An indentation-based framework for probing the glycosaminoglycan-mediated interactions of collagen fibrils.

Microscale deformation processes, such as reorientation, buckling, and sliding of collagen fibrils, determine the mechanical behavior and function of collagenous tissue. While changes in the structure and composition of tendon have been extensively studied, the deformation mechanisms that modulate the interaction of extracellular matrix (ECM) constituents are not well understood, partly due to the lack of appropriate techniques to probe the behavior. In particular, the role of glycosaminoglycans (GAGs) in modulating collagen fibril interactions has remained controversial. Some studies suggest that GAGs act as crosslinkers between the collagen fibrils, while others have not found such evidence and postulate that GAGs have other functions. Here, we introduce a new framework, relying on orientation-dependent indentation behavior of tissue and computational modeling, to evaluate the shear-mediated function of GAGs in modulating the collagen fibril interactions at a length scale more relevant to fibrils compared to bulk tests. Specifically, we use chondroitinase ABC to enzymatically deplete the GAGs in tendon; measure the orientation-dependent indentation response in transverse and longitudinal orientations; and infer the microscale deformation mechanisms and function of GAGs from a microstructural computational model and a modified shear-lag model. We validate the modeling approach experimentally and show that GAGs facilitate collagen fibril sliding with minimal crosslinking function. We suggest that the molecular reconfiguration of GAGs is a potential mechanism for their microscale, strain-dependent viscoelastic behavior. This study reveals the mechanisms that control the orientation-dependent indentation response by affecting the shear deformation and provides new insights into the mechanical function of GAGs and collagen crosslinkers in collagenous tissue.

Orientation-dependent indentation reveals the crosslink-mediated deformation mechanisms of collagen fibrils.

The spatial arrangement and interactions of the extracellular matrix (ECM) components control the mechanical behavior of tissue at multiple length scales. Changes in microscale deformation mechanisms affect tissue function and are often hallmarks of remodeling and disease. Despite their importance, the deformation mechanisms that modulate the mechanical behavior of collagenous tissue, particularly in indentation and compression modes of deformation, remain poorly understood. Here, we develop an integrated computational and experimental approach to investigate the deformation mechanisms of collagenous tissue at the microscale. While the complex deformation arising from indentation with a spherical probe is often considered a pitfall rather than an opportunity, we leverage this orientation-dependent deformation to examine the shear-regulated interactions of collagen fibrils and the role of crosslinks in modulating these interactions. We specifically examine tendon and cervix, two tissues rich in collagen with quite different microstructures and mechanical functions. We find that interacting, crosslinked collagen fibrils resist microscale longitudinal compressive forces, while widely used constitutive models fail to capture this behavior. The reorientation of collagen fibrils tunes the compressive stiffness of complex tissues like cervix. This study offers new insights into the mechanical behavior of collagen fibrils during indentation, and more generally, under longitudinal compressive forces, and illustrates the mechanisms that contribute to the experimentally observed orientation-dependent mechanical behavior. STATEMENT OF SIGNIFICANCE: Remodeling and disease can affect the deformation and interaction of tissue constituents, and thus mechanical function of tissue. Yet, the microscale deformation mechanisms are not well characterized in many tissues. Here, we develop a combined experimental-computational approach to infer the microscale deformation mechanisms of collagenous tissues with very different functions: tendon and cervix. Results show that collagen fibrils resist microscale forces along their length, though widely-used constitutive models do not account for this mechanism. This deformation process partially modulates the compressive stiffness of complex tissues such as cervix. Computational modeling shows that crosslink-mediated shear deformations are central to this unexpected behavior. This study offers new insights into the deformation mechanisms of collagenous tissue and the function of collagen crosslinkers.

Heterogeneous microstructural changes of the cervix influence cervical funneling.

The cervix acts as a dynamic barrier between the uterus and vagina, retaining the fetus during pregnancy and allowing birth at term. Critical to this function, the physical properties of the cervix change, or remodel, but abnormal remodeling can lead to preterm birth (PTB). Although cervical remodeling has been studied, the complex 3D cervical microstructure has not been well-characterized. In this complex, dynamic, and heterogeneous tissue microenvironment, the microstructural changes are likely also heterogeneous. Using quantitative, 3D, second-harmonic generation microscopy, we demonstrate that rat cervical remodeling during pregnancy is not uniform across the cervix; the collagen fibers orient progressively more perpendicular to the cervical canals in the inner cervical zone, but do not reorient in other regions. Furthermore, regions that are microstructurally distinct early in pregnancy become more similar as pregnancy progresses. We use a finite element simulation to show that heterogeneous regional changes influence cervical funneling, an important marker of increased risk for PTB; the internal cervical os shows ∼6.5x larger radial displacement when fibers in the inner cervical zone are parallel to the cervical canals compared to when fibers are perpendicular to the canals. Our results provide new insights into the microstructural and tissue-level cervical changes that have been correlated with PTB and motivate further clinical studies exploring the origins of cervical funneling. STATEMENT OF SIGNIFICANCE: Cervical funneling, or dilation of the internal cervical os, is highly associated with increased risk of preterm birth. This study explores the 3D microstructural changes of the rat cervix during pregnancy and illustrates how these changes influence cervical funneling, assuming similar evolution in rats and humans. Quantitative imaging showed that microstructural remodeling during pregnancy is nonuniform across cervical regions and that initially distinct regions become more similar. We report, for the first time, that remodeling of the inner cervical zone can influence the dilation of the internal cervical os and allow the cervix to stay closed despite increased intrauterine pressure. Our results suggest a possible relationship between the microstructural changes of this zone and cervical funneling, motivating further clinical investigations.

An optomechanogram for assessment of the structural and mechanical properties of tissues.

The structural and mechanical properties of tissue and the interplay between them play a critical role in tissue function. We introduce the optomechanogram, a combined quantitative and qualitative visualization of spatially co-registered measurements of the microstructural and micromechanical properties of any tissue. Our approach relies on the co-registration of two independent platforms, second-harmonic generation (SHG) microscopy for quantitative assessment of 3D collagen-fiber microstructural organization, and nanoindentation (NI) for local micromechanical properties. We experimentally validate our method by applying to uterine cervix tissue, which exhibits structural and mechanical complexity. We find statistically significant agreement between the micromechanical and microstructural data, and confirm that the distinct tissue regions are distinguishable using either the SHG or NI measurements. Our method could potentially be used for research in pregnancy maintenance, mechanobiological studies of tissues and their constitutive modeling and more generally for the optomechanical metrology of materials.

Application of Fourier transform-second-harmonic generation imaging to the rat cervix.

We present the application of Fourier transform-second-harmonic generation (FT-SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid-cervix and near the external orifice of the cervix were analyzed in both two-dimensions (2D) and three-dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT-SHG imaging and observe collagen fibers oriented both in and out-of-plane in the outermost and the innermost layers, which cannot be observed using 2D FT-SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans.

Ultrasonic attenuation estimation of the pregnant cervix: a preliminary report.

OBJECTIVE: Estimates of ultrasonic attenuation (the loss of energy as an ultrasonic wave propagates through tissue) have been used to evaluate the structure and function of tissues in health and disease. The purpose of this research was to develop a method to estimate ultrasonic cervical attenuation during human pregnancy using a clinical ultrasound system. METHODS: Forty women underwent a cervical scan once during pregnancy with the Zonare z.one clinical ultrasound system using a 4-9-MHz endovaginal transducer. This ultrasound system provides access to radiofrequency (RF) image data for processing and analysis. In addition, a scan of a tissue-mimicking phantom with a known attenuation coefficient was acquired and used as a reference. The same settings and transducer used in the clinical scan were used in the reference scan. Digital data of the beam-formed image were saved in Digital Imaging and Communications in Medicine (DICOM) format on a flash drive and converted to RF data on a personal computer using a Matlab program supplied by Zonare. Attenuation estimates were obtained using an algorithm that was independently validated using tissue-mimicking ultrasonic phantoms. RESULTS: RF data were acquired and analyzed to estimate attenuation of the human pregnant cervix. Regression analysis revealed that attenuation was: a predictor of the interval from ultrasound examination to delivery (beta = 0.43, P = 0.01); not a predictor of gestational age at time of examination (beta = - 0.23, P = 0.15); and not a predictor of cervical length (beta = 0.077, P = 0.65). CONCLUSIONS: Ultrasonic attenuation estimates have the potential to be an early and objective non-invasive method to detect interval between examination and delivery. We hypothesize that a larger sample size and a longitudinal study design will be needed to detect gestational age-associated changes in cervical attenuation.

A national survey of herbal preparation use by nurse-midwives for labor stimulation. Review of the literature and recommendations for practice.

To document the use of herbal preparations for cervical ripening, induction, and augmentation of labor by certified nurse-midwives (CNMs) and nurse-midwifery education programs, a national survey of 500 members of the American College of Nurse-Midwives was conducted. Forty eight nurse-midwifery education programs were also surveyed to determine whether they were formally or informally educating students in the use of herbal preparations for cervical ripening, induction, or augmentation of labor. The results of this study, a review of the literature, professional issues, and recommendations for clinical practice are presented in this article. Of 500 questionnaires mailed to ACNM members, 90 were returned from CNMs who used herbal preparations to stimulate labor and 82 were returned from CNMs who did not use herbal preparations to stimulate labor. Three questionnaires were excluded due to incomplete data or blank questionnaires. No significant differences were noted in relations to geographical region, midwifery education, or highest level of education between the CNM respondents who did and those who did not use alternative methods to stimulate labor. Of the CNMs who used herbal preparations to stimulate labor, 64% used blue cohosh, 45% used black cohosh, 63% used red raspberry leaf, 93% used castor oil, and 60% used evening primrose oil. CNMs who used herbal preparations to stimulate labor were younger (43 versus 45 years, P < .01) and more likely to deliver at home or in an in-hospital or out-of-hospital birthing center (P < .0006), than CNMs who never used herbal preparations to stimulate labor. The most cited reason for using herbal preparations to stimulate labor was that they are "natural," whereas the most common reason for not using herbal preparations was the lack of research or experience with the safety of these substances. Sixty-nine percent of CNMs who used herbal preparations to stimulate labor learned about using them from other CNMs, 4% from formal research publications, and none from their formal education programs. Although 78% of the CNMs who used herbal preparations to stimulate labor directly prescribed them and 70% indirectly suggested them to clients, only 22% had included them within their written practice protocols. Seventy-five percent of the CNMs who used herbal preparations to stimulate labor used them first or instead of pitocin. Twenty-one percent reported complications including precipitous labor, tetanic uterine contractions, nausea, and vomiting. Sixty-four percent of the nurse-midwifery education programs included instruction in the use of herbal preparations to stimulate labor in their formal curricula, and 92% included informal discussions on the use of herbal preparations. Evening primrose oil was the most common herbal preparation discussed in nurse-midwifery education programs. Castor oil was the most commonly used herbal preparation used by nurse-midwives in clinical practice.

Maternal syphilis: the next pregnancy.

We sought to determine: (1) the perinatal outcomes in the subsequent pregnancy; (2) the natural history of serologic titers over the course of the two pregnancies; and (3) the incidence and risk factors for delivering an infant with congenital syphilis in the subsequent pregnancy. Over a five-year period, we reviewed the charts of 46 women with maternal syphilis during pregnancy who had a subsequent pregnancy and delivered in our institution. The initial and subsequent pregnancy outcomes were contrasted. To characterize the subsequent pregnancy risk of congenital syphilis, those women with recurrent congenital syphilis were contrasted to women who delivered infants without congenital syphilis. Rapid plasma reagin (RPR) titers in the initial and subsequent pregnancy were analyzed by neonatal outcome. Forty percent of the women who delivered an infant with congenital syphilis in their first pregnancy delivered yet another infant with congenital syphilis in the subsequent pregnancy. Continued cocaine use was the single most important risk factor for delivering another infant with congenital syphilis (p < 0.0001). Forty-two percent of the women who delivered an infant without congenital syphilis in the initial pregnancy delivered an infant with congenital syphilis in the subsequent pregnancy. Birthweight, the number of neonatal hospital days, and the incidence of respiratory distress syndrome (RDS), neonatal pneumonia, abruption, and positive meconium drug screens were not significantly different between the initial and subsequent pregnancies. As with the initial pregnancy, women continued to experience poor pregnancy outcomes in the subsequent pregnancy.

Maternal syphilis in Michigan: the challenge to prevent congenital syphilis.

Syphilis has re-emerged as a significant public health problem for pregnant women and their babies in Michigan, US, and in many areas of the world. In the US over 50,000 cases of primary and secondary syphilis were reported to the Centers for Disease Control (CDC) in 1990. Due to the current epidemic of maternal syphilis in pregnancy in our city, we studied 253 cases of maternal syphilis over a one-year period. Our objective was to determine maternal risk factors during pregnancy predicting congenital infection. Women with high venereal disease research laboratory (VDRL) titres during pregnancy and unknown duration of disease had the highest incidence of delivering an infant with congenital syphilis. Even with treatment according to the current CDC guidelines there was a 27% incidence of congenital syphilis if disease duration was < 1 year and a 49% incidence of congenital syphilis for unknown duration of disease. Maternal syphilis during pregnancy was associated with significant neonatal morbidity and a preterm delivery incidence of 28%. The clinical evaluation, management, and current treatment guidelines are reviewed in this paper.

The biophysical profile as a tool for prediction of preterm delivery.

Our objective was to determine if any of the following parameters is a predictor of preterm delivery: gestational age, cervical dilatation, or biophysical profile score.Sixty-two women with certain dates, intact membranes, and preterm contractions more frequent than 300 min underwent a 30-min ultrasonographic assessment of fetal breathing movements, fetal tone, fetal movement and amniotic fluid volume, each of which was scored as 0, 1, or 2 before any tocolytics were given. The total score (0-8) was calculated and cervical dilatation was recorded. All women were subsequently placed on tocolytics. Data were analyzed using survival analysis with stepwise regression of the covariates to determine the predictive values of the total score, its individual parameters, gestational age, and cervical dilatation to the outcome variable of hours to delivery. Gestational age, cervical dilatation and time interval to delivery were categorized and entered into a logistic regression model with the total score and its components to determine predictive odds.Total score (p < 0.0001) and cervical dilatation (p < 0.0001) were the most significant variables associated with time interval to delivery, followed by gestational age (p = 0.035). When the individual biophysical parameters were entered into the model, fetal tone (p < 0.0001), cervical dilatation (p < 0.0001) and gestational age (p < 0.014) were the only variables related to time interval to delivery. Predictive odds of delivery in less than 48 h in relation to fetal tone and cervical dilatation were calculated. Total score, fetal tone, and cervical dilatation were strongly associated with time interval to delivery. Our study did not support fetal breathing movements as a predictor of preterm delivery.

Intrauterine growth retardation. Etiology, diagnosis, and management.

Intrauterine growth retardation (IUGR) occurs in 3% to 10% of all pregnancies. Although it is an uncommon occurrence in a low-risk nurse-midwifery practice, as many as 65% of the cases of IUGR are not identified until after the birth of the infant. Identification of IUGR is important because it carries a high risk of antepartum fetal death, anomalies, intrapartum asphyxia, and long-term morbidity. Additionally, identification of IUGR is essential for appropriate prenatal and intrapartum management. A review of etiologic factors, current assessment, and management of the pregnancy complicated by IUGR is presented. The use and interpretation of new technologies that the clinician may use in diagnosing and managing IUGR is reviewed.

Epidemic syphilis: maternal factors associated with congenital infection.

OBJECTIVE: Little is known about which cases of maternal syphilis will affect the newborn. Because of the current epidemic of syphilis in pregnancy in our city, we sought to identify risk factors during pregnancy associated with congenital infection. STUDY DESIGN: We reviewed 253 cases of maternal syphilis prospectively identified over a 1-year period. On the basis of neonatal diagnosis, these data were divided into two groups, those without evidence of presumptive congenital syphilis and those with evidence of presumptive congenital syphilis. Presumptive congenital syphilis was defined according to the Centers for Disease Control and Prevention surveillance case definition. Cases with bloody spinal taps and cases of suspected congenital syphilis that did not meet these criteria were excluded. Venereal Disease Research Laboratory titers are given as the inverse of the geometric mean. RESULTS: Venereal Disease Research Laboratory titer at time of diagnosis and unknown duration of disease were risk factors for congenital syphilis. There was a significantly decreased rate of congenital syphilis with single-dose therapy if disease length was < 1 year (p < 0.005). Unknown duration of disease was associated with 67.9% and 48.6% rates of congenital syphilis with one- and three-dose therapy respectively. There was a 28% incidence of preterm birth. CONCLUSION: Our study suggests an alarming rate of failure of current therapy to prevent congenital syphilis. Venereal Disease Research Laboratory titer at time of diagnosis and unknown duration of disease are risk factors for congenital syphilis. The high rate of presumptive congenital syphilis in the unknown duration group indicates that identification before or earlier in pregnancy will be necessary to prevent devastating consequences for the neonate.

Fundal height measurement. Part 4--Accuracy of clinicians' identification of the uterine fundus during pregnancy.

The purpose of this study was to determine how accurately clinicians can identify the uppermost border of the uterine fundus when they obtain fundal height measurements. Clinicians were instructed to identify the uppermost border of the uterine fundus in their usual manner and make a small pen mark on the maternal abdomen at that point. Real-time ultrasonography was then used to locate the actual level of the fundus and measure the distance between the clinician's assessment and the actual level of the fundus. Measurements were obtained from 126 women. Six clinicians participated in the study, and the differences between the clinicians' errors were not statistically significant (F = 1.26; d.f. = 5,120; P = .2873). For the entire series, the mean error was -.45 cm (SD = 1.99 cm), the mean absolute error was 1.25 cm, the maximal error was 8.6 cm, the percentage of errors that exceeded 1 cm was 42.1%, and the percentage of errors that exceeded 2 cm was 20.6%. Examiner error was not associated with factors such as maternal height, prepregnancy weight, present weight, prepregnancy body mass index, parity, gestational weeks, the amount of fat on the anterior abdominal wall, or the presence of the placenta or fetal parts in the fundus. Examiner error was influenced by thickness of the uterine wall and fetal presentation. These findings indicate that clinicians make errors in identifying the uterine fundus in a significant number of cases and that the methods used by clinicians to identify the uterine fundus need to be evaluated and improved.

Fundal height measurement. Part 2--Intra- and interexaminer reliability of three measurement techniques.

The intra- and interexaminer reliability of three fundal height measurement techniques (two tape measure and one caliper) was studied in a convenience sample of 60 pregnant women. The three measurements were obtained twice by two examiners. Four examiners with varying levels of clinical experience participated in the study; one examiner obtained measurements in all 60 cases and the other three examiners each obtained measurements in approximately one-third of the cases. Examiners were blinded to their own and to other examiners' measurements. The mean absolute differences between individual examiner's first and second measurements varied from 0.68 to 1.74 cm, the percentage of differences < or = 1.0 cm varied from 45% to 77.8%, and the maximal differences varied from 1.6 to 7.5 cm. The caliper technique consistently demonstrated the smallest mean absolute intraexaminer differences (0.68 to 1.39 cm) and the highest percentage of intraexaminer differences < or = 1.0 cm (55% to 77.8%). The differences between pairs of examiners' measurements were consistently greater than the differences between individual examiner's measurements: Mean absolute differences varied from 1.36 to 3.60 cm, the percentage of differences < or = 1.0 cm varied from 9.1% to 55.6%, and the maximal differences varied from 3.1 to 11.5 cm. Interexaminer differences were smallest for the tape "over-the-curve" measurements and the caliper measurements. These findings indicate that, to ensure maximal reliability, fundal height measurements should be obtained by the same clinician throughout pregnancy. Additionally, these findings indicate that calipers may be the most reliable method of obtaining fundal height measurements and that the accuracy of caliper measurements in identifying fetal growth disturbances and other pregnancy complications merits further investigation.

Phosphorus 31 nuclear magnetic resonance examination of female reproductive tissues.

Nuclear magnetic resonance spectroscopy is a powerful method of investigating the relationship between metabolism and function in living tissues. We present evidence that the phosphorus 31 spectra of myometrium and placenta are functions of physiologic state and gestational age. Specific spectroscopic abnormalities are observed in association with disorders of pregnancy and gynecologic diseases. Our results suggest that noninvasive nuclear magnetic resonance spectroscopy examinations may sometimes be a useful addition to magnetic resonance imaging examinations, and that nuclear magnetic resonance spectroscopy of biopsy specimens could become a cost-effective method of evaluating certain biochemical abnormalities.