Charlotte Landbo Tofteng

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.

Hormonal replacement therapy reduces forearm fracture incidence in recent postmenopausal women — results of the Danish Osteoporosis Prevention Study

OBJECTIVES To study the fracture reducing potential of hormonal replacement therapy (HRT) in recent postmenopausal women in a primary preventive scenario. METHODS Prospective controlled comprehensive cohort trial: 2016 healthy women aged 45-58 years, from three to 24 months past last menstrual bleeding were recruited from a random sample of the background population. Mean age was 50. 8+/-2.8 years, and the number of person years followed was 9335.3. There were two main study arms: a randomised arm (randomised to HRT; n=502, or not; n=504) and a non-randomised arm (on HRT; n=221, or not; n=789 by own choice). First line HRT was oral sequential oestradiol/norethisterone in women with intact uterus and oral continuous oestradiol in hysterectomised women. RESULTS After five years, a total of 156 fractures were sustained by 140 women. There were 51 forearm fractures in 51 women. By intention-to-treat analysis (n=2016), overall fracture risk was borderline statistically significantly reduced (RR=0.73, 95% CI: 0.50-1.05), and forearm fracture risk was significantly reduced (RR=0.45, 95% CI: 0.22-0.90) with HRT. Restricting the analysis to women who had adhered to their initial allocation of either HRT (n=395) or no HRT (n=977) showed a significant reduction in both the overall fracture risk (RR=0.61, 95% CI: 0.39-0.97) and the risk of forearm fractures (RR=0.24, 95% CI: 0.09-0.69). Compliance with HRT was 65% after five years. CONCLUSIONS It is possible to reduce the number of forearm fractures and possibly the total number of fractures in recent postmenopausal women by use of HRT as primary prevention.

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.

Single nucleotide polymorphisms in the P2X7 gene are associated to fracture risk and to effect of estrogen treatment

Objectives The purinergic P2RX7 receptor (P2RX7) has been shown to play a role in the regulation of osteoblast and osteoclast activity. The aim of this study was to determine the presence of polymorphisms in exon 13 of the P2X7 gene and the association with osteoclast apoptosis in vitro and bone status in vivo. Methods A total of 1764 postmenopausal women were genotyped for three single nucleotide polymorphisms detected after sequencing of exon 13 of P2X7. Bone markers, bone mineral density of the hip and lumbar spine were determined at baseline and after 10 years, and vertebral fracture incidence after 10 years. In-vitro ATP-induced caspase-1 determinations were performed on osteoclasts from the different genotypes. Results Three polymorphisms were detected (Gln460Arg, Glu496Ala, and Ile568Asn). None of the polymorphisms was related to bone mineral density or changes in bone mineral density over 10 years in hormone replacement therapy naïve women. The Ile568Asn polymorphism was however, associated with effect of hormone replacement therapy. Furthermore, the 10-year fracture incidence was significantly associated with both the Glu496Ala and the Ile568Asn. The Glu496Ala polymorphism was closely related to ATP-induced osteoclast apoptosis in vitro, as osteoclasts from individuals homozygous for the C allele had significantly decreased apoptotic activity. Conclusion The P2X7 Glu496Ala and the Ile568Asn single nucleotide polymorphisms are associated with 10-year fracture risk in postmenopausal women and response to hormone replacement therapy treatment. Further, the Glu496Ala polymorphism is strongly influencing osteoclast apoptosis in vitro, which could contribute to increased fracture risk.

Polymorphisms in the CYP19 and AR genes--relation to bone mass and longitudinal bone changes in postmenopausal women with or without hormone replacement therapy: The Danish Osteoporosis Prevention Study.

Polymorphisms in the androgen receptor (AR) gene and genes encoding enzymes involved in synthesis of sex steroids (e.g., the CYP19 gene encoding aromatase) have recently received attention in osteoporosis research. In the Danish Osteoporosis Prevention Study, recent postmenopausal women were allocated to either hormone replacement therapy (HRT) or no treatment. We genotyped 1792 women for the CYP19 (TTTA)n repeat [short (TTTA)n ≤ 7 or long (TTTA)n > 7] the CYP19 C1558-T, and the AR (CAG)n repeat polymorphism [short (CAG)n < 22, long (CAG)n ≥ 22], and investigated associations with bone mineral density (BMD) and 5-year change in BMD. The CYP19 polymorphisms were in strong linkage disequilibrium. Perimenopausal bone mass or bone loss in untreated women was not associated with the CYP19 polymorphisms. In hormone-treated women, BMD increase in the femoral neck was highest (+0.3%/year) for long CYP19 alleles, lowest (−0.09%/year) for short alleles, and intermediate (−0.002%/year) in heterozygous women, P = 0.015. Differences were also significant in the lumbar spine, total hip, and ultradistal forearm. The C1558-T T-allele was associated with a more pronounced response to HRT (P = 0.04, total hip). AR genotype was not related to BMD, but a modifying effect of sex hormone-binding globulin (SHBG) was present. In the highest SHBG quartile (SHBG > 95 nmol/1, n = 222), AR genotype was associated with baseline BMD (femoral neck: P < 0.001, total hip: P = 0.008), but without a clear gene dosage effect. We have demonstrated that polymorphisms in the CYP19 gene are associated with the magnitude of bone gain in response to HRT and that the (CAG)n repeat polymorphism in the AR gene is associated with bone mass in women with high levels of SHBG. These findings emphasize the complexity of the genetics of bone mass and bone loss.

Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures

The purinergic P2X7 receptor has a major role in the regulation of osteoblast and osteoclast activity and changes in receptor function may therefore affect bone mass in vivo. The aim of this study was to determine the association of non-synonymous single-nucleotide polymorphisms in the P2RX7 gene to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45–58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral fracture incidence was documented at 10 years. We found that the rate of bone loss was clearly associated with the Arg307Gln amino acid substitution such that individuals heterozygous for this polymorphism had a 40% increased rate of bone loss. Furthermore, individuals carrying the Ile568Asn variant allele had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using this model, we found a clear association between the low-risk (high-P2X7 function) alleles and low rate of bone loss. Conversely, high-risk (reduced P2X7 function) alleles were associated with a high rate of bone loss. In conclusion, an association was demonstrated between variants that reduce P2X7 receptor function and increased rate of bone loss. These data support that the P2X7 receptor is important in regulation of bone mass.

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.

Discordance Between Changes in Bone Mineral Density Measured at Different Skeletal Sites in Perimenopausal Women—Implications for Assessment of Bone Loss and Response to Therapy: The Danish Osteoporosis Prevention Study

Assessing bone loss and gain is important in clinical decision‐making, both in evaluating treatment and in following untreated patients. The aim of this study was to correlate changes in bone mineral density (BMD) at different skeletal sites during the first 5 years after menopause and determine if forearm measurements can substitute for dual‐energy X‐ray absorptiometry (DXA) of the spine and hip. BMD was measured at 0, 1, 2, 3, and 5 years using Hologic 1000/W and 2000 densitometers in 2016 perimenopausal women participating in a national cohort study. This analysis comprises 1422 women remaining in the study after 5 years without changes to their initial treatment (hormone‐replacement therapy [HRT], n = 497, or none, n = 925). Despite correlated rates of change between forearm and spine (r2 = 0.11; p < 0.01), one‐half of those who experienced a significant decrease in spine BMD at 5 years showed no significant fall in forearm BMD (sensitivity, 50%; specificity, 85%; κ = 0.25). The total hip had significant better agreement with spine (sensitivity, 63%; specificity, 85%; κ = 0.37; p < 0.01). Analysis of quartiles of change also showed significant better agreement with spine and whole body for the total hip than for the femoral neck or ultradistal (UD) forearm. In a logistic regression analysis for identification of group (HRT or control), the prediction was best for whole body (82.6%) and spine (80.9%), followed by total hip (78.5%) and forearm (74.7%). In conclusion, changes at the commonly measured sites are discordant, and DXA of the forearm is less useful than DXA of the hip or spine in determining the overall skeletal response to therapy or assessing bone loss in untreated women.

A CAG Repeat Polymorphism in the Androgen Receptor Gene is Associated with Reduced Bone Mass and Increased Risk of Osteoporotic Fractures

Osteoporosis is a common disease with a strong genetic component. Hypogonadism results in low bone mass and it increases significantly the risk of osteoporosis in both sexes. The estrogen and androgen receptor genes are therefore strong candidates for mediating the genetic influence on bone mass and risk of osteoporosis. A CAG repeat in the first exon of the androgen receptor (AR) is associated with reduced transcriptional activity of the AR. We therefore examined whether this CAG repeat polymorphism is associated with changes in bone mass and risk of osteoporotic fractures in 284 osteoporotic patients with vertebral fractures and 327 normal individuals. The number of CAG repeats varied between 13 and 30 in men and between 7 and 34 in women. The short and long alleles comprised 19.2 ± 2.5 and 19.0 ± 2.3 repeats (ns) and 22.7 ± 2.4 and 21.9 ± 2.4 (P < 0.01) in women with vertebral fractures and normal women, respectively. This difference was also reflected in the average number of CAG repeats: 21.0 ± 2.0 in osteoporotic women vs. 20.5 ± 2.0 in normal women (P < 0.05). 54.8% of women with osteoporotic fractures vs. 45.9% of normal women had average number of CAG repeats of 21 and more (χ2 = 3.11, P = 0.08). Logistic regression analyses revealed that both the average number of CAG repeats and the length of the long allele were significant predictors of osteoporotic fractures in women (P < 0.05 and P < 0.01, respectively). Men with vertebral fractures had 20.0 ± 2.8 CAG repeats compared with 20.7 ± 2.5 CAG repeats in normal men (ns). Linear regression analysis revealed that the length of the long allele was negatively correlated with BMD of the lumbar spine (P < 0.05) and femoral neck (P < 0.05) in women. In men, linear regression analyses demonstrated that BMD of the lumbar spine (P < 0.05), femoral neck (P < 0.05) and total hip (P < 0.05) was positively correlated with length of the CAG repeat polymorphism. In conclusion, we have demonstrated that the CAG repeat polymorphism in the first exon of the AR gene is associated with reduced bone mass and increased risk of osteoporotic fractures in women.

Two polymorphisms in the vitamin D receptor gene--association with bone mass and 5-year change in bone mass with or without hormone-replacement therapy in postmenopausal women: the Danish Osteoporosis Prevention Study.

The significance of an interrelation between nongenetic factors and genotype effects in the regulation of bone mass is not clear. In this prospective study of 429 healthy early postmenopausal Danish women, we investigated the association between bone mineral density (BMD) and the FokI and BsmI polymorphisms in the vitamin D receptor (VDR) gene. Participants were allocated to either hormone‐replacement therapy (HRT) or no treatment by randomization or personal choice. After 5 years, 332 women with unchanged treatment status were available for analyses, 98 of these women were still on HRT. No association with initial BMD or 5‐year change in BMD was found for either polymorphism. In women with body mass index (BMI) < 25 (n = 282), the f allele was associated with lower BMD of the hip (p < 0.001) and forearm (p = 0.001), and the b allele was associated with lower spine BMD (p = 0.02). Comparing thin/normal weight women with overweight/obese women of the same genotype, FF women had similar BMD at all measured sites in contrast to Ff andff women in whom BMD, as expected, was higher in the overweight/obese women. Similar results were found for the BsmI polymorphism with no difference in BMD between BMI groups in BB women. Segregation into groups according to dietary calcium intake did not reveal any genotype association with BMD. These results provide some evidence of a modifying effect of nongenetic factors, specifically BMI, on the association between VDR genotype and BMD. High BMI may protect against lower BMD seen in association with the f or b alleles. In some genotypes (FF and BB), BMI had relatively little effect on BMD.