Hysteroscopic resectoscope-directed biopsies and outpatient endometrial sampling for assessment of tumor histology in women with endometrial cancer or atypical hyperplasia.

OBJECTIVE: To evaluate and compare the diagnostic efficiency of outpatient endometrial sampling (OES) and hysteroscopic resectoscope-directed biopsies (HYbiopsy) to distinguish between endometrial cancer (EC) and atypical hyperplasia (AH) and to assess tumor type and grade (histotype) in women with EC. DESIGN: Patients with AH or EC (n = 266) among 1013 patients consecutively referred because of postmenopausal bleeding were included. Identification of EC versus AH, and unfavorable tumor types (endometrioid grade 3 or non-endometrioid tumors) using OES and HYbiopsy was compared to final histopathology at hysterectomy. AH or EC were identified by OES in 184 patients and by HYbiopsy in212. RESULTS: OES had only sufficient tissue samples in 72.7% of intended samples. Even when OES did provide sufficient material, addition of HYbiopsy was a better technique than OES alone to distinguish between EC and AH, with an AUC of 95.9% and 79.8%; sensitivity of 97.4% and 64.6% and a specificity of 94.4% and 95.0%, respectively (p = 0.008). AH was falsely diagnosed with OES in 58 (35.4%) of 164 women with a final diagnose of EC. A final diagnosis of stage 1b or more was seen in 22 of these 58 women, while 5 of 194 patients with EC all stage 1a grade 1 had AH by HYbiopsy. HYbiopsy had higher correlation in assessment of tumor type and grade than OES, but OES and HYbiopsy had comparable AUC of 90.3% and 92.4% for identification of unfavorable tumors when tumor histotype was successfully identified. Regarding identification of unfavorable tumors (n = 57), a successfully assessment of histotype by OES combined with HYbiopsy in women without successfully diagnosed histotype by OES alone had AUC of 91.3%. CONCLUSION: Addition of HYbiopsy may improve diagnosis when preoperative OES identifies AH or is insufficient for explicit diagnosis of tumor type and grade. However, there is limited benefit of the addition of HYbiopsy in the presence of definite diagnosis of grade 1-2 endometrioid tumors by OES.

Identification of endometrial cancers and atypical hyperplasia: Development and validation of a simplified system for ultrasound scoring of endometrial pattern.

OBJECTIVES: To derive and validate a practical scoring system for identification of endometrial cancer (EC) or atypical hyperplasia (AH) using transvaginal ultrasonography (TVS) and gel infusion sonography (GIS) in women with postmenopausal bleeding (PMB). STUDY DESIGN: Endometrial pattern was correlated with endometrial pathology in consecutive women with PMB in both a derivation study (N = 164) and a validation study (N = 711). Logistic regression was used to derive and validate two scoring systems (A and B) for prediction of EC/AH: scoring system A was Doppler score + interrupted endo-myometrial junction (IEJ) (2 points); and scoring system B was Doppler score + IEJ (1 point) + Irregular Endometrial Outline (IESO) by GIS (1 point); the Doppler score was based on the presence of more than one single or double vessel (1 point) + multiple vessels (1 point) + large vessels (1 point). OUTCOME MEASURES: Diagnostic performance and calibration curves for identification of EC/AH. RESULTS: Both scoring systems had good observer agreement. VALIDATION DATA: Scoring was most effective with endometrial thickness (ET) ≥ 8 mm. Both scoring systems were well calibrated and performed satisfactorily in women with ET ≥ 8 mm. The sensitivity and specificity of a score of ≥ 2 points in system A were 92% and 84%; the respective values were 89% and 88% in system B. CONCLUSIONS: Scoring was highly efficient in identifying EC/AH. Four risk groups of EC/AH may guide the management of women with PMB: very low (ET < 4 mm), low (ET 4-7.9 mm), intermediate (ET ≥ 8 mm and score < 2 points) and high risk (ET ≥ 8 mm and score ≥ 2 points).

Cervical conization doubles the risk of preterm and very preterm birth in assisted reproductive technology twin pregnancies.

STUDY QUESTION: Does cervical conization add an additional risk of preterm birth (PTB) in assisted reproduction technology (ART) singleton and twin pregnancies? SUMMARY ANSWER: Cervical conization doubles the risk of preterm and very PTB in ART twin pregnancies. WHAT IS KNOWN ALREADY: ART and cervical conization are both risk factors for PTB. STUDY DESIGN, SIZE, DURATION: In this national population-based controlled cohort study, we included all ART singletons and twin deliveries from 1995 to 2009 in Denmark by cross-linkage of maternal and child data from the National IVF register and the Medical Birth register. Furthermore, control groups of naturally conceived (NC) singletons and twins were extracted. Cervical diagnoses were obtained from the Danish Pathology register. Cervical conization included both cold knife cone and LEEP (loop electrosurgical excision procedure) but not cervical biopsies. The main outcomes measures were PTB (PTB ≤ 37 + 0 gestational weeks), very preterm birth (VPTB ≤ 32 + 0 gestational weeks) and preterm premature rupture of membranes (PPROM). PARTICIPANTS/MATERIALS, SETTING, METHODS: In all 16 923 ART singletons and 4829 ART twin deliveries were included. A random sample of NC singletons, 2-fold the size of the ART singleton group matched by date and year of birth (n = 33 835) and all NC twin deliveries (n = 15 112), was also extracted. Multiple logistic regression analyses were performed to adjust for the following confounders: maternal age, parity, year of child birth and sex of child. MAIN RESULTS AND THE ROLE OF CHANCE: Cervical morbidity (dysplasia and conization) was more often observed in ART pregnancies (6.2% of ART singletons and 5.4% ART twins) than in NC pregnancies (4.2% for NC singletons and 4.5% for NC twins), both for singletons and twins. In ART singleton deliveries, the PTB rate was 13.1 versus 8.2% in women with and without conization, respectively, with an adjusted odds ratio (aOR) of 1.56 [95% confidence interval (CI) 1.21-2.01]. In ART twin deliveries, the prevalence of PTB was 58.2 versus 41.3% in women with and without conization, respectively, with an aOR 1.94 (95% CI 1.36-2.77), and the risk of VPTB was also doubled. Furthermore, previous dysplasia (without conization) increased the risk of VPTB in ART twins (aOR 1.74, 95% CI 1.04-2.94). Cervical dysplasia did not increase the risk of any of the other adverse outcomes in ART singletons or twins. The risk of PPROM was increased in both in ART and NC singleton deliveries with conization versus no conization; however, this increased risk of PPROM after conization was not observed in either ART or NC twin pregnancies. LIMITATIONS, REASONS FOR CAUTION: We were not able to adjust for the height of the cervical cone or the severity of the cervical intraepithelial neoplasia (CIN) or the time window between diagnosis of CIN and ART treatment. The finding on an increased risk of VPTB in ART twin pregnancies after dysplasia without conization may be random as we found no other increased risk after dysplasia alone either in singletons or in twins. WIDER IMPLICATIONS OF THE FINDINGS: After ART and prior conization, 58% of twin pregnancies versus 13% of ART singleton pregnancies result in PTB. There is a doubled risk of preterm delivery in ART twins with conization versus ART twins with no prior conization. Single-embryo transfer should always be recommended in women with prior conization irrespective of female age, embryo quality and prior number of ART attempts. STUDY FUNDING/COMPETING INTERESTS: No external funding was achieved for this project.

Two- and three-dimensional transvaginal ultrasound with power Doppler angiography and gel infusion sonography for diagnosis of endometrial malignancy.

OBJECTIVES: To evaluate the diagnostic efficiency of two-dimensional (2D) and three-dimensional (3D) transvaginal ultrasonography, power Doppler angiography (PDA) and gel infusion sonography (GIS) at offline analysis for recognition of malignant endometrium compared with real-time evaluation during scanning, and to determine optimal image parameters at 3D analysis. METHODS: One hundred and sixty-nine consecutive women with postmenopausal bleeding and endometrial thickness ≥ 5 mm underwent systematic evaluation of endometrial pattern on 2D imaging, and 2D videoclips and 3D volumes were later analyzed offline. Histopathological findings at hysteroscopy or hysterectomy were used as the reference standard. The efficiency of the different techniques for diagnosis of malignancy was calculated and compared. 3D image parameters, endometrial volume and 3D vascular indices were assessed. Optimal 3D image parameters were transformed by logistic regression into a risk of endometrial cancer (REC) score, including scores for body mass index, endometrial thickness and endometrial morphology at gray-scale and PDA and GIS. RESULTS: Offline 2D and 3D analysis were equivalent, but had lower diagnostic performance compared with real-time evaluation during scanning. Their diagnostic performance was not markedly improved by the addition of PDA or GIS, but their efficiency was comparable with that of real-time 2D-GIS in offline examinations of good image quality. On logistic regression, the 3D parameters from the REC-score system had the highest diagnostic efficiency. The area under the curve of the REC-score system at 3D-GIS (0.89) was not improved by inclusion of vascular indices or endometrial volume calculations. CONCLUSION: Real-time evaluation during scanning is most efficient, but offline 2D and 3D analysis is useful for prediction of endometrial cancer when good image quality can be obtained. The diagnostic efficiency at 3D analysis may be improved by use of REC-scoring systems, without the need for calculation of vascular indices or endometrial volume. The optimal imaging modality appears to be real-time 2D-GIS.

An ultrasound algorithm for identification of endometrial cancer.

OBJECTIVE: To propose a scoring system to predict endometrial cancer using different ultrasound image characteristics at gray-scale, with and without enhancement by gel infusion, and Doppler transvaginal sonography (TVS) and to evaluate intra- and interobserver variability in assessment of these characteristics. METHOD: Unenhanced TVS, Doppler examinations and gel infusion sonography (GIS) were performed prospectively in 174 consecutive postmenopausal women with endometrial thickness ≥ 5 mm. The reference standard in all women was hysteroscopy or hysterectomy with pathological evaluation of the malignancy. The presence of various ultrasound pattern characteristics indicative of endometrial malignancy and intra- and interobserver variability in their assessment were evaluated. Multivariate logistic regression was used to correlate image and clinical parameters to presence of endometrial cancer. RESULTS: A simple Doppler flow score (which considered only presence of vascularity and not presence of single/double dominant vessel, multiple vessels, large vessels, color splash or densely packed vessels) had an area under the receiver-operating characteristics curve (AUC) of 0.83 in the prediction of endometrial cancer. Models including endometrial thickness, Doppler score and interrupted endomyometrial junction on unenhanced TVS predicted endometrial cancer with an AUC of 0.95 (95% CI, 0.92-0.99) and, with addition of irregular surface on GIS, the AUC was 0.97 (95% CI, 0.94-0.99). A risk of endometrial cancer (REC) scoring system based on body mass index, Doppler score, endometrial thickness and interrupted endomyometrial junction on unenhanced TVS and irregular surface at GIS performed very well at identifying endometrial cancer; at a REC-score of ≥ 4 the sensitivity for detection of endometrial cancer was 91% and specificity was 94%. Observers agreed in 82.3% of cases (kappa, 0.63 (0.48-0.78)) when subjective parameters were analyzed in stored videoclips. CONCLUSION: Our observer-dependent proposed scoring system seems to perform well in the prediction of endometrial cancer and should be tested in future studies.

After conisation of the cervix, the perinatal mortality as a result of preterm delivery increases in subsequent pregnancy.

OBJECTIVE: To determine the effects of one or two conisations on preterm delivery and perinatal mortality in subsequent pregnancies. DESIGN: A population-based cohort study. SETTING: Aarhus University Hospital. POPULATION: Preterm delivery and mortality rates were evaluated in 721 deliveries after one conisation, and in 37 deliveries after two conisations, and were compared with 390 deliveries after dysplasia and 74 552 deliveries that were not preceded by conisation or dysplasia. METHODS: Cox regression was used to evaluate preterm delivery rates and perinatal mortality. MAIN OUTCOME MEASURES: Birthweight, gestational age (prior to 28, 32, and 37 weeks of gestation, respectively) and perinatal mortality. RESULTS: The risk of preterm delivery was increased after one conisation [adjusted hazard ratios (95% CI): <37 weeks, 2.8 (2.3-3.5); <28 weeks, 4.9 (2.5-9.7)], and was further increased after two conisations [adjusted hazard ratios (95% CI): <37 weeks, 9.9 (6-17); <28 weeks, 9.8 (1.4-70)], compared with no conisation. One conisation was associated with an increased perinatal mortality [<28 weeks, 9.9 (4.0-25)]. All three methods of conisation [large loop excision of the transformation zone, electroknife and cold knife] increased the risk of preterm delivery. CONCLUSIONS: A single conisation was associated with a 2.8-fold increased risk of perinatal death, most likely because of a 4.9-fold increase in extreme preterm delivery. Only 37 patients had two conisations, and the results showed a ten-fold increase in the risk of preterm delivery.

The anabolic effect of PTH on bone is attenuated by simultaneous glucocorticoid treatment.

Glucocorticoids (GC) are used for the treatment of a wide spectrum of diseases because of their potent anti-inflammatory and immunosuppressive effects, and they are serious and common causes of secondary osteoporosis. Administration of intermittent parathyroid hormone (PTH) may induce formation of new bone and may counteract the bone loss induced by GC treatment. Effects of simultaneous PTH and GC treatment were investigated on bone biomechanics, static and dynamic histomorphometry, and bone metabolism. Twenty-seven-month-old female rats were divided randomly into the following groups: baseline, vehicle, PTH, GC, and PTH + GC. PTH (1-34) 25 mug/kg and GC (methylprednisolone) 2.5 mg/kg were injected subcutaneously each day for a treatment period of 8 weeks. The rats were labeled with fluorochromes 3 times during the experiment. Bone sections were studied by fluorescence microscopy. The PTH injections resulted in a 5-fold increase in cancellous bone volume. At the proximal tibia, PTH induced a pronounced formation of new cancellous bone which originated from the endocortical bone surfaces and from thin trabeculae. Formation and modeling of connections between trabeculae were observed. Similar but less pronounced structural changes were seen in the PTH + GC group. The compressive strength of the cancellous bone was increased by 6-fold in the PTH group compared with the vehicle group. GC partially inhibited the increase in compressive strength induced by PTH. Concerning cortical bone, PTH induced a pronounced increase in the endocortical bone formation rate (BFR) and a smaller increase in periosteal BFR. The combination of PTH + GC resulted in a partial inhibition of the PTH-induced increase in bone formation. Serum-osteocalcin was increased by 65% in the PTH group and reduced by 39% in the GC group. The pronounced anabolic effect of PTH injections on the endocortical and trabecular bone surfaces and less pronounced anabolic effect on periosteal surfaces were partially inhibited, but not prevented, by simultaneous GC treatment in old rats. Both cortical and cancellous bone possessed full mechanical competence after treatment with PTH + GC.

Low-intensity, high-frequency vibration appears to prevent the decrease in strength of the femur and tibia associated with ovariectomy of adult rats.

The effect of low-intensity, high-frequency vibration on bone mass, bone strength, and skeletal muscle mass was studied in an adult ovariectomized (OVX) rat model. One-year-old female rats were allocated randomly to the following groups: start control, sham OVX, OVX without vibration, OVX with vibration at 17 Hz (0.5g), OVX with vibration at 30 Hz (1.5g), OVX with vibration at 45 Hz (3.0g). Vibrations were given 30 min/day for 90 days. During vibration each group of rats was placed in a box on top of the vibration motor. The amplitude of the vibration motor was 1.0 mm. The animals were labeled with calcein at day 63 and with tetracycline at day 84. The tibia middiaphysis was studied by mechanical testing and dynamic histomorphometry and the femur distal metaphysis by mechanical compression. OVX without vibration increased the periosteal bone formation rate and increased the medullary cross-sectional area, i.e., increased the endocortical resorption and outward anteromedial and lateral drifts of cortical bone at the tibia middiaphysis. OVX also resulted in a reduced maximum bending stress of the tibia diaphysis and a reduced compressive stress of the femur distal metaphysis. Vibration at the highest intensity, i.e., 45 Hz, of OVX rats induced a further increase in periosteal bone formation rate and inhibited the endocortical resorption seen in OVX rats. Furthermore, vibration at 45 Hz inhibited the decline in maximum bending stress and compressive stress induced by OVX. Neither OVX nor OVX with vibration influenced skeletal muscle mass. In conclusion, the results support the idea of a possible beneficial effect of passive physical loading on the preservation of bone in OVX animals.

The growth hormone secretagogue ipamorelin counteracts glucocorticoid-induced decrease in bone formation of adult rats.

The ability of the growth hormone secretagogue (GHS) Ipamorelin to counteract the catabolic effects of glucocorticoid (GC) on skeletal muscles and bone was investigated in vivo in an adult rat model. Groups of 8-month-old female rats were injected subcutaneously for 3 months with GC (methylprednisolone) 9 mg/kg/day or GHS (Ipamorelin) 100 microg/kg three times daily, or both GC and GHS in combination. The maximum tetanic tension of the calf muscles was determined in vivo in a materials testing machine. The maximum tetanic tension was increased significantly, and the periosteal bone formation rate increased four-fold in animals injected with GC and GHS in combination, compared with the group injected with GC alone. In conclusion, the decrease in muscle strength and bone formation found in GC-injected rats was counteracted by simultaneous administration of the growth hormone secretagogue.

Growth hormone increases cortical and cancellous bone mass in young growing rats with glucocorticoid-induced osteopenia.

The effects of growth hormone (GH) on linear growth, bone formation, and bone mass have been examined in glucocorticoid (GC)-injected young growing rats. Two-month-old female Wistar rats were injected for 90 days with 1, 3, 6, or 9 mg of methylprednisolone alone or in combination with 5 mg of GH. Bone mass and bone formation parameters were examined in the femoral cortical bone and in cortical bone and cancellous bone of the lumbar vertebra. GC administration dose dependently decreased growth, longitudinal growth of the vertebra, as well as the modeling drift of the cortical bone of the vertebral body and femoral diaphysis. In the vertebral cancellous bone, GC also decreased the mineralizing surface and inhibited the growth-related increase in cancellous bone volume. GH increased growth, longitudinal growth of the vertebra, as well as the modeling drift of the vertebral body and the femoral diaphysis, resulting in an increased cortical bone mass. GH also increased cancellous bone volume and the mineralizing surface of the vertebral body. In GC-injected animals, GH normalized and further increased growth, longitudinal growth, and the modeling drift of both the femoral diaphysis and the vertebral body, resulting in an increased cortical bone mass at both locations. GH also increased cancellous bone volume of the vertebral body in GC-injected animals, but GH did not, however, reverse the decreased mineralizing surface of cancellous bone induced by GC injections. In conclusion, GC administration to growing rats retards normal growth, longitudinal growth, and cortical bone modeling drift. It also decreases the cancellous bone mineralizing surface and inhibits the normal age-related increase in cancellous bone volume of the vertebral body. In the growing rat skeleton, GH can counteract these GC-induced side effects, except for the GC-induced decrease in the mineralizing surface of cancellous bone of the vertebral body, which remained unaffected by GH administration.

Inhibition of cross-links in collagen is associated with reduced stiffness of the aorta in young rats.

Collagen and elastin fibres are of major importance in providing the aorta with tensile strength and elasticity. The presence of cross-links in collagen and elastin is essential for the mechanical stability of collagen and elastin fibres. beta-aminopropionitrile (BAPN) reduces the formation of cross-links by inhibiting the enzyme lysyloxidase. Young rats were injected with BAPN to inhibit the formation of cross-links, and the changes in the biomechanical and biochemical properties of the thoracic aorta were studied. The biomechanical analyses of aortic samples from BAPN-treated rats showed a significantly increased diameter (1.64 +/-0.02 mm), a significantly reduced maximum load (1.08+/-0.08 N), and a significantly reduced maximum stiffness (3.34+/-0.10 N) compared with controls (1.57+/-0.02 mm, 1.55+/-0.04 N and 4.49 +/-0.14 N, respectively). No changes in the concentrations of collagen and elastin were found. The content of pyridinoline, a mature collagen cross-link, was significantly decreased by 49% in the BAPN-treated group compared with controls. No changes in the concentration of desmosine + isodesmosine, the major cross-links of elastin. were found. The present study shows that cross-links are essential in providing mechanical stability of the aorta. Even a partial inhibition of the cross-linking processes results in a destabilisation of the aortic wall with increased diameter and reduced strength and stiffness.

Administration of a glucocorticoid with depot effect counteracts the stimulating effect of growth hormone on cancellous and cortical bone of the vertebral body in rats.

Our earlier studies have shown that growth hormone administration could not counteract decreased longitudinal growth and cortical osteopenia of rat femora induced by a glucocorticoid with depot effect. In the present study we examined the effects of glucocorticoid on vertebral bone as well as the effect of growth hormone on vertebral bone in young growing animals also given glucocorticoid injections. Five groups of female rats (3 1/2 months) were treated for 80 days as follows: (1) saline, (2) prednisolone: Delcortol 5 mg/kg/day, (3) growth hormone: 5 mg/kg/day, (4) prednisolone and growth hormone, (5) food restriction. Vertebral dimensions, histomorphometry, and mechanical competence of the vertebral bone were examined. Growth hormone administration increased body weight, vertebral height, cross-sectional area, and volume. The compressive strength of the L4-corpus cylinder was also increased due to an increase in cancellous bone volume and an increase in the area of cortical bone surrounding the vertebral body. Glucocorticoid administration decreased body weight, height, and volume of the intact vertebrae. Histological examination revealed that glucocorticoid administration decreased the area of cortical bone surrounding the vertebral body but had no effect on the cancellous bone volume. No effect of glucocorticoid administration on mechanical strength of the L4 corpus cylinder could be detected. In agreement with our findings in cortical bone, we found no effect of growth hormone on vertebral bone when given to animals also receiving glucocorticoid injections. Growth hormone increases longitudinal growth, cortical and cancellous bone mass, and mechanical competence of the vertebral body. Glucocorticoid administration decreases longitudinal growth of the vertebrae and cortical bone mass without affecting cancellous bone mass of the vertebral body. Despite this, administration of a glucocorticoid with depot effect totally inhibits the effect of growth hormone on vertebral bone.

Growth hormone and mild exercise in combination markedly enhance cortical bone formation and strength in old rats.

The effects of a combination of mild exercise and GH injections on bone were studied in old female rats. Biosynthetic human GH, 2.7 mg/kg/day, was injected s.c. for 73 days. Exercised rats ran 8 m/min on a treadmill for 1 h/day. All rats (age 21 months old) were labeled with a tetracycline injection 56 days and a calcein injection 11 days before killing. The GH injections resulted in an 11-fold increase in femoral middiaphyseal bone formation rate and a 12% increase in cross-sectional area compared with the saline-injected group. The mild exercise doubled the mineralizing surface but did not influence the bone formation rate significantly. The combination of GH injections plus exercise, however, resulted in a further increase of 39% in bone formation rate, primarily at the anterolateral aspects, and an increase of 5% in cross-sectional area compared with the group injected with GH only. The femur ultimate breaking load was increased by 37% and the stiffness by 42% in the group injected with GH compared with the saline-injected group. Exercise alone did not influence the femur mechanical properties. The combination of GH injections plus exercise induced a 4% further increase in ultimate breaking load and 7% further increase in stiffness compared with the group injected with GH alone. The GH injections induced a 117% increase in serum insulin-like growth factor I. The GH-insulin-like growth factor I axis stimulates recruitment of osteoblast precursor cells, resulting in increased bone formation at the periosteal surface. GH injections and mild excercise in combination modulate and increase further the formation and strength of cortical bone in old female rats.

Growth hormone administration can improve growth in glucocorticoid-injected rats without affecting the lymphocytopenic effect of the glucocorticoid.

Long-term glucocorticoid therapy as it is found in children with kidney transplants results in retarded longitudinal growth. The aim of the present study was to evaluate if growth hormone could improve longitudinal growth in glucocorticoid-injected experimental animals without affecting the immuno-suppressive effect of the glucocorticoid. 117 female Wistar rats were injected from the ages of 2-5 months with either saline, growth hormone (5 mg/kg/day), or glucocorticoid (methylprednisolone 1,3,6 or 9 mg/kg/day), alone or in combination with growth hormone (5 mg/kg/day). Body weight, nose-tail length and length of the lower extremity were measured continuously during the study. After death, femoral and tibial lengths, growth at the proximal, epiphyseal growth plate, muscle mass and immunological parameters were examined. Glucocorticoid administration dose-dependently decreased weight gain and growth (nose-tail length, growth of the lower extremity), lengths of femur and tibia, growth at the proximal, epiphyseal growth plate and muscle mass. Glucocorticoid administration decreased spleen and thymus weight as well as the white blood cell count (WBC count), mainly due to a decrease in lymphocyte number. For all glucocorticoid doses examined, growth hormone increased weight gain and growth (nose-tail length, growth of the lower extremity), lengths of femur and tibia, and muscle mass. The effects of growth hormone were, however, dose-dependently decreased by glucocorticoid administration. Growth hormone injection alone increased the WBC count due to an increase in the number of lymphocytes and monocytes. When the two hormones were administered concomitantly, growth hormone did not, however, reverse the lymphocytopenic effect induced by glucocorticoid administration. In conclusion, growth hormone can increase longitudinal growth and increase muscle mass in glucocorticoid-injected rats, if a glucocorticoid preparation of a short half-life is used. Growth hormone does not reverse the lymphocytopenic effect of glucocorticoid injections.

Reduced concentration of collagen reducible cross links in human trabecular bone with respect to age and osteoporosis.

The decrease of bone strength in relation to age and osteoporosis is more pronounced than would be expected from the relative deficit in the amount of bone. Besides bone mass, the mechanical properties of cancellous bone also depend on the microarchitecture and possibly on the molecular structure of inorganic and organic components. The present study examines the bone collagen, especially the collagen cross links, in relation to age and osteoporosis. Samples of vertebral trabecular bone were taken at autopsy from 43 normal individuals, aged 15-90 years. Eleven of these served as sex- and age-matched controls for similar samples from 11 osteoporotic individuals, 70-90 years. The volume of each trabecular bone sample was estimated. After removal of the marrow, the trabeculae were ground to powder and decalcified. The extractability of the bone collagen was studied by repeated extractions with acetic acid and pepsin. The divalent reducible collagen cross-links, dehydro-dihydroxylysinonorleucine (DHLNL) and dehydro-hydroxylysinonorleucine (HLNL), were determined by reducing the bone collagen with tritiated potassium borohydride followed by ion-exchange chromatography. The mature trivalent pyridinium cross links were determined by reverse-phase HPLC with fluorescence detection. The extractability of collagen prepared from the vertebral trabecular bone of control individuals was increased with age. Bone collagen of osteoporotic individuals showed increased extractability and a substantial decrease in the concentration of the divalent reducible collagen cross links (DHLNL reduced by 30% and HLNL by 24%) compared with the sex- and age-matched controls. No alterations were observed in the concentration of the pyridinolines. The divalent reducible cross-links are the most frequent known cross links in bone (2-4 times the concentration of the pyridinium cross links). These changes would therefore be expected to reduce the strength of the bone trabeculae and could explain why the osteoporotic individuals had bone fractures even though the collagen density (mg/cm3) did not differ from that of the sex- and age-matched controls. The microarchitecture of the cancellous bone was not assessed. The osteoporotic and control individuals seemed to have the same amount of trabecular bone, but the quality of the osteoporotic bone collagen was reduced.

Qualitative alterations of cortical bone in female rats after long-term administration of growth hormone and glucocorticoid.

A serious side effect of glucocorticoid treatment is the development of osteoporosis. We have earlier shown that long-term glucocorticoid administration results in a decrease in longitudinal bone growth, cortical bone mass, and biomechanical strength, while growth hormone administration increases these parameters. The result of biomechanical testing also indicates that glucocorticoid administration reduces the quality of bone. The glucocorticoid-induced osteopenia could not be inhibited by concomitant administration of large doses of growth hormone. The aim of the present study was to evaluate why glucocorticoid administration decreases the quality of cortical bone and why growth hormone administration had no beneficial effect on glucocorticoid-induced osteopenia. Five groups of female rats (3 1/2 months old) were treated for 80 days as follows: (1) glucocorticoid (prednisolone: Delcortol 5 mg/kg/day); (2) glucocorticoid and growth hormone; (3) saline; (4) growth hormone (recombinant human growth hormone 5 mg/kg/day); (5) Food restriction (consisting of restricted access to food to reduce their weight gain to match with that of the glucocorticoid injected rats). The animals were injected with tetracycline (15 mg/kg), 18 and 3 days before sacrifice, respectively. Furthermore, a baseline group (3 1/2-month-old female rats) was examined in order to enable us to differentiate between age-related changes and changes due to the hormone administration. Cortical mid-diaphysial cross sections of the femora were prepared and used for histological examination including determination of bone porosity, bone formation rate, and determination of the area of endosteal cavities as an indication of bone resorption. Furthermore, a cortical bone cylinder was cut from the mid-diaphysis and used for examinations of wet weight, dry weight, ash weight, volume, collagen content, and apparent density. Glucocorticoid administration resulted in an almost complete arrest of bone formation as shown by a decreased bone formation rate and a decreased periosteal mineralizing surface. Glucocorticoid administration also increased the porosity of bone indicating increased osteoclast activity. The increased porosity was due to a glucocorticoid-induced increase in the number of endosteal cavities in the mid-diaphysial cross section of the femora. The decreased bone formation and the increased bone resorption can explain the decrease in bone mass (volume and ash weight) found after glucocorticoid administration. Growth hormone administration, on the other hand, resulted in a marked increase in bone formation as shown by a marked increase in bone formation rate and periosteal mineralizing surface. In agreement with this, we found an increase in cortical bone mass (volume and ash weight). When the two hormones were given concomitantly, growth hormone administration did not increase bone formation. Our findings indicate the reason why growth hormone has no beneficial effect on cortical osteopenia induced by a high dose of glucocorticoid with protracted effect.

Growth hormone is not able to counteract osteopenia of rat cortical bone induced by glucocorticoid with protracted effect.

UNLABELLED: Osteopenia and inhibited longitudinal growth in childhood are serious side effects during glucocorticoid therapy. The effects of glucocorticoids on bone have been confirmed in animal experiments. Long-term glucocorticoid administration to rats results in reduced body weights, reduced bone growth (length and cross-sectional area), and bone strength. Glucocorticoid treatment also resulted in a reduced bending stress, indicating reduced bone quality. Growth hormone, on the other hand, increased body weights, bone dimensions, and bone strength. The aim of the present study was to evaluate if growth hormone administration would have an anabolic effect on rat bone when given to animals also receiving a high dosage of glucocorticoid. Five groups of female rats, 3.5 months old, were treated as follows: (1) saline control; (2) glucocorticoid (prednisolone: Delcortol 5 mg/kg/day); (3) growth hormone (recombinant human growth hormone 5 mg/kg/day); (4) glucocorticoid and growth hormone; and (5) food restriction, consisting of restricted access to food to reduce their weight gain to match that of the glucocorticoid injected rats. After 80 days of hormone administration the animals were sacrificed. The right femur was removed and tested biomechanically in a three-point bending procedure. The left femur was used for determination of bone dimensions. Biomechanical parameters (ultimate load and ultimate stiffness) were then normalized to diaphyseal cross-sectional diameters of the femur, giving the values of ultimate bending stress and Young's modulus. RESULTS: administration of both hormones simultaneously could not reverse the decrease in body weights, bone length, and diameters, or the decreased bone strength induced by glucocorticoid administration. In conclusion, growth hormone cannot prevent cortical osteopenia in female rats induced by a high dose of glucocorticoid with protracted effect.

Reduced concentrations of collagen cross-links are associated with reduced strength of bone.

The known cross-links of bone collagen are derived from lysine and hydroxylysine. The first step in the enzymatic cross-linking process is a deamination by lysyl oxidase producing an aldehyde which then may condense with a lysyl or hydroxylysyl residue of a neighbouring collagen molecule. Some of the resulting divalent aldimine and oxo-imine cross-links may later on be incorporated in trivalent hydroxylysyl-pyridinoline and lysyl-pyridinoline cross-links. In bone collagen prepared from the cancellous bone of vertebral bodies of osteoporotic individuals we found a reduced stability towards acetic acid and pepsin, and a substantial reduction in the concentration of the divalent collagen cross-links compared with sex- and age-matched controls. To what extent do the collagen cross-links influence the mechanical properties of bone? beta-amino-propionitrile (BAPN) irreversibly inhibits the enzyme lysyl oxidase and therefore, the formation of cross-links between the collagen molecules. In the present study female rats, 70 days old, injected subcutaneously two times daily with BAPN (333 mg/kg/day) for 1 month and saline injected control rats were studied. The concentration of the hydroxypyridinium cross-links of femoral mid-diaphyseal cortical bone was determined by HPLC with fluorescence detection and the mechanical properties of the rat femoral diaphyses were analyzed by a materials testing machine. The BAPN injections resulted in a 45% reduction in the concentration of the hydroxypyridinium cross-links and a 31% decrease in the stability of the bone collagen towards acetic acid and pepsin compared with the control rats. No changes were found in ash or collagen concentrations of the cortical bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of vertebral body strength by dual photon absorptiometry in elderly individuals: comparison between measurements of total vertebral and vertebral body bone mineral.

The aim of the present study was to investigate the predictive value of bone mineral measurements by dual photon absorptiometry (DPA) in vitro for strength and ash weight of lumbar vertebral bodies in elderly, otherwise nonselected individuals. The material comprised 46 individuals: 26 males (43-95 years) and 20 females (63-95 years) without malignant diseases. Spinal segments, including L2, L3, and L4, were removed en bloc at autopsy. Bone mineral content (BMC) measurements imitating the normal DPA procedure were performed on the segments suspended in a water bath. The segments were measured in toto (BMCT) and remeasured after removal of the posterior elements (BMCB). The second lumbar vertebral body (L2) was then dissected and sawed below the endplates to obtain samples with planoparallel ends before compression in a materials testing machine. Finally, the bone specimens were incinerated for ash weight estimations. BMCT showed significant correlations to vertebral body ash weight (r = 0.79), compressive strength (load, r = 0.69), and stress (load per unit area, r = 0.47). The correlations were improved by removing the posterior elements (BMCB-ash weight, r = 0.86, BMCB-load, r = 0.74, BMCB-stress, r = 0.49). Correction of BMC for differences in vertebral body height (BMC/cm) further increased the correlation coefficients (BMCB/cm-ash weight, r = 0.92, BMCB/cm-load, r = 0.78, BMCB/cm-stress, r = 0.55). We conclude that lumbar BMC is predictive for lumbar vertebral body compressive strength in vitro and ash weight. The correlation coefficient is improved by removing the posterior non-weight-bearing element.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid treatment or food deprivation counteract the stimulating effect of growth hormone on rat cortical bone strength.

Growth hormone (GH) has been found to increase the length, thickness and bending strength of rat femora. The present study was designed to investigate if glucocorticoid treatment or food restriction would interfere with the effect of exogenous GH on bone growth. Male rats treated with GH for 30 days experienced a weight gain of 30-35% and longitudinal and periosteal femoral growth. A dose-related increase in the bending strength of the femora was found and was explained by an increased thickness of the femora. In spite of a reduced real density, biomechanical competence was preserved after GH treatment. GH treatment combined with a relatively small dose of glucocorticoid, which in itself had no significant effect on bone growth and strength, reduced the stimulating effect of GH on body weight gain, femoral growth and strength. GH-treated rats that were food restricted, so as to limit their body weight gain to that of the saline group, experienced significant longitudinal and periosteal femoral growth. Bone strength, however, was not increased, which conforms to a reduced mineralization and increased porosity of the femora. Young's modulus (normalized bone stiffness) was significantly decreased in this group, probably as a result of decreased mineralization. Furthermore, the combination of GH treatment and food restriction resulted in a reduced apparent density indicating increased bone resorption.