three and six months, 43 for 12 months. The objective cure rate at 12 months postoperatively was approximately 93.5%. In evaluating quality of life, we observed a significant improvement in 12 months postoperatively compared with the preoperative period. There was no no urethral/bladder injury. As adverse results, we had one persistent urinary retention (2.3%), who was submitted to urethrolysis, currently without incontinence. CONCLUSION: The proposed procedure is safe as for the risk of bladder or urethral injuries, promoting significant improvement in quality of life and objective cure.

Manifestation of estrous behavior and subsequent progesterone concentration at timed-embryo transfer in cattle are positively associated with pregnancy success of recipients.

Plasma estradiol and progesterone (P4) concentrations during the peri-ovulatory period are positively correlated with pregnancy success in cattle. The aims of this study were to assess the effects of estrus occurrence and early diestrus P4 concentrations on pregnancy per timed-embryo transfer (P/TET). A total of 267 crossbred beef heifers [222 with corpus luteum (CL) and 45 without a CL but with a follicle >8mm at beginning of the estrous synchronization protocol) received an intra-vaginal P4 device and intramuscular administration of estradiol benzoate. Progesterone devices were removed 8 days later (Day 0), and heifers received d-cloprostenol, eCG and estradiol cypionate. Estrous behavior was monitored twice daily for 3 days after P4 device removal. Plasma P4 concentration was measured by radioimmunoassay at Day 7 and Day 9. At Day 9, heifers with a CL (n=236; i.e. submission rate of 85.5%; 236/276) undergoing TET received an in vitro-produced embryo. Heifers expressing a standing behavioral estrus had a greater P/TET than heifers that did not express a standing estrus [62.4% (106/170) compared with 47.0% (31/66)]. The probability of pregnancy was positively correlated with plasma P4 concentration at TET. When heifers were grouped by quartiles of P4 concentration at TET (Q1=0.64±0.16, Q2=1.70±0.04, Q3=2.90±0.07 and Q4=5.52±0.27ng/mL) the P/TET were 45.8% (Q1; 27/59)(c), 52.25% (Q2; 31/59)(bc), 66.1% (Q3; 39/59)(ab) and 67.8% (Q4; 40/59)(a). Additionally, heifers that became pregnant had greater P4 concentrations at TET (2.87±0.16ng/mL; n=137) than heifers that did not become pregnant (2.45±0.24ng/mL; n=99). No statistical difference was observed regarding P4 concentrations on Day 7, regardless of standing estrus or pregnancy status. In cattle, manifestation of estrous behavior and plasma P4 concentration at TET increase the probability of pregnancy in in vitro-produced embryo recipients.

Programmed cell death-associated gene transcripts in bovine embryos exposed to bovine Herpesvirus type 5.

In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MTT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos.

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.

Susceptibility of neuron-like cells derived from bovine Wharton's jelly to bovine herpesvirus type 5 infections.

BACKGROUND: Bovine herpesvirus type 5 (BoHV-5), frequently lethal in cattle, is associated with significant agricultural economic losses due to neurological disease. Cattle and rabbits are frequently used as models to study the biology and pathogenesis of BoHV-5 infection. In particular, neural invasion and proliferation are two of the factors important in BoHV-5 infection. The present study investigated the potential of bovine Wharton's jelly mesenchymal stromal cells (bWJ-MSCs) to differentiate into a neuronal phenotype and support robust BoHV-5 replication. RESULTS: Upon inducing differentiation within a defined neuronal specific medium, most bWJ-MSCs acquired the distinctive neuronal morphological features and stained positively for the neuronal/glial markers MAP2 (neuronal microtubule associated protein 2), N200 (neurofilament 200), NT3 (neutrophin 3), tau and GFAP (glial fibrillary acidic protein). Expression of nestin, N200, ß-tubulin III (TuJI) and GFAP was further demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR). Following BoHV-5 inoculation, there were low rates of cell detachment, good cell viability at 96 h post-infection (p.i.), and small vesicles developed along neuronal branches. Levels of BoHV-5 antigens and DNA were associated with the peak in viral titres at 72 h p.i. BoHV-5 glycoprotein C mRNA expression was significantly correlated with production of progeny virus at 72 h p.i. (p < 0.05). CONCLUSION: The results demonstrated the ability of bWJ-MSCs to differentiate into a neuronal phenotype in vitro and support productive BoHV-5 replication. These findings constitute a remarkable contribution to the in vitro study of neurotropic viruses. This work may pave the way for bWJ-MSCs to be used as an alternative to animal models in the study of BoHV-5 biology.

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5.

BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system.

BACKGROUND: The possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton's jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. RESULTS: Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline(®) mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105(+), CD29(+), CD73(+) and CD90(+) in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells was included in the culture which demonstrated the immunossupression profile typically observed among isolated mesenchymal cells from other species. After classified the UC-WJ cells as mesenchymal stromal phenotype the in vitro 3D cultures was performed using the AlgiMatrix(®) protocol. Based on the size of spheroids (283,07 µm ± 43,10 µm) we found that three weeks of culture was the best period to growth the UC-WJ cells on 3D dimension. The initial cell density was measured and the best value was 1.5 × 10(6) cells/well. CONCLUSIONS: We described for the first time the isolation and characterization of UC-WJ cells in a serum-free condition and maintenance of primitive mesenchymal phenotype. The culture was stable under 60 consecutive passages with no genetic abnormalities and proliferating ratios. Taken together all results, it was possible to demonstrate an easy way to isolate and culture of bovine-derived UC-WJ cells under 2D and 3D serum-free condition, from fetal adnexa with a great potential in cell therapy and biotechnology.

Bull semen variables after experimental exposure with Bovine Herpesvirus type 5.

The influence of Bovine Herpesvirus type 5 (BoHV-5) infection on semen variables and sperm morphology collected from healthy bulls with no reproductive disorder was evaluated in ten ejaculates distributed into two experimental groups: group I, bull semen exposed to 10(2.3) (tissue culture infectious dose) TCID(50)/50 µl of a Brazilian strain of BoHV-5 (US9/BR/2007; GU9457818) and group II, unexposed bull control semen. After experimental infection, the semen was frozen-thawed prior to computerized analysis (CASA) of sperm motility and movement. Also analyzed were sperm phosphatidylserine transposition, acrosomal integrity, mitochondrial function, plasma membrane integrity and Annexin V expression. Viable BoHV-5 particles and their DNA were detected in infected semen after virus isolation and in situ hybridization (ISH) assay. The ISH revealed the BoHV-5 US9 gene in the acrosome and tail of infected spermatozoa. The only remarkable differences between groups I and II were the sperm kinetic variables, whereby infected sperm had a lesser mean velocity (VAP) and curvilinear velocity (VCL) values as compared to controls (P≤0.05). However, the straightness coefficient (STR) and beat cross frequency (BCF) values were higher in infected sperm. These results indicate that BoHV-5 can be found in infected sperm but induces no functional and morphological damage even after freeze-thawing, and, importantly, BoHV-5 can be spread via in vitro and in vivo reproductive biotechnology procedures.

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 µl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

A novel in situ polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues.

An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3'-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.

Estudo urodinâmico da pressão de perda ao esforço, nas posições ortostática e sentada, em mulheres com incontinência urinária / Urodynamic evaluation of leak point pressure under stress, in orthostatic and seating position, in women with urinary incontinency

OBJETIVOS: comparar os resultados obtidos durante o estudo urodinâmico realizado em duas diferentes posições em relação às pressões de perda urinária sob esforço e discutir sua relevância clínica. MÉTODOS: sessenta e quatro pacientes com queixa de incontinência urinária de esforço (IUE) com idades variando entre 25 e 80 anos, atendidas no ambulatório de uroginecologia e cirurgia vaginal, no período de junho 2003 a setembro 2005 foram incluídas neste estudo. As pacientes foram inicialmente submetidas ao estudo urodinâmico de acordo com a técnica preconizada pela International Continency Society (ICS) na posição ortostática e logo depois foram avaliadas na posição sentada. RESULTADOS: diferença significante foi obtida após a avaliação das pressões de perda obtidas nas diferentes posições (99,8 ± 33,3 versus 102,9 ± 32,4; respectivamente, posição sentada e em pé, p < 0,05). Testes de regressão linear com análise de freqüência foram realizados com a finalidade de verificar a porcentagem de pacientes que ficaram dentro dos limites de confiança em relação às PP nas posições sentada e em pé. Uma taxa de 90,6 por cento de compatibilidade foi obtida nesses resultados. Quando três unidades foram somadas aos valores das pressões obtidas no estudo urodinâmico realizado na posição sentada, percebeu-se que 92,2 por cento ficaram inseridas neste intervalo. CONCLUSÕES: estes achados sugerem que o estudo urodinâmico pode ser realizado na posição sentada sem comprometimento diagnóstico e terapêutico proporcionando maior conforto e comodidade às pacientes.

PURPOSE: compare the outcomes verified during urodynamic investigation realized in two different positions related to urinary leak point pressure under stress and to discuss its clinical relevance. METHODS: sixty-four patients with stress urinary incontinency (SUI) aged 25-80 years old, attended, during June 2003 to September 2005 were included in this study. Patients were initially submitted to urodynamic investigation in accordance with International Continence Society (ICS) techniques in orthostatic position and just after were evaluated in seating position. RESULTS: statistical significance was obtained after evaluation of Vasalva leak point pressure (VLPP) obtained in two positions (99,8 ± 33,3 versus 102,9 ± 32,4; respectivamente, posição sentada e em pé, p<0,05). Linear regression test based on frequency analyses was applied with the purpose to verify the patient percentage allocated in confidence interval in terms of Valsalva leak point pressure in seating or orthostatic positions. A rate of 90.6 percent of compatibility was gotten in these results. When three unities were added to VLPP values after urodynamic investigation in seating position, it was noted that 92.2 percent of patients was included in this interval. CONCLUSIONS: these findings suggest that the urodynamic investigation can be realized in seating position without diagnostic a therapeutic impairment allowing higher comfort to the patients.

Optimizing pelvic organ prolapse research.

For many years, researchers on this field have suffered from the lack of an efficient method for describing pelvic organ prolapse. Struggling to solve this problem, the International Continence Society has proposed a pelvic organ prolapse quantification (POP-Q) system [Bump RC, Mattiasson A, Bo K, Brubaker LP, DeLancey JO, Klarskov P, Shull B, Smith ARB, Am J Obstet Gynecol, 175(1):1956-1962, 1996], which was validated as a precise and reproducible technique for describing pelvic organ position. However, even though very precise at describing pelvic organ position, our critic to this system is its limited ability to quantify the prolapse itself, since it still classifies prolapse into four grades, almost the same way as Baden and Walker did in 1972. As a result, the same grade can include a wide prolapse intensity range. The objective of this paper is to propose a method that makes POP research more efficient by directly measuring prolapse as a continuous variable that requires lesser number of subjects in order to achieve statistical significance.