Head-to-Head Comparison of Tc-99m-sestamibi SPECT/CT and C-11-L-Methionin PET/CT in Parathyroid Scanning Before Operation for Primary Hyperparathyroidism.

PURPOSE: The preferred nuclear medicine method for identification of hyperfunctioning parathyroid glands in hyperparathyroidism (HPT) develops continuously in relation to the technological progress. Diagnostic methods based on PET/CT have during recent years evolved with new tracer possibilities competing with traditional scintigraphic methods. This investigation is a head-to-head comparison of Tc-99m-sestamibi SPECT/CT gamma camera scintigraphy (sestamibi SPECT/CT) and C-11-L-methionin PET/CT imaging (methionine PET/CT) for preoperative identification of hyperfunctioning parathyroid glands. PROCEDURES: The study is a prospective cohort study including 27 patients diagnosed with primary hyperparathyroidism (PHPT). Two nuclear medicine physicians assessed all examinations independently and blinded. All scanning assessments were matched to the final surgical diagnosis as confirmed by histopathology. Biochemical monitoring of the therapeutical effects was performed preoperatively by PTH-measurements and followed postoperatively for up to 12 months. Comparisons were made for differences in sensitivity and positive predictive value (PPV). RESULTS: Twenty-seven patients (18 females, 9 males; mean age (range): 58.9 years (34.1-79)) were enrolled into the study. The 27 patients had a total of 33 identified sites of lesions of which 28 (85%) turned out to be histopathological verified hyperfunctioning parathyroid glands. The sensitivity and PPV for sestamibi SPECT/CT were 0.71 and 0.95; that of methionine PET/CT was 0.82 and 1, respectively. Both sensitivity and PPV were slightly lower for sestamibi SPECT/CT than for methionine PET PET/CT (-0.11, 95% confidence interval (95% CI): -0.29 to 0.08; -0.05, 95% CI: -0.14 to 0.04, respectively), but not to a statistically significant extent (p=0.38 and p=0.31). The sensitivity and PPV for diagnostic CT were 0.64 (95% CI: 0.44 to 0.81) and 1 (95% CI: 0.81 to 1). CONCLUSIONS: Methionine PET/CT performed comparable to sestamibi SPECT/CT with respect to identification and localization of hyperfunctioning parathyroid glands prior to surgery.

A pragmatic real-life study of flash glucose monitoring versus self-monitoring of blood glucose.

INTRODUCTION: The aim of this study was to investigate if the flash glucose level monitoring system (FGM) is better than traditional self-monitoring of blood glucose level (SMBG) in helping patients in the outpatient clinic control their blood glucose and improve glycaemic control measured by the concentration of glycated haemoglobin (HbA1c). METHODS: This was an observational real-life study based on data retrieved from a regional diabetes database and conducted in patients with Type 1 diabetes. HbA1c levels at baseline, and at six, nine and 12 months were compared in and between two groups counting 128 patients each. One group included patients who had recently started using the FGM system; the other patients who were using SMBG and otherwise following the routine protocol of the outpatient clinic. RESULTS: We found no difference between the FGM group and the SMBG group with respect to age, sex, weight, diabetes duration or HbA1c at baseline. After six months, HbA1c had been reduced from 64 to 60 mmol/mol (p = 0.00) in the FGM group, whereas it remained unchanged in the SMBG group (from 63 to 63 mmol/mol, p = 0.66). According to the ANOVA repeated measures test, HbA1c measures showed a significant trend of reduction from 65 to 60 mmol/mol (p = 0.002) over 12 months in the FGM group and no trend of reduction in the SMBG group (from 63 to 64 mmol/mol, p = 0.386) Conclusions: Changing the blood glucose measuring method from SMBG to FGM helped patients with Type 1 diabetes in an outpatient clinic reduce their HbA1c. FUNDING: none. TRIAL REGISTRATION: not relevant.

Bone Matrix Levels of Dickkopf and Sclerostin are Positively Correlated with Bone Mass and Strength in Postmenopausal Osteoporosis.

Wnt signaling plays a pivotal role in maintaining bone mass. Secreted pathway modulators such as sclerostin (SOST) and Dickkopfs (DKKs) may influence bone mass inhibiting the canonical Wnt pathway. We evaluated whether bone protein content of secreted Wnt antagonists is related to age, bone mass, and strength in postmenopausal osteoporosis. We measured cortical and trabecular bone contents of SOST and Dickkopf-1 (DKK1) in combined extracts obtained after ethylenediaminetetraacetic acid and guanidine hydrochloride extraction in 56 postmenopausal women aged 47-74 (mean, 63) yr with a previous distal forearm fracture and a hip or spine Z-score less than 0. Our findings were (i) SOST and DKK1 protein levels were higher in trabecular bone, (ii) cortical and trabecular DKK1 and trabecular SOST correlated positively with bone matrix levels of osteocalcin (r between 0.28 and 0.45, p < 0.05), (iii) cortical DKK1 correlated with lumbar spine bone mineral density (BMD) (r = 0.32, p < 0.05) and femoral neck BMD (r = 0.41, p < 0.01), and (iv) cortical DKK1 and SOST correlated with apparent bone volumetric density and compressive strength (r between 0.34 and 0.51, p < 0.01). In conclusion, cortical bone matrix levels of DKK1 and SOST were positively correlated with bone mass and bone strength in postmenopausal osteoporotic women.

Effect of hormone replacement therapy on cardiovascular events in recently postmenopausal women: randomised trial.

OBJECTIVE: To investigate the long term effect of hormone replacement therapy on cardiovascular outcomes in recently postmenopausal women. DESIGN: Open label, randomised controlled trial. SETTING: Denmark, 1990-93. PARTICIPANTS: 1006 healthy women aged 45-58 who were recently postmenopausal or had perimenopausal symptoms in combination with recorded postmenopausal serum follicle stimulating hormone values. 502 women were randomly allocated to receive hormone replacement therapy and 504 to receive no treatment (control). Women who had undergone hysterectomy were included if they were aged 45-52 and had recorded values for postmenopausal serum follicle stimulating hormone. INTERVENTIONS: In the treatment group, women with an intact uterus were treated with triphasic estradiol and norethisterone acetate and women who had undergone hysterectomy received 2 mg estradiol a day. Intervention was stopped after about 11 years owing to adverse reports from other trials, but participants were followed for death, cardiovascular disease, and cancer for up to 16 years. Sensitivity analyses were carried out on women who took more than 80% of the prescribed treatment for five years. MAIN OUTCOME MEASURE: The primary endpoint was a composite of death, admission to hospital for heart failure, and myocardial infarction. RESULTS: At inclusion the women on average were aged 50 and had been postmenopausal for seven months. After 10 years of intervention, 16 women in the treatment group experienced the primary composite endpoint compared with 33 in the control group (hazard ratio 0.48, 95% confidence interval 0.26 to 0.87; P=0.015) and 15 died compared with 26 (0.57, 0.30 to 1.08; P=0.084). The reduction in cardiovascular events was not associated with an increase in any cancer (36 in treated group v 39 in control group, 0.92, 0.58 to 1.45; P=0.71) or in breast cancer (10 in treated group v 17 in control group, 0.58, 0.27 to 1.27; P=0.17). The hazard ratio for deep vein thrombosis (2 in treated group v 1 in control group) was 2.01 (0.18 to 22.16) and for stroke (11 in treated group v 14 in control group) was 0.77 (0.35 to 1.70). After 16 years the reduction in the primary composite outcome was still present and not associated with an increase in any cancer. CONCLUSIONS: After 10 years of randomised treatment, women receiving hormone replacement therapy early after menopause had a significantly reduced risk of mortality, heart failure, or myocardial infarction, without any apparent increase in risk of cancer, venous thromboembolism, or stroke. TRIAL REGISTRATION: ClinicalTrials.gov NCT00252408.

Vitamin D deficiency in postmenopausal, healthy women predicts increased cardiovascular events: a 16-year follow-up study.

OBJECTIVE: To investigate the relationship between vitamin D status in healthy women and cardiovascular outcome. DESIGN AND METHODS: Between 1990 and 1993, 2016 healthy, recently postmenopausal women were enrolled in the Danish Osteoporosis Prevention Study. Serum levels of 25-hydroxyvitamin D (25(OH)D, nmol/l) were measured at baseline. Participants were followed for 16 years. The primary end point was a combination of death, heart failure, myocardial infarction (MI) and stroke. Vitamin D deficiency was defined as serum 25(OH)D<50 nmol/l. The primary end point was adjusted for other risk factors of adverse cardiovascular events (age, smoking, blood pressure, hip-waist ratio, education and family history of MI). RESULTS: At baseline, mean age was 50 years and BMI 25. Women with vitamin D deficiency (n=788) had more cardiovascular risk factors than vitamin D-replete women (n=1225). Compared with vitamin D-replete women, women with low 25(OH)D levels had significantly higher BMI and triglycerides, lower HDL and hip-waist ratio and less education. More were smokers among the vitamin D deficient (47 vs 38%). A primary end point was experienced by 118 (15%) with vitamin D deficiency and by 125 (10%) of the vitamin D replete. Hazard ratio (HR) was 1.49 (95% confidence interval: 1.16-1.92; P=0.002) in the vitamin D deficient. Adjusted HR was 1.32 (1.02-1.71; P=0.03). In total, 135 women died; of these, 65 (8%) were of the vitamin D deficient and 70 (6%) in the vitamin D-replete group; unadjusted HR was 1.44 (1.02-2.01; P=0.04) for vitamin D deficiency. CONCLUSION: Healthy women with vitamin D deficiency have increased risk of adverse cardiovascular outcome.

Abnormal muscle and hematopoietic gene expression may be important for clinical morbidity in primary hyperparathyroidism.

In primary hyperparathyroidism (PHPT), excess PTH secretion by adenomatous or hyperplastic parathyroid glands leads to elevated serum [Ca(2+)]. Patients present complex symptoms of muscular fatigue, various neuropsychiatric, neuromuscular, and cardiovascular manifestations, and, in advanced disease, kidney stones and metabolic bone disease. Our objective was to characterize changes in muscle and hematopoietic gene expression in patients with reversible mild PHPT after parathyroidectomy and possibly link molecular pathology to symptoms. Global mRNA profiling using Affymetrix gene chips was carried out in biopsies obtained before and 1 yr after parathyroidectomy in seven patients discovered by routine blood [Ca(2+)] screening. The tissue distribution of PTH receptor (PTHR1 and PTHR2) mRNAs were quantitated using real-time RT-PCR in unrelated persons to define PTH target tissues. Of about 10,000 expressed genes, 175 muscle, 169 hematological, and 99 bone-associated mRNAs were affected. Notably, the major part of muscle-related mRNAs was increased whereas hematological mRNAs were predominantly decreased during disease. Functional and molecular network analysis demonstrated major alterations of several tissue characteristic groups of mRNAs as well as those belonging to common cell signaling and major metabolic pathways. PTHR1 and PTHR2 mRNAs were more abundantly expressed in muscle and brain than in hematopoietic cells. We suggest that sustained stimulation of PTH receptors present in brain, muscle, and hematopoietic cells have to be considered as one independent, important cause of molecular disease in PHPT leading to profound alterations in gene expression that may help explain symptoms like muscle fatigue, cardiovascular pathology, and precipitation of psychiatric illness.

Gene expression profiles give insight into the molecular pathology of bone in primary hyperparathyroidism.

Global gene expression profiling has been used to study the molecular mechanisms of increased bone remodeling caused by PHPT. This disease is a model for chronic over-stimulation of target organs by PTH due to an inappropriate overproduction of the hormone. Hyperactivity of osteoblasts and osteoclasts lead to increased calcium and phosphate mobilization from the skeleton and hypercalcaemia. The ensemble of genes that alter expression and thus is responsible for the effects of chronic PTH stimulation is today largely unknown. The differentiated gene expression profiles revealed characteristic molecular disease modalities which define the bone remodeling abnormalities occurring in PTH dependent osteodystrophy. We analyzed mRNAs in transiliacal bone biopsies from 7 patients with PHPT using Affymetrix HG-U133A Gene Chips containing more than 22000 different probe sets. Similar analyses of the global transcriptional activity were repeated in a second bone biopsy from the same patient taken one year after surgery and reversal of disease parameters. Real time PCR was carried out on many genes for corroboration of the results. Out of more than 14500 different genes examined, 99 which were related to bone and extra-cellular matrix, showed altered expression. Of these were 85 up- and 14 down-regulated before operation. The majority of regulated genes represented structural and adhesion proteins, but included also proteases and protease regulators which promote resorption. Increased expressions of collagen type 1 and osteocalcin mRNAs in disease reflecting the PTH anabolic action were paralleled by increased concentrations of these proteins in serum. In addition, genes encoding transcriptional factors and their regulators as well as cellular signal molecules were up-regulated during disease. The identified genetic signature represents the first extensive description of the ensemble of bone and matrix related mRNAs, which are regulated by chronic PTH action. These results identify the molecular basis for this skeletal disease, and provide new insight into this clinical condition with potential bearing on future treatment.

Circulating amounts of osteoprotegerin and RANK ligand: genetic influence and relationship with BMD assessed in female twins.

UNLABELLED: Osteoprotegerin (OPG) is a circulating receptor that inhibits osteoclastogenesis by binding to RANK ligand (RANKL). OPG knock-out animals develop severe osteoporosis. Treatment with OPG lowers bone resorption and increases BMD. OPG production is influenced by a wide range of hormones and cytokines. The influence of genetic factors on circulating amounts of OPG and RANKL is not known. BMD has been demonstrated to have a high heritability and there is evidence also that bone turnover and bone loss rates are controlled at least in part by genetic factors. OBJECTIVE: Assessing the genetic impact on serum OPG and RANKL in women and estimation of the relative contribution of this inheritance to the total heritability of BMD. METHODS: 188 female twins (52 DZ and 42 MZ pairs) from the Danish Twin Registry were included in the study. Mean age was 35 years (range 19-64 years), average spine BMD was 1.04 +/- 0.11 g/cm2. Serum levels of OPG and RANKL were measured by ELISA (Biomedica, Vienna, Austria). This register covers twins born in Denmark since 1870. Heritability and environmental influence was assessed using a maximum-likelihood model for genetic pleiotropy. RESULTS: RANKL levels showed a negative correlation with age and lower values in smokers. OPG levels were higher in postmenopausal women. Heritability (h(2)) was 85% for spine BMD and 52% for serum RANKL after adjustment for age, smoking and BMI. By contrast, there was no significant genetic influence on OPG levels (h(2) = 0, 95% CI: 0 to 0.31). Serum OPG was determined almost exclusively by individual environment (e(2) = 0.79), with a small, non-significant contribution from shared environment (c(2) = 0.21). Restricting analyses to the 158 premenopausal twins did not alter the findings. CONCLUSIONS: Serum OPG and RANKL levels have only a weak relation to BMD in healthy women. Phenotype correlations indicate that the genes that contribute to twin similarity for BMD are not genes regulating serum levels of RANKL or OPG. The weak correlation with BMD appears to consist in shared environmental factors.

Are effects of MTHFR (C677T) genotype on BMD confined to women with low folate and riboflavin intake? Analysis of food records from the Danish osteoporosis prevention study.

We have previously found BMD and fracture risk to be significantly associated with the MTHFR (C677T) polymorphism in healthy postmenopausal women in the first years after menopause. Since then, other cohort studies have suggested that sufficient intake of riboflavin and/or folate may have the potential to prevent development of low BMD in women with the TT genotype. This could to some extent explain why this polymorphism is associated with low BMD or fracture in some study populations and not in others. It would also indicate that fractures associated with the TT genotype could be preventable by vitamin B supplementation. We have, therefore, reviewed baseline food record data from our original study to determine if BMD and fracture associations with the MTHFR genotype depended on the intake of folate, riboflavin, or other members of the vitamin B complex, associated with homocysteine metabolism. We analyzed genotype, BMD, and dietary records from 1700 healthy postmenopausal women who participated in the DOPS study. For the assessment of fracture risk, we used longitudinal observations from 854 women in the control group who remained compliant with their initial allocation of no treatment. Riboflavin intake was significantly correlated with femoral neck (FN) BMD in women with the TT genotype (r = 0.24, P < 0.01). FN and lumbar spine (LS) BMD were only associated with the MTHFR genotype in the lowest quartile of riboflavin intake. At the FN, similar threshold effects were shown for folate, vitamin B12, and vitamin B6. Among these vitamin B complex members, stepwise regression analysis identified riboflavin as the only significant predictor of FN BMD in the TT genotype. In conclusion, we confirm reports that BMD in the MTHFR TT genotype is only significantly reduced in the lowest quartile of riboflavin, B12, B6, and folate intake, at least at the time of menopause. Vitamin B supplementation would only be expected to benefit BMD in about 2% of the population, i.e., those with the TT genotype and low vitamin B intake.

Increased RANKL/OPG mRNA ratio in iliac bone biopsies from women with hip fractures.

RANKL (receptor activator of NF-kappaB) is a potent physiological inducer of osteoclastogenesis. Its actions are blocked by the decoy receptor osteoprotegerin (OPG), and treatment with OPG blocks bone resorption in postmenopausal women. Both positive and negative associations between serum OPG and bone mineral density (BMD) have been reported in the literature. We hypothesized that decreased OPG production relative to RANKL within bone itself could lead to increased risk of osteoporotic fracture. We included ten women with hip fracture (age 76.3 +/- 8.0 years, N.S, : hip BMD 0.686 +/- 1.3 g/cm2, P < 0.05) and 24 women with osteoarthrosis of the hip (age 72.8 +/- 7.2 years, hip BMD 0.832 +/- 1.1 g/cm(2)). Transiliac biopsies were obtained at the time of surgery. Total RNA was extracted from biopsies and reverse-transcribed. Real-time quantification of mRNA was performed with a SYBR Green I real time PCR assay, calculating relative gene expression with normalization of results for beta actin mRNA. Actin normalized mRNA levels for OPG and interleukin (IL)-6 were significantly lower in fracture patients, with a significantly higher RANKL/OPG ratio in patients with fractures. There was no significant difference in tumor necrosis factor (TNF), IL-1, IL-1ra, or IL-7 expression. IL-6 mRNA levels were lower in fracture patients (P < 0.05). The effect of increased RANKL/OPG ratio (Z = 2.08, P < 0.05) on fracture risk was additive to that of hip BMD T score (Z = -1.95, P < 0.05) when assessed using logistic regression. Elderly women with hip fractures exhibit an increased RANKL/OPG mRNA content of iliac bone. This is associated with increased fracture susceptibility, which is not in itself explained by low BMD.

Effects of 5 years of hormonal replacement therapy on menopausal symptoms and blood pressure-a randomised controlled study.

OBJECTIVES: To study the effects of hormonal replacement on hot flushes, other symptoms linked to menopause, and blood pressure. METHODS: The study included 1006 early postmenopausal women aged 45-58 years, participating in the Danish Osteoporosis Prevention Study (DOPS) randomised to Hormonal replacement therapy (HRT) (n=502) or no HRT (n=504) in an open label trial. Symptom scores were recorded at baseline, after 6 month, 1, 2, and 5 years on a modified Greene scale (range 0-4 with 0 equalling no symptoms, and 4 maximal symptoms). RESULTS: HRT efficiently alleviated hot flushes (mean+/-S.E.M. score 0.48+/-0.04 in HRT vs. 0.83+/-0.05 in no HRT after 5 years, P<0.01 by repeated measures ANOVA), sleeping difficulties associated with hot flushes (0.21+/-0.60 vs. 0.37+/-0.86, P<0.01), vaginal dryness (0.45+/-0.04 vs. 0.73+/-0.05, P<0.01), dyspareunia (0.27+/-0.04 vs. 0.39+/-0.04, P<0.01), and libido (0.48+/-0.05 vs. 0.59+/-0.05, P=0.08). In the untreated group the occurrence of mood swings (from 0.77+/-0.05 at baseline to 0.45+/-0.04 after 5 years, 2P<0.01) and oedemas (from 0.59+/-0.04 to 0.43+/-0.04, 2P=0.02) decreased with age while the occurrence of incontinence increased (from 0.43+/-0.03 to 0.52+/-0.04, 2P<0.01). These changes were not influenced by HRT. Furthermore, HRT had no influence on presence of headache (0.54+/-0.05 vs. 0.58+/-0.05 after 5 years), voiding pattern (0.49+/-0.04 vs. 0.53+/-0.04), or blood pressure (mean systolic pressure 123+/-18 vs. 123+/-19, diastolic pressure 77+/-10 vs. 77+/-11). CONCLUSIONS: HRT is efficient in controlling hot flushes and vaginal dryness, and symptoms related to these conditions. However, no effect on blood pressure or other menopause symptoms was recorded.

Standardization of BMD T-Scores in the first five years after the menopause: do femoral neck-equivalent and older normative range T-Scores improve diagnostic agreement?

Calculating T-scores using an older reference population reduces inconsistency between measurement sites when osteoporosis is diagnosed in the elderly. The present analysis in a younger, early postmenopausal cohort examined 5-yr consistency of normalization by local and femoral neck-equivalent T-scores. NHANES (femur) and Hologic (spine and forearm) references were applied to baseline, 1-, 2-, 3-, and 5-yr scans in 925 untreated women in a national cohort study, and alternative local and neck-equivalent scores calculated. The baseline prevalence of osteopenia/osteoporosis was 35.5%/4.1% (spine), 31.0%/1.2% (neck), 31.3%/1.2% (total hip), and 37.2%/2.5% (forearm). It increased to 54.6%/7% by combining sites. The prevalences at 5-yr were 57.2%/12.4% (spine), 51.9%/5.0% (neck), 46.6%/3.7% (total hip), 52.5%/7.4% (forearm), and 77.3%/17.8% (any). A T-score cut-off at the lowest of four sites of -1.65 for osteopenia and -3.37 for osteoporosis was equivalent in patient numbers to T<-1 and T<-2.5 at the femoral neck. The proportion of inconsistently classified subjects decreased from 48% to 42% (p<0.05) with neck-equivalent scores. No improvement remained after 5 yr. Kappa scores did not improve by the use of local or femoral neck scores. In conclusion, adjusted thresholds cannot remove the anatomic discrepancy between T-scores. To overcome this problem, risk-based diagnostic cut-offs must therefore be calculated separately for each measurement site and fracture localization.

A common methylenetetrahydrofolate reductase (C677T) polymorphism is associated with low bone mineral density and increased fracture incidence after menopause: longitudinal data from the Danish osteoporosis prevention study.

A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) has recently been associated with bone mineral density (BMD) in postmenopausal Japanese women. It is not known whether this effect is also present in European populations and whether it is caused by lower peak bone mass or accelerated postmenopausal bone loss. MTHFR genotyping was done in 1748 healthy postmenopausal Danish women participating in a prospective study of risk factors for osteoporosis. At the time of enrollment, 3-24 months after last menstrual period, the less prevalent genotype (TT, 8.7% of the population) was associated with significantly lower BMD at the femoral neck (ANOVA, p < 0.05), total hip (p < 0.01), and spine (p < 0.05 adjusted for lifestyle covariates, p = 0.06 without adjustment). The mean difference was between 0.1 and 0.3 SD, depending on measurement site. MTHFR genotype added significantly to prediction of BMD by weight and age. Fracture incidence was increased more than 2-fold in subjects with the TT genotype (risk ratio [RR], 2.6; 95% CI 1.2-5.6). This remained significant when the Cox analysis was controlled for BMD (RR, 2.4; 95% CI 1.1-5.2). No differences in serum osteocalcin, bone-specific alkaline phosphatase, and 25-OH-vitamin D were found between genotypes. The response to hormone replacement therapy (HRT) did not differ, but the association of the TT genotype with reduced BMD was maintained at the total hip after 5 years of HRT. The MTHFR TT genotype is associated with low BMD and increased fracture incidence in early postmenopausal women.

Effect of long-term hormone replacement therapy on plasma homocysteine in postmenopausal women: a randomized controlled study.

OBJECTIVE: The purpose of this study was to investigate the long-term effect of hormone replacement therapy on total homocysteine and to study whether there was any difference in effect between opposed and unopposed hormone replacement therapy or whether the methylenetetrahydrofolate reductase C677T polymorphism was associated with the effect of hormone replacement therapy on total homocysteine. STUDY DESIGN: Two hundred nine healthy postmenopausal women were randomized to hormone replacement therapy (n = 103) or no substitution (n = 106) 5 to 7 years earlier. RESULTS: Women who received hormone replacement therapy had significantly lower total homocysteine concentrations than women in the control group; median total homocysteine values were 8.6 micromol/L and 9.7 micromol/L, respectively, in a per-protocol analysis (P =.02). The effect was comparable in all methylenetetrahydrofolate reductase genotypes, and no difference between unopposed and opposed hormone replacement therapy could be demonstrated. Similar results were obtained when an intention-to-treat analysis was performed. CONCLUSION: Long-term hormone replacement therapy results in lower total homocysteine concentrations in all methylenetetrahydrofolate reductase genotypes without demonstrable difference in effect between unopposed and opposed hormone replacement therapy.