3D ultrasound strain imaging of puborectal muscle with and without unilateral avulsion.

INTRODUCTION AND HYPOTHESIS: The puborectal muscle (PRM), one of the female pelvic floor (PF) muscles, can get damaged during vaginal delivery, leading to disorders such as pelvic organ prolapse. Current diagnosis involves ultrasound (US) imaging of the female PF muscles, but functional information is limited. Previously, we developed a method for strain imaging of the PRM from US images in order to obtain functional information. In this article, we hypothesize that strain in the PRM would differ from intact to the avulsed end. METHODS: We calculated strain in PRMs at maximum contraction, along their muscle fiber direction, from US images of two groups of women, which consisted of women with intact (n1 = 8) and avulsed PRMs (unilateral) (n2 = 10). Normalized strain ratios between both ends of the PRM (avulsed or intact) and the mid region were calculated. Subsequently, the difference in ratio between the avulsed and intact PRMs was determined. RESULTS: We observe from the obtained results that the contraction/strain pattern of intact and undamaged PRMs is different from PRMs with unilateral avulsion. Normalized strain ratios between avulsed and intact PRMs were statistically significant (p = 0.04). CONCLUSION: In this pilot study, we were able to show that US strain imaging of PRMs can show differences between intact PRMs and PRMs with unilateral avulsion.

Pessary fitting for pelvic organ prolapse: parameters associated with specific reasons for failure.

INTRODUCTION AND HYPOTHESIS: The objective was to assess if specific reasons for unsuccessful pessary fitting have different predictive parameters. METHODS: This is a prospective observational case-control study of women with symptomatic pelvic organ prolapse (POP) choosing pessary treatment. All women underwent an interview, clinical examination, and 3D/4D transperineal ultrasound (TPUS). Groups were defined based on fitting outcome: successful, pessary dislodgment, failure to relieve POP symptoms, pain/discomfort, increased/de novo urinary incontinence, or other reasons. Clinical, demographic, and TPUS parameters were assessed in the prediction of different reasons for unsuccessful fitting and receiver operating characteristic (ROC) curves were constructed. RESULTS: A total of 162 women were assessed and 130 were included. Levator hiatal area (HA) on maximum Valsalva divided by ring pessary size ("Valsalva HARP ratio") was a predictor of unsuccessful fitting (OR 3.00, 95% CI 1.15-7.81, p = 0.025) with an area under the ROC curve (AUC) of 0.62 (95% CI 0.50-0.74, p = 0.04). Predictors of pessary dislodgment were: complete avulsion (OR 24.20, 95% CI 2.46-237.84, p value 0.01) and Valsalva HARP ratio (OR 2.94, 95% CI 1.32-6.55, p value 0.01) with an area under the ROC curve (AUC) of 0.92 (95% CI 0.84-0.99, p = 0.00). No significant parameter was identified in the prediction of pain/discomfort. Solitary predominant posterior compartment POP was a predictor of failure to relieve POP symptoms (OR 20.00, 95% CI 3.48-115.02, p value 0.00; AUC 0.75, 95% CI 0.53-0.98, p = 0.03). CONCLUSION: Complete avulsion and a small ring pessary with respect to the levator HA in Valsalva are predictors of pessary dislodgment, whereas solitary predominant posterior compartment POP is a predictor of failure to relieve POP symptoms.

Parameters associated with unsuccessful pessary fitting for pelvic organ prolapse up to three months follow-up: a systematic review and meta-analysis.

OBJECTIVES: To clarify which parameters are associated with unsuccessful pessary fitting for pelvic organ prolapse (POP) at up to 3 months follow-up. METHODS: Embase, PubMed and Cochrane CENTRAL library were searched in May 2020. Inclusion criteria were: (1) pessary fitting attempted in women with symptomatic POP; (2) pessary fitting success among the study outcomes with a maximal follow-up of 3 months; (3) baseline parameters compared between successful and unsuccessful group. A meta-analysis was performed using the random effects model. MAIN RESULTS: Twenty-four studies were included in the meta-analysis. Parameters associated with unsuccessful pessary fitting were: age (OR 0.70, 95% CI 0.56-0.86); BMI (OR 1.35, 95% CI 1.08-1.70); menopause (OR 0.65 95% CI 0.47-0.88); de novo stress urinary incontinence (OR 5.59, 95% CI 2.24-13.99); prior surgery, i.e. hysterectomy (OR 1.88, 95% CI 1.48-2.40), POP surgery (OR 2.13, 95% CI 1.34-3.38), pelvic surgery (OR 1.81, 05% CI 1.01-3.26) and incontinence surgery (OR 1.87, 95% CI 1.08-3.25); Colorectal-Anal Distress Inventory-8 scores (OR 1.92, 95% CI 1.22-3.02); solitary predominant posterior compartment POP (OR 1.59, 95% CI 1.08-2.35); total vaginal length (OR 0.56, 95% CI 0.32-0.97); wide introitus (OR 4.85, 95% CI 1.60-14.68); levator ani avulsion (OR 2.47, 95% CI 1.35-4.53) and hiatal area on maximum Valsalva (OR 1.89, 95% CI 1.27-2.80). CONCLUSION: During counselling for pessary treatment a higher risk of failure due to the aforementioned parameters should be discussed and modifiable parameters should be addressed. More research is needed on the association between anatomical parameters and specific reasons for unsuccessful pessary fitting.

Transperineal ultrasound to estimate the appropriate ring pessary size for women with pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: The objective was to predict the successful ring pessary size based on the levator hiatal area (HA). METHODS: This is a prospective case-control study. Women with symptomatic pelvic organ prolapse (POP) choosing pessary treatment were included. All women underwent an interview, clinical examination, and 3D/4D transperineal ultrasound (TPUS). The ring pessary size used in each trial and the reason for unsuccessful trials were recorded. In addition, levator hiatal area divided by ring pessary size (HARP ratio) was measured at rest, maximum contraction, and maximum Valsalva. The HARP ratios of successful and unsuccessful trials were compared, receiver operating characteristic curves in the prediction of successful trials were constructed, and the cut-off optimizing sensitivity and specificity was identified. RESULTS: A total of 162 women were assessed and 106 were included with 77 successful trials, 49 unsuccessful trials owing to dislodgment or failure to relieve POP symptoms, and 20 unsuccessful trials owing to pain/discomfort. Rest HARP ratio and Valsalva HARP ratio were significantly smaller in the successful trials versus dislodgment/failure to relieve POP symptoms trials (mean rest HARP ratio [SD]: 2.93 [0.59] vs 3.24 [0.67], p = 0.021; median Valsalva HARP ratio (IQR): 4.65 (1.56) vs 5.32 (2.08), p = 0.004). No significant difference was observed between pain/discomfort trials and successful trials. The best cut-off for the prediction of successful trials was Valsalva HARP ratio ≤ 5.00. CONCLUSIONS: Unsuccessful fitting trials due to dislodgment/failure to relieve POP symptoms are associated with a small ring pessary with respect to the levator HA. A ring pessary that produces a Valsalva HARP ratio > 5.00 has a higher risk of dislodgment/failure to relieve POP symptoms.

The effect of pessary treatment on puborectalis muscle function.

INTRODUCTION AND HYPOTHESIS: The objective was to assess if puborectalis muscle (PRM) function changes in women with pelvic organ prolapse (POP) undergoing pessary treatment. METHODS: This was a prospective cohort study of women with symptomatic POP choosing pessary treatment. An interview, clinical examination and 3D/4D transperineal ultrasound were performed at baseline and at 3-month follow-up. POP was assessed using the Pelvic Organ Prolapse Quantification system (POPQ). Parameters compared between baseline and follow-up were: hiatal area at rest (HArest), maximal contraction (HActx), and maximal Valsalva maneuver (HAVal), displacement in contraction (DISPL-ctx, i.e., relative difference between HArest and HActx), and displacement in Valsalva (DISPL-Val, i.e., relative difference between and HAVal and HArest). Parameters were compared in women with and those without complete avulsion. RESULTS: A total of 162 women were assessed and 34 were included. Mean age was 64 years (SD 11.4), and mean BMI 24 kg/m2 (SD 3.1). Thirty-one women had a cystocele, 8 a uterine prolapse, and 12 had a posterior compartment prolapse. Twenty-one women (61.8%) had a POP stage II, and 13 (38.2%) a POP stage III. Ring pessaries were most frequently used (97%). In the entire group a statistically significant increase in DISPL-ctx was observed (mean difference 2.1%, p = 0.017). In the no avulsion group HArest and DISPL-ctx increased significantly (mean difference 4.1%, p = 0.016 and 2.7%, p = 0.016 respectively) and the increase in DISPL-ctx was higher than in the avulsion group (mean difference 2.7% vs 0.2%, p = 0.056). CONCLUSION: Our results show that PRM function changes in women with POP undergoing pessary treatment and suggest that such change occurs mainly in the absence of complete avulsion.

Hypoxia downregulates the expression of activating receptors involved in NK-cell-mediated target cell killing without affecting ADCC.

In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus-infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic environment by upregulating the hypoxia-inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK-cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL-2 (or other activating cytokines, including IL-15, IL-12, and IL-21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc-γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody-dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody-based immunotherapy of cancer.

Melanoma cells become resistant to NK-cell-mediated killing when exposed to NK-cell numbers compatible with NK-cell infiltration in the tumor.

During the past few years, a number of studies reported that different melanoma cell lines could be extensively lysed in vitro by IL-2-activated NK cells at appropriate effector/target ratios. Here, we show, by histological evaluation of different melanoma lesions, that NK/target-cell ratios compatible with those allowing efficient melanoma cell killing in vitro are hardly reached at the tumor site. We then investigated the outcome of cocultures established at low NK/melanoma cell ratios. After initial NK-mediated lysis, residual melanoma cells acquired resistance to IL-2-activated NK cells. This reflected primarily an increased expression, on melanoma cells, of classical and nonclassical HLA class I molecules, accompanied by a partial downregulation of NKG2D-ligands, and was dependent on NK-mediated IFN-γ release. Consistently, melanoma lesions showed a higher HLA class I expression on tumor cells that were proximal to infiltrating NK cells. In long-term cocultures, the "protective phenotype" acquired by melanoma cells was lost over time. However, this phenomenon was counteracted by downregulation of relevant activating receptors in cocultured NK cells. Analysis of different NK-cell-activating cytokines indicated that IL-15 can partially overcome this novel tumor escape mechanism suggesting that IL-15, rather than IL-2, may be eligible for NK-cell-based immunotherapy.

Unsupervised convolutional autoencoders for 4D transperineal ultrasound classification.

Purpose: 4D Transperineal ultrasound (TPUS) is used to examine female pelvic floor disorders. Muscle movement, like performing a muscle contraction or a Valsalva maneuver, can be captured on TPUS. Our work investigates the possibility for unsupervised analysis and classification of the TPUS data. Approach: An unsupervised 3D-convolutional autoencoder is trained to compress TPUS volume frames into a latent feature vector (LFV) of 128 elements. The (co)variance of the features are analyzed and statistical tests are performed to analyze how features contribute in storing contraction and Valsalva information. Further dimensionality reduction is applied (principal component analysis or a 2D-convolutional autoencoder) to the LFVs of the frames of the TPUS movie to compress the data and analyze the interframe movement. Clustering algorithms ( K -means clustering and Gaussian mixture models) are applied to this representation of the data to investigate the possibilities of unsupervised classification. Results: The majority of the features show a significant difference between contraction and Valsalva. The (co)variance of the features from the LFVs was investigated and features most prominent in capturing muscle movement were identified. Furthermore, the first principal component of the frames from a single TPUS movie can be used to identify movement between the frames. The best classification results were obtained after applying principal component analysis and Gaussian mixture models to the LFVs of the TPUS movies, yielding a 91.2% accuracy. Conclusion: Unsupervised analysis and classification of TPUS data yields relevant information about the type and amount of muscle movement present.

Tissue Characterization of Puborectalis Muscle From 3-D Ultrasound.

Pelvic floor (PF) muscles have the role of preventing pelvic organ descent. The puborectalis muscle (PRM), which is one of the female PF muscles, can be damaged during child delivery. This damage can potentially cause irreversible muscle trauma and even lead to an avulsion, which is disconnection of the muscle from its insertion point, the pubic bone. Ultrasound imaging allows diagnosis of such trauma based on comparison of geometric features of a damaged muscle with the geometric features of a healthy muscle. Although avulsion, which is considered severe damage, can be diagnosed, microdamage within the muscle itself leading to structural changes cannot be diagnosed by visual inspection through imaging only. Therefore, we developed a quantitative ultrasound tissue characterization method to obtain information on the state of the tissue of the PRM and the presence of microdamage in avulsed PRMs. The muscle was segmented as the region of interest (ROI) and further subdivided into six regions of interest (sub-ROIs). Mean echogenicity, entropy and shape parameter of the statistical distribution of gray values were analyzed on two of these sub-ROIs nearest to the bone. The regions nearest to the bones are also the most likely regions to exhibit damage in case of disconnection or avulsion. This analysis was performed for both the muscle at rest and the muscle in contraction. We found that, for PRMs with unilateral avulsion compared with undamaged PRMs, the mean echogenicity (p = 0.02) and shape parameter (p < 0.01) were higher, whereas the entropy was lower (p < 0.01). This method might be applicable to quantification of PRM damage within the muscle.

Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity.

Although the role of the tumor microenvironment in the process of cancer progression has been extensively investigated, the contribution of different stromal components to tumor growth and/or evasion from immune surveillance is still only partially defined. In this study we analyzed fibroblasts derived from metastatic melanomas and provide evidence for their strong immunosuppressive activity. In coculture experiments, melanoma-derived fibroblasts sharply interfered with NK cell functions including cytotoxicity and cytokine production. Thus, both the IL-2-induced up-regulation of the surface expression of NKp44, NKp30, and DNAM-1 triggering receptors and the acquisition of cytolytic granules were inhibited in NK cells. This resulted in an impairment of the NK cell-mediated killing of melanoma target cells. Transwell cocultures and the use of specific inhibitors suggested that cell-to-cell contact was required for inducing DNAM-1 modulation. In contrast, modulation of NKp44 and NKp30 was due to PGE(2) released by fibroblasts during coculture. Normal skin fibroblasts could also partially affect NK cell phenotype and function. However, the inhibitory effect of tumor-derived fibroblasts was far stronger and directly correlated with their ability to produce PGE(2) either constitutively or upon induction by NK cells.

Appearance of the levator ani muscle subdivisions on 3D transperineal ultrasound.

BACKGROUND: The levator ani muscle (LAM) consists of different subdivisions, which play a specific role in the pelvic floor mechanics. The aim of this study is to identify and describe the appearance of these subdivisions on 3-Dimensional (3D) transperineal ultrasound (TPUS). To do so, a study designed in three phases was performed in which twenty 3D TPUS scans of vaginally nulliparous women were assessed. The first phase was aimed at getting acquainted with the anatomy of the LAM subdivisions and its appearance on TPUS: relevant literature was consulted, and the TPUS scan of one patient was analyzed to identify the puborectal, iliococcygeal, puboperineal, pubovaginal, and puboanal muscle. In the second phase, the five LAM subdivisions and the pubic bone and external sphincter, used as reference structures, were manually segmented in volume data obtained from five nulliparous women at rest. In the third phase, intra- and inter-observer reproducibility were assessed on twenty TPUS scans by measuring the Dice Similarity Index (DSI). RESULTS: The mean inter-observer and median intra-observer DSI values (with interquartile range) were: puborectal 0.83 (0.13)/0.83 (0.10), puboanal 0.70 (0.16)/0.79 (0.09), iliococcygeal 0.73 (0.14)/0.79 (0.10), puboperineal 0.63 (0.25)/0.75 (0.22), pubovaginal muscle 0.62 (0.22)/0.71 (0.16), and the external sphincter 0.81 (0.12)/0.89 (0.03). CONCLUSION: Our results show that the LAM subdivisions of nulliparous women can be reproducibly identified on 3D TPUS data.

3D Ultrasound Strain Imaging of Puborectalis Muscle.

The female pelvic floor (PF) muscles provide support to the pelvic organs. During delivery, some of these muscles have to stretch up to three times their original length to allow passage of the baby, leading frequently to damage and consequently later-life PF dysfunction (PFD). Three-dimensional (3D) ultrasound (US) imaging can be used to image these muscles and to diagnose the damage by assessing quantitative, geometric and functional information of the muscles through strain imaging. In this study we developed 3D US strain imaging of the PF muscles and explored its application to the puborectalis muscle (PRM), which is one of the major PF muscles.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells.

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133(+) cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

The emerging role of HLA-E-restricted CD8+ T lymphocytes in the adaptive immune response to pathogens and tumors.

Human leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule of limited sequence variability that is expressed by most tissues albeit at low levels. HLA-E has been first described as the ligand of CD94/NKG2 receptors expressed mainly by natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. However, recent evidences obtained by our and other groups indicate that HLA-E complexed with peptides can interact with alphabeta T-cell receptor (TCR) expressed on CD8(+) T cells. Although, HLA-E displays a selective preference for nonameric peptides, derived from the leader sequence of various HLA class I alleles, several reports indicate that it can present also "noncanonical" peptides derived from both stress-related and pathogen-associated proteins. Because HLA-E displays binding specificity for innate CD94/NKG2 receptors, as well as all the features of an antigen-presenting molecule, its role in both natural and acquired immune responses has recently been re-evaluated.

Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration.

Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues.

Cytokines can counteract the inhibitory effect of MEK-i on NK-cell function.

Oncogene-targeted therapies based on mutated BRAF- and/or MEK-specific inhibitors have been developed for melanoma treatment. Although these drugs induce tumor regression in a high percentage of patients, clinical responses are frequently limited in time and tumors often recur. Recent studies suggested that the combination of BRAF/MEK inhibition with immunotherapy could represent a promising strategy for the cure of melanoma. NK cells are suitable effectors for tumor immunotherapy. Here we show that PLX4032 (a mutant BRAFV600 inhibitor) had no effect on the functional properties of NK cells cultured in the presence of IL-2 or IL-15. In contrast, PD0325901 (a MEK inhibitor) induced the down-regulation of the main activating NK receptors and inhibited NK cell function. Importantly, PD0325901 did not affect the anti-tumor activity of NK cells that had been exposed to a combination of IL-15 and IL-18. In addition, both PLX4032 and PD0325901 did not exert any inhibitory effect on in vitro IL-2 or IL-15 pre-activated NK cells.Our data may provide a rationale for future clinical protocols that combine IL-15/IL-18 cytokine administration with MEK inhibitors. In addition, they suggest that oncogene-targeting drugs are compatible with NK-based adoptive therapy.

Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity.

Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.