Hysteroscopic Sterilization Device Follow-Up Rate: Hysterosalpingogram Versus Transvaginal Ultrasound

Study Objective To assess if follow-up confirmation testing 3 months after hysteroscopic sterilization with the Essure device improved with recommendation for transvaginal ultrasound (TVUS) versus hysterosalpingogram (HSG). Design Retrospective, observational case-controlled cohort study (Canadian Task Force classification II-2) Setting Two physician private practice in Evansville, Indiana Patients Compliance rates for a TVUS confirmation test on 100 women who underwent hysteroscopic sterilization compared to a previously published cohort of 1004 women who were scheduled to undergo HSG confirmation test. Intervention Acquisition of 3 month confirmation testing after Essure hysteroscopic sterilization Measurement and Main Results All women who underwent Essure hysteroscopic sterilization with recommendation for TVUS confirmation testing between July 2015 and January 2017 were compared to a previously published cohort of 1004 patients with recommendation for HSG confirmation testing (HSG cohort). In addition, an HSG subgroup cohort (HSG subgroup) similar in size and closest chronology to the TVUS cohort was drawn from the original 1004 patients and analyzed for HSG follow-up. Records for all patients were reviewed for demographic, procedural, confirmation testing, and outcome data. One hundred patients were identified with successful Essure device placement and a recommendation for TVUS confirmation testing. Eighty-eight (88.0%) patients returned for TVUS at 3 months. In the HSG cohort, 1004 successful Essure devices were placed and 778 patients returned for the recommended HSG follow-up (77.5%). There was a significantly higher follow-up rate for TVUS compared to the HSG cohort (88.0% vs 77.5%, p = 0.008). In the HSG subgroup, 184 patients were identified and 133 patients presented for HSG follow-up (72.3%) indicating a significantly higher follow-up rate in the TVUS cohort (88.0% vs 72.3%, p = 0.001). No pregnancies after any confirmation testing were noted. Conclusion Confirmation testing with transvaginal ultrasound rather than hysterosalpingogram 3 months after Essure device placement results in increased patient compliance that may lead to improved patient outcomes with reduction of unintended pregnancy

The Phosphate/Amide I ratio is Reduced by in vitro Glycation and may Correlate with Fracture Toughness

poster abstractIntroduction: Advanced glycation end products (AGEs) form when reducing sugars react with proteins. In bone AGEs can form in type I collagen which results in non-enzymatically derived crosslinks. While enzymatic crosslinks play an important role in strengthening the collagen matrix, non-enzymatic crosslinks are believed to reduce toughness. AGEs accumulate in bone over time and play an important role in reducing bone quality particularly in aging and diabetic patients who accumulate AGEs more rapidly due to increases in circulating glucose. Non-enzymatic glycation of bone can be modeled experimentally by soaking samples in a sugar solution which allows decades worth of AGE accumulation to occur in a short time. AGEs are primarily measured using fluorescence measurements or high performance liquid chromatography (HPLC). Spectroscopic techniques have been developed to determine enzymatic crosslinking maturity by detecting perturbations in collagen structure in the Amide I region and it may be possible to detect similar changes caused by AGEs. We hypothesized that the formation of AGEs in collagen would perturb the Amide I band of Raman spectra causing changes to the mineral to matrix ratio (MMR) which would correlate with AGE-induced mechanical changes in an in vitro ribose soaking experiment. If changes due to non-enzymatic glycation can be detected in the Amide I band, Raman spectroscopic techniques could be developed to assess the presence of AGEs in a non-destructive and widely available manner. Methods: Five femurs were harvested from male hounds from a previous IACUC approved study. From the mid-diaphysis, six beams ~1.4 x 4 x 24 mm were sectioned from each bone. Two beams from each sample were randomly assigned to one of three groups. One of those beams was sanded to 1.4 x 2 x 20 mm for fracture toughness testing while the other was used for Raman spectroscopy and Reference Point Indentation (RPI). All beams were soaked for 14 consecutive days at 37°C in solutions containing 1% Pen-Strep, 1.3mM CaCl2 and either no ribose (Control), 0.2M ribose (Low), or 0.6M ribose (High) in Hank’s Balanced Salt Solution with solutions changed every other day. After soaking, a notch was started in the sanded beam with a diamond wire sectioning saw and then sharpened by hand with a razor using a 1μm diamond suspension. Notched beams were submerged in fluid and loaded in displacement control to 0.03mm, unloaded to 0.015mm, held for 10s, then cycled until failure with a 0.05mm load, a 0.02 unload, and a 10s hold. J-R curves were calculated using ASTM E1820-5a to obtain initiation stress intensity factor (KIc) and maximum stress intensity factor (Kmax). Raman spectra were acquired at five points along the length of the second beam using a LabRAM HR 800 with a 660nm laser focused to a spot size of ~10μm. After baseline correction, OriginPro 8.6 was used to calculate MMR as the area of the PO43- ν1 peak over the area of the Amide I band. Following Raman spectroscopy, co-localized RPI was performed at each Raman location using 10 cycles of a 5N force at 2Hz. One-way ANOVA tested mean differences between samples. Pearson product-moment correlation coefficients were calculated between MMR and parameters from RPI and fracture toughness. All values are presented as mean ± standard deviation and all statistics were carried out using SAS 9.4. Results: Raman spectroscopy and RPI were not performed on one sample from the Low group. Data were not available for one Control sample and Kmax was excluded for one High sample. Neither KIc nor Kmax were significantly different between groups (Control: 6.59 ± 0.42, 13.55 ± 1.38 MPa√m; Low: 6.19 ± 1.98, 14.80 ± 2.00 MPa√m; High: 6.84 ± 1.18, 15.25 ± 2.35 MPa√m). MMR was significantly different between groups (p=0.039). Tukey HSD post-hoc tests revealed that Control (2.45 ± 0.37) was significantly greater than High (1.85 ± 0.20) while Low was intermediate (2.18 ± 0.37) but not significantly different. No significant differences were observed with RPI. A weak positive correlation was observed between average creep indentation increase (CID) and MMR (R2=0.079, p=0.0185) but no other RPI measurements were correlated with MMR. Two influential points, determined by a Cook’s distance > 4/n, were excluded from the regression KIc to MMR. A mild trend was observed between KIc and MMR but the fit did not reach significance (R2=0.334, p=0.0628). Discussion: Because samples were all from the same 5 animals and randomized into groups, any differences between groups arose from the soaking in solutions of different concentrations of ribose. AGEs were not measured to confirm the expected dose-dependent increase, but noticeable browning occurred in the High group which was less pronounced in the Low group and not present in Control. The soaking protocol and ribose concentrations were chosen based on previous literature showing increases in AGEs. Therefore, we are confident changes noted here are due to the presence of AGEs and the resulting non-enzymatic crosslinks. Because soaking was performed in appropriately buffered solutions, decreased MMR in the High group relative to Control are expected to occur due to glycation of collagen rather than changes in mineral content. We suspect that perturbations in collagen structure due to the presence of non-enzymatic crosslinks are causing the differences in the area of the Amide I band between groups. Given the changes in MMR with glycation, future studies investigating models where AGEs are likely present should be cautious in their interpretation of MMR if it is not supported by other measures of mineralization. The lack of significant differences between groups for RPI and fracture toughness parameters may be due to the small sample size (n=4-5 per group) and biological variations associated with mechanical techniques. However, the sample size was adequate to assess correlations between Raman and RPI due to the co-localized measurements in each sample (n=70). The positive correlation between CID and MMR was expected given AGEs have been shown to reduce creep behavior and since MMR is decreased by AGEs. However, the correlation is weak which is likely due to the overall small non-significant effect in CID compared to its variation. The correlation between MMR and initiation toughness similarly suggests that as AGEs reduce MMR, KIc decreases which is known to occur with glycation. While the correlation did not reach significance (p=0.063), the trend is compelling given the small sample size (n=11) and the use of Raman data from an adjacent beam from the same sample rather than the beam used to measure KIc. In conclusion, MMR changes in response to in vitro glycation and these changes are correlated to CID and possibly to KIc. Deconvolution of the Amide I region into sub-peaks to determine which peak(s) are altered in the presence of AGEs is an important next step to developing a spectroscopic technique that can assess the presence of AGEs and is recommended in future work. Significance: Correlations were performed between Raman spectroscopy, Reference Point Indentation, and fracture toughness measurements to evaluate the ability of perturbations in the Amide I band to explain glycation-induced changes in tissue mechanics. Non-enzymatic glycation is an important determinant of bone quality especially in aging and diabetic patients and understanding the specific roles composition and microscale mechanics play in determining how non-enzymatic glycation affects fracture toughness may lead to new therapeutic targets

Incorporating tissue anisotropy and heterogeneity in finite element models of trabecular bone altered predicted local stress distributions

Trabecular bone is composed of organized mineralized collagen fibrils, which results in heterogeneous and anisotropic mechanical properties at the tissue level. Recently, biomechanical models computing stresses and strains in trabecular bone have indicated a significant effect of tissue heterogeneity on predicted stresses and strains. However, the effect of the tissue-level mechanical anisotropy on the trabecular bone biomechanical response is unknown. Here, a computational method was established to automatically impose physiologically relevant orientation inherent in trabecular bone tissue on a trabecular bone microscale finite element model. Spatially varying tissue-level anisotropic elastic properties were then applied according to the bone mineral density and the local tissue orientation. The model was used to test the hypothesis that anisotropy in both homogeneous and heterogeneous models alters the predicted distribution of stress invariants. Linear elastic finite element computations were performed on a 3 mm cube model isolated from a microcomputed tomography scan of human trabecular bone from the distal femur. Hydrostatic stress and von Mises equivalent stress were recorded at every element, and the distributions of these values were analyzed. Anisotropy reduced the range of hydrostatic stress in both tension and compression more strongly than the associated increase in von Mises equivalent stress. The effect of anisotropy was independent of the spatial redistribution high compressive stresses due to tissue elastic heterogeneity. Tissue anisotropy and heterogeneity are likely important mechanisms to protect bone from failure and should be included for stress analyses in trabecular bone

Treadmill Exercise Improves Fracture Toughness and Indentation Modulus without Altering the Nanoscale Morphology of Collagen in Mice.

The specifics of how the nanoscale properties of collagen (e.g., the crosslinking profile) affect the mechanical integrity of bone at larger length scales is poorly understood despite growing evidence that collagen’s nanoscale properties are altered with disease. Additionally, mass independent increases in postyield displacement due to exercise suggest loading-induced improvements in bone quality associated with collagen. To test whether disease-induced reductions in bone quality driven by alterations in collagen can be rescued or prevented via exercise-mediated changes to collagen’s nanoscale morphology and mechanical properties, the effects of treadmill exercise and β-aminopropionitrile treatment were investigated. Eight week old female C57BL/6 mice were given a daily subcutaneous injection of either 164 mg/kg β-aminopropionitrile or phosphate buffered saline while experiencing either normal cage activity or 30 min of treadmill exercise for 21 consecutive days. Despite differences in D-spacing distribution (P = 0.003) and increased cortical area (tibial: P = 0.005 and femoral: P = 0.015) due to β-aminopropionitrile treatment, an overt mechanical disease state was not achieved as there were no differences in fracture toughness or 4 point bending due to β-aminopropionitrile treatment. While exercise did not alter (P = 0.058) the D-spacing distribution of collagen or prevent (P < 0.001) the β-aminopropionitrile-induced changes present in the unexercised animals, there were differential effects in the distribution of the reduced elastic modulus due to exercise between control and β-aminopropionitrile-treated animals (P < 0.001). Fracture toughness was increased (P = 0.043) as a main effect of exercise, but no significant differences due to exercise were observed using 4 point bending. Future studies should examine the potential for sex specific differences in the dose of β-aminopropionitrile required to induce mechanical effects in mice and the contributions of other nanoscale aspects of bone (e.g., the mineral–collagen interface) to elucidate the mechanism for the exercise-based improvements in fracture toughness observed here and the increased postyield deformation observed in other studies

Even With Rehydration, Preservation in Ethanol Influences the Mechanical Properties of Bone and How Bone Responds to Experimental Manipulation

Typically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0M or 0.6M ribose for 1week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (-16.3%), stiffness (+24.5%), strain to yield (-13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%-68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation)

Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function

Surficial Geologic Map of the Flaherty 7.5-Minute Quadrangle, Kentucky

The Flaherty 7.5-minute quadrangle is located southwest of Louisville and northwest of Elizabethtown along the boundary between Hardin and Meade Counties. The quadrangle includes mostly the Pennyroyal region of the Mississippian Plateau and also smaller areas of the Mammoth Cave plateau and the highly dissected Dripping Springs escarpment in the western half of the map area (McDowell, 1986). Topography is mostly characterized by pervasive sinkhole development in a lower elevation and low-relief plain, and high-relief plateaus, ridges, and knobs of the Dripping Springs escarpment scattered along the west side of the quadrangle. Swadley (1963) mapped the bedrock geology of the quadrangle, which was later digitized by Crawford (2002). Mississippian bedrock is exposed throughout the quadrangle and is cut by several normal faults in the south. The St. Louis Limestone and overlying Ste. Genevieve Limestone are the oldest and lowest (stratigraphy and elevation) map units in Flaherty, and underlie the Pennyroyal region. The higher elevation landforms characterizing the Dripping Springs escarpment are predominantly underlain by the Paoli Limestone, Beaver Bend Limestone, and Sample Sandstone, from oldest to youngest respectively. The Mooretown Formation is stratigraphically above the Paoli Limestone and below the Beaver Bend Limestone, and is only exposed along Sand Ridge, a prominent landform in the quadrangle trending northeast to southwest. Previously mapped surficial deposits include alluvium in Otter Creek, Flippin Creek, and a large karst basin, and “slumped sandstone” (colluvium) along Sand Ridge and other smaller areas throughout the quadrangle (Swadley, 1963)

Surficial Geologic Map of the Big Clifty 7.5-Minute Quadrangle, Kentucky

The Big Clifty 7.5-minute quadrangle is located south of Louisville and west of Elizabethtown along the boundary between Hardin and Grayson Counties. The quadrangle lies within the Mammoth Cave plateau of the Mississippian Plateau physiographic region (McDowell, 1986). Topography is characterized by a low relief plain sitting at elevations between 650 to 850 ft above sea level, which is dissected and incised by Rough River, Meeting Creek, Clifty Creek, and their tributaries to below 500 ft. Swadley (1962) mapped the bedrock geology of the quadrangle, which was later digitized by Conley (2002). Mississippian bedrock is exposed throughout the quadrangle and is cut by several vertical faults in the southeast. The oldest bedrock units include the Beaver Bend and Paoli Limestones, Sample Sandstone, and Reelsville Limestone from oldest to youngest, respectively, and are exposed in the lowest sections of river valleys on the west side of the quadrangle. The Golconda Formation (Beech Creek Limestone, Big Clifty Sandstone, and Haney Limestone Members) is primarily exposed along steep slopes of those same river valleys, which lead up to the top of the plateau. The majority of the broad Mammoth Cave plateau is underlain by Hardinsburg Sandstone with local exposures of Glen Dean Limestone and Leitchfield Formation occurring in the southwest corner of the quadrangle. Previously mapped surficial deposits include scattered areas of alluvium in Meeting Creek, Little Meeting Creek, and Clifty Creek (Swadley, 1962)

Surficial Geologic Map of the Millerstown 7.5-Minute Quadrangle, Kentucky

The Millerstown 7.5-minute quadrangle is located south of Louisville and southwest of Elizabethtown along the boundaries between Hardin, Grayson, and Hart Counties and within the Mississippian Plateau physiographic region (McDowell, 1986). Topography is characterized by the low relief Pennyroyal plain that sits at altitudes below about 650 ft above sea level, the low relief Mammoth Cave plateau at altitudes above about 650 ft, and steep slopes of and isolated knobs of the incised Dripping Springs escarpment that separates the two plains. Moore (1965) mapped the bedrock geology of the quadrangle, which was later digitized by Johnson (2006). Mississippian bedrock and local areas of Pennsylvanian bedrock are exposed throughout most of the quadrangle, all, of which, are cut by several vertical faults. The Ste. Genevieve Limestone is the oldest lithology and underlies the Pennyroyal region in the northeast and southwest corners of the quadrangle, and locally along the Nolin River. The Beaver Bend Limestone, Mooretown Formation, Paoli Limestone, Sample Sandstone, and Reelsville Limestone stratigraphic sequence underlie most of the remaining Pennyroyal plain and several steep slopes of the Dripping Springs escarpment. The Beech Creek Limestone, Big Clifty Sandstone, and Haney Limestone Members of the Golconda Formation are exposed along the Dripping Springs escarpment the edges of the Mammoth Cave plateau region. Most of the Mammoth Cave plateau is underlain by the Hardinsburg Limestone, and, locally in the southwest corner of the quadrangle, the Glen Dean Limestone, Leitchfield Formation, and Pennsylvanian Caseyville Formation. Previously mapped surficial deposits include minor areas of alluvium in tributaries across the Millerstown quadrangle (Moore, 1964)

Surficial Geologic Map of the Summit 7.5-Minute Quadrangle, Kentucky

The Summit 7.5-minute quadrangle is located south of Louisville and west of Elizabethtown along the boundary between Hardin and Grayson Counties and within the Mississippian Plateau physiographic region (McDowell, 1986). Topography is characterized by the low relief Pennyroyal region that sits at elevations between 560 to 650 ft above sea level, the low relief Mammoth Cave plateau at elevations between 750 to 900 ft, and the steep Dripping Springs escarpment that separates the two plains. Moore (1964) mapped the bedrock geology of the quadrangle, which was later digitized by Conley (2002). Mississippian bedrock is exposed throughout the quadrangle and is cut by several vertical faults. The St. Louis Limestone and overlying Ste. Genevieve Limestone underlie the Pennyroyal region and are the oldest bedrock units in the quadrangle. The Beaver Bend and Paoli Limestones, Sample Sandstone, Reelsville Limestone, and Beech Creek Limestone Member of the Golconda Formation are exposed along the Dripping Springs escarpment. The Mammoth Cave plateau region is underlain by the Big Clifty Sandstone and, locally, Haney Limestone Members of the Golconda Formation east of the Summit Fault, and Hardinsburg Sandstone west of the fault. Previously mapped surficial deposits include minor areas of alluvium in tributaries across the Summit quadrangle, and areas of “slumped sandstone” (colluvium) in the Pennyroyal region (Moore, 1964)