Efficacy and safety of estrogen therapy for prevention of osteoporosis in postmenopausal women

Introduction

Osteoporosis is a common bone disease that is characterized by loss of bone mass and structural deterioration of bone tissue. Osteoporosis has significant physical, emotional, medical, and financial consequences. Fractures related to osteoporosis can lead to diminished quality of life, disability, and even death. Also, the loss of height, deformity of the spine, or prolonged immobilization due to osteoporotic fractures often have adverse consequences, leading to feelings of anxiety or depression. ¹ In addition, direct and indirect costs of osteoporosis and the associated fractures are huge. In a study by Lewiecki and coworkers in 2019, annual fractures among U.S. women were projected to increase from 1.9 to 3.2 million (68%) from 2018 to 2040, with related costs rising from $57 billion to over $95 billion. ²

Prevention of osteoporosis and its increased fracture risk prior to the need for treatment is a clinical priority. A recent publication reported that among women ≥65 years osteoporosis drug treatment (ODT) fill rates increased significantly between 2011 and 2022 from 36.3% to 50.1% in women without fragility fractures and from 30.8% to 43.7% in those with fragility fractures. ³ Similar trends were observed in women aged 50-64 years. Bone resorption inhibitors such as bisphosphonates are used for the prevention and treatment of osteoporosis and denosumab for treatment of osteoporosis. A rare complication of bisphosphonates and denosumab is an atypical femoral fracture. ⁴ In addition, each of these 2 medications has been reported to cause osteonecrosis of the jaw. ⁵ The risk of developing either of these two rare adverse effects increases with increasing time of bisphosphonate administration. Although the serious adverse effects of denosumab are considered to be rare, our experience is that many patients are apprehensive about using it because it is administered long-term by injection. Prevention of osteoporosis and fracture risk with estrogen use is an alternative to prevention with bisphosphonates, the goal being avoiding potential osteoporosis treatment.

Over 75 years ago, it was reported that osteoporosis in postmenopausal women is largely the result of the significant decrease in estrogen production following menopause, and that treatment of postmenopausal women with estrogen was efficient to maintain bone mass and thus reduce fracture risk.^6,7 Since that time many studies have shown that lack of estrogen in postmenopausal women is associated with bone loss and consequent osteoporosis, and researchers have proposed mechanisms by which estrogen prevents bone loss.

Multiple randomized controlled trials (RCTs) and observational studies have demonstrated that estrogen alone or combined with a progestogen, to prevent endometrial cancer, reduces the risk of osteoporotic-related fractures, including hip, vertebral, and nonvertebral fractures. ⁸ A meta-analysis of 13 RCTs, carried out during the years 1990-2001, in which postmenopausal women were randomized to at least 12 months of hormone therapy (HT) or no HT, showed that there was an overall 33% reduction in vertebral fractures with HT use. ⁹ Also, a meta-analysis of 22 RCTs, carried out during the years 1993-2001, in which postmenopausal women were randomized to at least 12 months of HT showed an overall 27% reduction in nonvertebral fractures. ¹⁰ A subsequent meta-analysis of 28 RCTs, 9 of which were carried out after 2001, involved studies on the effect of HT on bone fractures. ¹¹ The duration of the studies was 0.5-15 years. The results consistently show that HT is associated with a reduced risk of total, hip, and vertebral fractures. The authors of the study pointed out that in sensitivity analysis, the first arm of the large-scale, randomized, prospective, placebo-controlled Women’s Health Initiative (WHI) trial accounted for a significant weight in the meta-analysis. ¹² In that arm, in which women were treated with an estrogen plus a progestin and compared to a placebo group, it was shown that bone mineral density (BMD) increased, and the risk of fractures was reduced. ¹³ In a subsequent second arm of the WHI trial, in which the women received estrogen alone, the fracture risk was also reduced significantly. ¹⁴ In a longitudinal observational study of 80,955 postmenopausal women using HT for 2.5 years of follow-up, women who discontinued HT had significantly increased risk of hip fracture and lower BMD compared to women who continued taking HT. ¹⁵ The protective effect of HT with hip fracture disappeared within 2 years of cessation of HT.

Estrogen therapy, either oral or transdermal, alone or in combination with a progestogen or bazedoxifene, is approved by the FDA for the prevention of postmenopausal osteoporosis. According to the latest HT statement of the Menopause Society, ¹⁶ initiating systemic HT is an appropriate therapy to protect against bone loss in women younger than 60 years or within 10 years of menopause onset, in the absence of contraindications. This recommendation differs from that stated in the guidelines of the Endocrine Society and Bone Health and Osteoporosis Foundation in which estrogen therapy and selective estrogen receptor (ER) modulators (SERMs) are recommended only in selected populations or circumstances, owing to adverse effects of the treatment. ¹⁷

The fear about adverse effects of HT stems mostly from large RCTs that reported excess cardiovascular and/or breast cancer risks from HT use. The findings of the WHI trial have been given prominent consideration of the overall risks and benefits of HT because it is the largest RCT of HT in postmenopausal women aged 50-79 years. The conjugated equine estrogen (CEE)/medroxyprogesterone acetate (MPA) arm of the WHI trial was terminated in 2002 after a median of 5.6 years after the data and safety monitoring board concluded that the evidence for breast cancer harm, along with evidence for some increase in coronary heart disease (CHD), stroke, and pulmonary embolism (PE) outweighed the evidence of benefit for fractures. ¹² Also, the CEE arm of the trial was terminated in 2004 after a median of 7.2 years, primarily owing to an increase in stroke risk. ¹⁴ After release of the WHI findings, many postmenopausal women stopped taking HT and many clinicians stopped prescribing it because of the perceived increased risks reported by the WHI investigators. By 2003, use of HT in postmenopausal women in the U.S. was reported to have dropped by an estimated 25% to 70% compared to levels in 2000-2001 before the WHI trial. ¹⁸ However, subsequent findings from reanalysis of data and postintervention follow-up involving studies of HT and cardiovascular disease (CVD) as well as breast cancer have provided a better understanding of the risks and benefits of HT. As such, major medical societies now recommend HT in appropriate patients for management of menopausal symptoms and prevention of bone loss. ¹⁹

Although experts on bone health in women believe that measurement of estradiol (E₂) levels is not important in managing menopausal HT for preventing osteoporosis because FDA-approved estrogen treatment doses that work for bone protection are known, there is still interest in determining a cut-off value for circulating E₂. ²⁰ Since there has been a trend to use lower E₂ doses for HT, the clinical need for knowing the E₂ concentration for a bone density response is highly desirable. It is accepted that there is a dose response effect of HT for prevention of bone loss, for both oral and transdermal delivery, with lower doses having an impact. ²¹ However, individuals may vary in their absorption and metabolism of estrogen, necessitating the determination of E₂ concentrations for clinical management. Studies over the past 30 years or so have not established a valid threshold E₂ concentration to protect against osteoporosis. Notable limitations in those studies include insufficient numbers of study participants and use of inaccurate E₂ assays.

The focus of this paper will be to review the importance of estrogen in preventing bone loss and consequent osteoporosis in postmenopausal women. The following topics will be addressed: 1) biology of bone; 2) estrogen signaling in bone; 3) effect of exogenous estrogens and progestogens on bone; 4) safety of HT use regarding breast cancer and CVD risks; 5) minimum effective E₂ dose for preventing osteoporosis, and the challenges of determining a reliable E₂ threshold.

Biology of bone

Bone is composed of approximately 60% minerals, 30% organic matter, and 10% cells. ²² Ossification occurs through the action of 3 types of cells, namely osteoblasts, osteoclasts, and osteocytes.^22,23 Osteoblasts are bone-forming cells that account for 4-6% of bone cells, whereas osteoclasts are responsible for bone resorption and represent <1% of bone cells. Osteocytes are the last stage of osteoblastic differentiation and account for about 95% of bone cells. These 3 cell types are involved not only in ossification, but also in the growth, modeling, and remodeling of bones.

Bone growth occurs at the epiphyseal plate where the cartilage continues to grow by mitosis until osteoblasts move in and ossify the matrix to form bone. ²⁴ This process continues throughout childhood and the adolescent years until the cartilage growth slows down and finally stops, usually in the early twenties. ²⁴ At that time the epiphyseal plate completely ossifies and bone can no longer grow in length. The growth process of bone is under the influence of the GH/IGF axis and sex steroid hormones. Estrogens have a biphasic effect on long bone development. ²⁵ They induce long bone development at low doses but promote epiphyseal closure and stop further growth with higher doses. ²⁶ The absence of estrogen is associated with unfused growth plates and intermediate growth.^27,28

Mature bone consists of 2 types of tissue: cortical bone and trabecular bone, which represent 80% and 20% of the skeleton, respectively. ²⁵ Cortical bone comprises the compact external wall of bone and the diaphysis of long bones. In contrast, trabecular bone constitutes the end of long and flat bones and is made up of interconnected trabeculae surrounded by bone marrow.

Bone modeling determines the development and maintenance of bone shape during skeletal growth. ²⁵ It begins from the start of fetal bone formation and continues into old age, albeit comprising only a small percentage of total bone formation. ²⁹ Peak bone mass is achieved by the end of skeletal development. It was shown that peak BMD occurs between 20.5-23.6 years in females. ³⁰

In contrast to bone modeling, bone remodeling is a life-long process in which old or damaged bone is replaced with new bone by a sequence of cellular events that occur on the surface of bone, without any change in bone shape. ²³ For many years, a 2-stage process was considered to be responsible for bone remodeling. This process involved bone resorption by osteoclasts followed by bone formation with osteoblasts to achieve a net mass bone equilibrium upon physiological maturity of bone. ³¹ However, recent findings have shown that bone remodeling is a physiological process that involves a continuous flow of cellular signaling and connected events, and not a process comprising discrete stages as has been represented historically. ²³ The recent discoveries indicate that bone cells participate in the basic multicellular unit responsible for bone remodeling by interacting simultaneously and at different stages of differentiation with their progenitors, other cells, and matrix constituents.

Osteocytes play a master role in bone remodeling. ²³ It has been estimated that the human skeleton contains about 42 billion osteocytes. ³² Osteocytes have a stellate body connected by multiple slender, long, neuron-like dendrite processes that project through the lacunar canalicular system in bone. ²² These cells translate external signals, such as hormonal changes and mechanical stresses, into alterations in bone remodeling by secreting paracrine-acting factors that regular osteoblast and osteoclast activity. ³³ In addition, the osteocyte network of dendrites allows for extensive communication among osteocytes and adjacent cells on bone surfaces. ³⁴

It has been estimated that the human skeleton contains 23 trillion osteocyte connections with each other and bone surface cells. ³⁵ During aging, this network deteriorates. A large and linear reduction in dendrite number and bone mass has been observed throughout aging. ³⁶ It has been suggested that dendrite loss may contribute to diminished osteocyte viability.

Estrogen signaling in bone

In postmenopausal women with osteoporosis, rapid bone loss results from an increase in bone turnover with an imbalance between bone resorption and bone formation. Estrogen is critical for regulating bone remodeling and skeletal homeostasis. Although the cellular mechanisms by which estrogen acts on bone are still controversial, recent evidence shows that estrogen regulates bone turnover primarily by modulating the expression of the receptor activator of NF-κB ligand (RANKL) via estrogen receptor (ER)α-mediated signaling. RANKL is an essential cytokine for the differentiation, activation, and survival of osteoclasts. ³⁷ RANKL acts through the receptor RANK, which is expressed in the cell membrane of osteoclast and osteoclast precursor cells. ³⁸ It has been shown that the increase in bone resorption found in states of estrogen deficiency in mice is caused mainly by lack of ERα-regulated suppression of RANKL expression in bone lining cells. ³⁷

In addition to upregulation of RANKL expression by estrogen to inhibit osteoclast formation and bone resorption activity, various other mechanisms involving estrogen that may contribute to the imbalance of bone remodeling have been proposed. Estrogen can also activate Wnt/β-catenin signaling to increase osteogenesis, and to upregulate bone morphogenic protein (BMP) signaling which promotes mesenchymal stem cell differentiation from pre-osteoblasts to osteoblasts. ³⁹ In addition, estrogen deficiency leads to an increase in interleukin (IL)-7 which promotes the activation of T cells. ³⁹ These cells then induce pro-inflammatory molecules such as IL-1, IL-6, and tumor necrosis factor (TNFα), resulting in osteoclast formation. Interference in these signaling pathways inhibits the maturation of osteoblasts and increases osteoclastogenesis, resulting in excess bone resorption relative to bone formation, and eventually osteoporosis.

Effect of exogenous estrogens and progestogens on bone

Numerous RCTs, including the Women’s Health Initiative (WHI) and the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial have established that estrogen attenuates or prevents the decrease in bone density in postmenopausal women, resulting in a lower risk of fracture.^{12,14,40–44} The WHI trials provide the best randomized prospective evidence that estrogen reduces fracture risk in the general population. In the two WHI trials, it was shown that fracture risk is reduced by both combined CEE (0.625 mg) + MPA (5 mg) ¹² and unopposed estrogen (0.625 mg CEE). ¹⁴ The estrogen + progestin cohort experienced low hip fracture rates (10 per 10,000 person-years vs. 15 per 10,000 person-years in the placebo group). The observed hip and clinical vertebral fractures in the treatment group were reduced significantly by one-third compared to placebo. Also, the reduction in other osteoporotic fractures (23%) and total fractures (24%) were statistically significant. Treatment with CEE alone reduced the fracture risk significantly by 30-39%. The rates were 11 vs. 17 for hip and clinical vertebral fractures, and 129 vs. 195 for total osteoporotic fractures per 10,000 person-years.

It is important to realize that postmenopausal women with a uterus require addition of a progestogen when using estrogen treatment in order to prevent endometrial hyperplasia and endometrial cancer. However, there are limited data on effects of progestogens used for HT on bone; this includes the progestogens combined with an estrogen, namely, MPA and progesterone. In one study, ⁴⁵ it was shown that, compared to placebo, MPA reduced the rate of loss in cortical areas of the skeleton, but not in the spine, in postmenopausal women. In the same study, CEE reduced the rate of bone loss in both cortical and trabecular areas of the skeleton. In contrast, clinical investigations of the effect of progesterone on bone have been inconclusive. ⁴⁶

Safety of HT regarding breast cancer and cardiovascular disease risks

As women reach menopause, the dramatic decrease in circulating estrogen concentrations affects almost all organ systems, including urogenital, reproductive, cardiovascular, neurologic, skeletal, dermatologic, immune, and digestive systems. ⁴⁷ It is clear from the basic science literature that ERs are found in those organ systems and that they are associated with uncountable beneficial actions in both women and men, even as they age. Lack of estrogen can have a profound effect on organ systems, resulting in an overall negative impact on the quality and quantity of life of women. Because estrogen and ERs are critical to the functioning of many systems of the body, there is great utility in replacing the decreased supply of estrogen in postmenopausal women.

During the past 60 years or so, controversies have arisen regarding the safety of estrogens for treatment of postmenopausal women. ⁴⁸ However, data from RCTs and observational studies during the past 25 years have provided us with valuable information regarding their safety, including their effects on breast cancer and CVD risks, which are discussed below.

Application of findings from HT/breast cancer and cardiovascular risk studies to clinical practice

The findings just described from the studies on the effect of breast cancer and CVD risk are consistent with the Menopause Society position statement regarding use of HT in postmenopausal women. ¹⁶ They state that the benefit-risk ratio is favorable to initiate treatment for vasomotor symptoms (VMS) and to prevent bone loss with HT for women who are younger than 60 years or are within 10 years of menopause onset and have no contraindications. The benefit-risk ratio appears to be less favorable in women over 60 years of age or more than 10 years from menopause onset, because of the greater absolute risks of CHD, stroke, VTE, and dementia. Nevertheless, some investigators believe that initiating HT only for women who are within 10 years of menopause or under age 60 years is too restrictive, and denies many women the benefits of HT, especially regarding bone health. ⁷⁶

It is important to realize that findings from most of the studies on the effect of HT on breast cancer and CVD risk are based on the administration of a standard oral dose of CEE or E₂. There is general agreement that the doses used to prevent osteoporosis are similar to those used to reduce VMS. However, there are various other HT formulations and doses that can be used, not only orally but also parenterally, as shown in Table 1. Also, different routes of administration offer different therapeutic benefits and risks. For example, as mentioned earlier, using transdermal estrogen avoids first hepatic pass metabolism, thereby minimizing effects on estrogen-sensitive hepatic proteins, including coagulation, anticoagulation, and fibrinolytic factors.

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Table 1.

Commonly used estrogen formulations and doses used for postmenopausal hormone therapy.

Formulation		Dose
Oral	Estradiol (micronized)	0.5, 1, 2 mg
	Conjugated equine estrogens	0.3, 0.45, 0.625, 0.9, 1.25 mg
	Esterified estrogens	0.3, 0.625, 1.25 mg
Transdermal	Estradiol patch	0.014, 0.025, 0.0375, 0.05, 0.06, 0.075, 0.1 mg/day
	Topical estradiol gel	0.25, 0.5, 0.75, 1 mg
	Topical estradiol spray	1.53 mg
Vaginal	Estradiol ring	0.0075, 0.05, 0.1 mg/day
	Estradiol tablet	0.01 mg
	Estradiol insert	0.004, 0.01 mg
	Estradiol cream	0.1 mg
	Conjugated equine estrogen cream	0.625 mg
Injectable	Estradiol valerate	2–10 mg
	Estradiol cypionate	2-10 mg

Minimal effective estradiol dose for preventing osteoporosis

It is generally accepted that estrogen treatment for preventing bone loss and osteoporosis should be based on clinical status, age, medical and surgical history, and bone density findings in postmenopausal women. Experts on bone health in women believe that measurement of E₂ levels is not important in managing menopausal HT for the prevention of osteoporosis because the estrogen doses that work for bone protection are known for FDA-approved formulations. Neither the Menopause Society nor the Endocrine Society in the United States recommend checking circulating E₂ concentrations related to bone health.^16,81

Although knowing the serum E₂ concentration in a patient is not essential to initiate treatment, E₂ testing can play an important role in the treatment process. This is especially important to identify and guide dose customization in women who absorb E₂ poorly. E₂ tests can also be useful when the clinical response to estrogen treatment is suboptimal to determine whether a change in formulation is likely to be beneficial. In addition, a cornerstone of menopausal HT, endorsed by authoritative bodies such as the Menopause Society and the American College of Obstetrics and Gynecology (ACOG), is the principle of using the minimal effective dose of a drug for the shortest duration of time to achieve treatment goals.^16,82 This principle emphasizes a careful titration process of the drug, in which a health care provider assesses an individual’s symptoms, health history, and response to treatment. A standard dose can be a starting point for titration.

Over the past 33 years or so, a number of studies have reported threshold circulating E₂ concentrations for preventing postmenopausal bone loss and osteoporosis. An often-cited study on this topic was carried out by Reginster and coworkers in 1992. ⁸³ In their study, 158 postmenopausal women received estrogen therapy for at least 6 months; the estrogens used were 0.625 mg/day oral CEE (N=35); 1 mg/day oral E₂ valerate (N=14); 2 mg/day oral E₂ valerate (N=18); 2 mg/day oral estriol hemisuccinate (N=45); and 1.5 mg/day percutaneous E₂ gel (N=46). A single blood sample was obtained for measurement of serum E₂ concentrations using a commercial direct radioimmunoassay (RIA) (without a preceding purification step). In addition, a spot urine sample was obtained for measurement of biochemical parameters reflecting bone resorption, specifically the calcium/creatinine ratio and hydroxyproline/creatinine ratio. Women with serum E₂ concentrations between 60-90 pg/mL had a significant reduction in both ratios when compared to lower E₂ concentrations. The authors concluded that a significant reduction in bone resorption is achieved when E₂ concentrations reach a value of 60 pg/mL, and they considered this value to be the minimum serum E₂ concentration to prevent postmenopausal bone loss. The study by Reginster and coworkers ⁸³ has major deficiencies that notably include an insufficient number of serum samples obtained in each group, use of an inaccurate assay to measure E₂, and use of biochemical markers of bone.

Minimum E₂ concentrations to prevent osteoporosis in postmenopausal women have been proposed in other studies over the last 33 years or so. They include E₂ concentrations of 40-50 pg/mL, based on data obtained from two different studies on the pharmacokinetics of E₂ following transdermal application of two different E₂ patches.^84,85 However, there is insufficient evidence in the two studies to support the proposed threshold concentrations. Similarly, a recently proposed serum E₂ threshold value of >54 pg/mL for bone protection was not supported by sufficient evidence. ²⁰ To date, there is no study supporting a valid concentration of circulating E₂ to protect against ongoing bone loss and osteoporosis. This is especially important in the young premature ovarian insufficiency patients who are treated with high doses of estrogen. They sometimes do not tolerate the recommended estrogen doses and would benefit from more intense evaluation of the dose needed based on their circulating estrogen levels.

Despite the limitations in studies attempting to determine a cut-off value for circulating E₂ to prevent osteoporosis, the trend to use lower E₂ doses for HT means that the clinical need for establishing a bone density response is highly desirable. A reliable E₂ assay is crucial because it would be incorporated into management discussions regarding the efficacy of estrogen formulations being used.

It is well recognized that direct E₂ immunoassays overestimate E₂ measurements in serum because they do not utilize a preceding purification step (e.g., organic solvent extraction, chromatography) to remove potential E₂ metabolites that may cross-react with the E₂ antibody in the assay. ⁸⁶ E₂ can theoretically be converted to over 100 metabolites (including unconjugated, sulfated, and glucuronidated estrogens), many of which have been identified. This is evident in postmenopausal women using exogenous estrogens; serum E₂ levels can be overestimated by as much as 10-fold. ⁸⁷ It is also well known that direct E₂ immunoassays lack the sensitivity to measure E₂ reliably in baseline postmenopausal serum samples (<10 pg/mL) obtained from postmenopausal women and women using low-dose transdermal E₂ formulations (≤0.0375 mg/day). The limit of quantitation in most direct E₂ immunoassays is 10-20 pg/mL. It is important for clinicians to know that a direct E₂ immunoassay may give misleading results because it lacks specificity and sensitivity. It is now well accepted that mass spectrometry assay methodology is the gold standard for accurate measurement of E₂. If an E₂ immunoassay must be used, it should be standardized using an E₂ mass spectrometry assay. This can be done by submitting serum samples to the Centers for Disease Control (CDC) Hormone Standardization Program. ⁸⁸ The CDC evaluates the accuracy and precision of the results and technical assistance is offered to resolve potential problems.

In addition to use of inaccurate assays in studies attempting to obtain threshold E₂ levels, a major limitation is use of biochemical markers of bone resorption. Showing a reduction in these markers is not evidence that an estrogen dose prevents bone loss, especially when these markers are non-specific and imprecise. What is needed is a comparison of serum E₂ levels with their effect on BMD. It has been shown that increasing serum E₂ levels in postmenopausal women are positively correlated with BMD. ⁸⁹

Limitations of review

While this review provides a comprehensive analysis of estrogen’s role in postmenopausal bone health, several limitations must be acknowledged. A primary challenge lies in the significant variability across studies, particularly regarding HT formulations, delivery methods, and the timing of intervention. This heterogeneity complicates establishing a universal minimum effective dose for E₂, as individual metabolic responses and baseline BMD vary widely across postmenopausal populations. In addition, bone metabolism is a complex process influenced by a myriad of other factors, such as vitamin D status, parathyroid hormone (PTH) signaling, and lifestyle variables that were outside the primary scope of this review.

Conclusions

It is now well recognized that estrogen reduces the risk of osteoporotic-related fractures, including hip and vertebral and nonvertebral fractures. This is achieved via the ERs in conjunction with signaling pathways, resulting in wide effects of estrogen on bone. Although numerous RCTs and observational studies have shown beneficial effects of estrogen on bone, many clinicians have concerns about potential serious adverse effects of estrogen, particularly those related to breast cancer and CVD. The concerns stem largely from the initial negative findings of the WHI trials regarding breast cancer and CVD. However, the recent decision by the FDA to remove the “black box warning” on estrogen products has dramatically reversed these concerns. There is now an emphasis on the long list of benefits of HT, including effectively reducing short- and long-term menopausal symptoms and protecting bone. The FDA argued that in an analysis of 30 trials with 26,708 women, HT was not associated with increased cancer mortality. In fact, women who start HT before age 60 appear to have a decreased mortality risk. Lastly, the FDA stated that benefits associated with HT include a reduction in risk of bone fractures, CVD, and Alzheimer’s disease. There is even some concern that limiting the recommendation to initiating menopausal HT only to women who are within 10 years of menopause or under age 60 years denies many women the benefits of HT, especially regarding bone health. Further research examining the value of HT protection on the older bones, arteries and brain tissue in women starting HT outside of those age parameters is needed.

Although it is generally accepted that knowing serum E₂ concentrations in a patient is not essential to initiate treatment for preventing bone loss and osteoporosis, E₂ testing can be useful when the clinical response to estrogen treatment is suboptimal and a change in estrogen formulation and/or dose is likely to be beneficial. E₂ measurements have also been performed in serum to determine threshold E₂ concentrations for preventing bone loss and osteoporosis. However, inaccurate E₂ assays have generally been used to obtain threshold values, and there is presently no valid cutoff E₂ concentration to protect against osteoporosis.

Although findings from most studies on efficacy and safety of estrogen are based on oral administration of standard doses of CEE or E₂, a variety of low-dose estrogens are also available for menopausal HT. In addition, transdermal E₂ formulations are available. They avoid the first hepatic pass, thereby minimizing effects on hemostatic factors and potentially providing even safer estrogen treatment. It is important to realize that more than 50 years of study has failed to prove conclusively a cause-and-effect between HT and breast cancer. The significant reduction in bone fractures, dementia, CVD, all-cause mortality, and relief of short- and long-term menopausal symptoms with HT is well supported by the evidence.