**Biology of the Perimenopause: Impact on Health and Aging**

Bethesda, Maryland

May 26--27, 2004

**Summary of an NIH Workshop**

WORKSHOP SPONSORS:

National Institute on Aging

Office of Research on Women's Health

*National Institutes of Health*

**Table of Contents**

**Executive Summary iii**

**List of Acronyms x**

**Introduction 1**

**Premenopausal Protection against Chronic Diseases of Aging:**

**Re-thinking the Paradigm 2**

**Neurobiology of Ovarian Steroids 4**

**Dynamics of the Female Reproductive System and Changes with Aging 7**

**Relating Aging and Outcomes of Menopause 9**

**Animal Models Discussion Session 10**

**Rodent Models 11**

**Mouse Models 12**

**Rodent Models Discussion 12**

**Macaque Models 12**

**Peri/postmenopausal Baboon Models 13**

**Monkey Models Discussion 14**

**Adipose Discussion Session 14**

**Estrogen Insufficiency and the Metabolic Syndrome 15**

**Menopausal Changes in Body Fat Distribution and Metabolic Syndrome
15**

**Obesity and Premenopausal Risk Factors 16**

**Interaction between GnRH and Diet 17**

**Discussion 17**

**Immune System Discussion Session 17**

**Hormonal Effects 18**

**Autoreactivity in the Immune System 18**

**Inflammatory Markers and Mechanisms 19**

**Discussion 20**

**Bone Discussion Session 20**

**Menopause Transition 21**

**Anti-Osteoporotic Effects of Sex Steroids 21**

**Environment, Hormones, and Bones 21**

**Changes in Bone Mass with Age 22**

**Discussion 22**

**Cardiovascular Discussion Session 22**

**Epidemiological Factors 23**

**Hormonal Effects 23**

**Longitudinal Effects 24**

**Changes in Hemostasis with Menopause and with HT 25**

**Discussion 26**

**Cognition, Mood and Brain Function Discussion Session 26**

**Brain Organization Effects of Hormones 27**

**Menstrual Cycle Studies 27**

**Mood Alterations and Depression 28**

**CNS Effects of Estrogen 28**

**Neural Targets of Estrogen and Underlying Mechanisms of Action 28**

**Altered CNS Mechanisms during Perimenopause 29**

**Discussion 29**

**A Paradigm on Staging the Menopause Transition 29**

**Wrap Up Session 30**

**Research Agenda Items presented by Discussion Session Chairs 32**

**Adipose 32**

**Animal Models 32**

**Immune System 32**

**Bone 32**

**Cognition, Mood and Brain Function 33**

**Cardiovascular 33**

**Research Agenda Items from Workshop Participants (presented either at
the workshop or contributed following the workshop) 33**

**References 35**

**Subject
Index......................................................................................................\...43**

**Author
Index..........................................................................................................52**

# EXECUTIVE SUMMARY

# 

**Introduction**

The intricate and dynamic interplay of hypothalamic-pituitary-ovarian
(HPO) hormones during the normal menstrual cycle may serve to protect
women from a variety of health problems and conditions associated with
the menopause, including cardiovascular disease, osteoporosis, loss of
cognitive function, and menopausal symptoms. The role of aging certainly
plays a role in this loss of protection as women traverse the menopause
in middle-age. A workshop was convened to explore the current state of
knowledge regarding the role of HPO axis hormones and their dynamic
interrelationships during the normal menstrual cycle in this apparent
protection, and the loss of protection in non-reproductive somatic and
neuronal tissues as the menopausal process proceeds and menstrual cycles
terminate. This workshop was convened in Bethesda, MD from May 26-27,
2004, with sponsorship from the National Institute on Aging and the NIH
Office of Research on Women's Health. It consisted of four overview
presentations focused on HPO axis hormones, their dynamic
interrelationships preceding, during and following the menopausal
transition, and aging-related processes. These were followed by a series
of discussion sessions focused on animal model systems and on
non-reproductive somatic and neuronal tissues and their responses to HPO
axis hormone changes across the menopausal transition.

Overview presentations:

Nanette Santoro -- Albert Einstein College of Medicine: Premenopausal
protection against chronic diseases of aging: rethinking the paradigms

In light of what we know today, the concept that "replacing" estrogen, a
major hormone that declines at menopause, in postmenopausal (PM) women
in order to provide the level of health and cognition protection
seemingly apparent in premenopausal women is far too simple. Rather, we
need to better understand the interactions among the multiple changes in
HPO axis hormones across the menopausal transition and the processes of
aging. Regarding specific organ systems, the annual incidence of
osteoporotic fracture in women of all ages is about twice that of the
second most common disease, heart attack. Although older women are at
greater fracture risk than middle-aged women with the same bone mineral
density (BMD), probably reflective of differences in bone quality,
nevertheless bone is a simple model in terms of estrogen treatment. More
complex models are type II diabetes and cardiovascular disease (CVD).
The onset of type II diabetes in women is age-dependent, increasing
sharply over age 45. Rates of heart disease in women increase sharply
over age 55. In older postmenopausal women hormone therapy (HT) appears
to reduce the incidence of diabetes by a small but significant effect,
while not protecting them from CVD. These observations raise questions
regarding the relative roles of the individual hormones or collections
of hormones that change across the menopause and during aging in both
the apparent protection from PM health problems and conditions in
premenopausal women, and the biology underlying the development of
health problems and conditions after menopause. There are two conditions
in younger women where menstrual cycles terminate (premature ovarian
failure (POF)) or become irregular (functional hypothalamic amenorrhea
(FHA)); CVD risk is increased for both conditions. More research is
needed to elucidate relationships between aging and hormonal changes
associated with the menopause on disease risk

Bruce McEwen -- Rockefeller University: Neurobiology of ovarian
hormones: "It ain't just sex anymore!"

Brain regions outside of that involved in reproduction have only
recently been considered a target organ of sex steroids. It is now well
documented that gonadal steroids affect general brain structure and
function during development and reproductive life stages. Neuronal
plasticity is regulated by steroid hormones, but it isn't yet known if
plasticity is decreased with age, or if gonadal steroids are required to
maintain plasticity at older ages. Ovarian hormones have been shown to
have many effects on non-reproductive functions, including motor
function, mood disorders, damage due to stroke, pain, and cognition.
Although numerous brain regions are responsive to estrogen (E2), most
responsive brain areas don't have well-defined nuclear estrogen
receptors (ER). Thus, non-genomic E2 pathways are probably very
important in explaining the E2 response, including interconnections of
brain regions not usually connected and involving activation of
intracellular signaling systems. Male rats don't usually respond to E2,
but do respond to the testosterone (T) metabolite dihydrotestosterone
(DHT) for synapse induction. Brain tissue can synthesize reproductive
steroid hormones from cholesterol, including E2. Thus E2 effects in the
brain do not depend on systemic E2 levels. In addition to further
defining steroid hormone action in non-reproductive functions of the
brain, extensive further exploration is needed on the interaction of
gonadal hormones with cortisol, catecholamines, and other stress
mediators. Thus gonadal steroids have widespread influences on the
brain, including many non-reproductive functions.

Janet Hall -- Massachusetts General Hospital: Dynamics of the female
reproductive system and changes with aging

The female reproductive cycle in premenopausal women is a highly
interactive, exquisitely timed process involving dynamic secretion of
hormones and regulatory peptides from the entire HPO axis. As
middle-aged women transition through the menopause, numbers of ovarian
follicles decline and peak levels of HPO axis hormones change
substantially (declines in inhibin A and B, progesterone, and luteal
phase E2; increases in follicle stimulating hormone (FSH) and follicular
phase E2). In so doing the dynamic interactive relationships among these
hormones also change, leading to the irregular cycles during the
perimenopause, and eventually cycle cessation at menopause. At menopause
the previous dynamic interactions among HPO hormones becomes static.
Steroid sex hormones are at low basal levels and remain low as women
age; gonadotrophic hormones are high in early PM and decline with
increasing age. Studies have utilized this static condition of HPO
hormones during the PM to explore age-changes in hypothalamic
gonadotropin releasing hormone (GnRH) pulsatility, and pituitary
response to GnRH and to steroid feedback. Results from these studies
suggest with aging there are increased levels of secreted hypothalamic
GnRH, but at a reduced pulse frequency; the pituitary shows an
age-related reduction in gonadotropin output in response to GnRH.
Gonadotropin secretion remains responsive to steroid feedback with
increasing age. Studies have also noted menopause-associated changes in
glycosylation content of gonadotropins to more acidic forms. Very few
studies have addressed effects outside of the reproductive system of
these age- and menopause-related changes in HPO axis peptide and steroid
hormones.

Linda Fried -- Johns Hopkins School of Public Health: Relating Aging and
Outcomes of Menopause

While aging and menopause are distinctly different processses, the
consequences of each are similar: for example, decrements in
cardiovascular and muscle function, body composition changes,
osteoporosis, and cognition. Is there a relationship or are these two
processes independent? What might explain the sex differences observed
in the Cardiovascular Health Study, where males had a significantly
greater risk from cardiovascular mortality than females, while women
live longer with chronic disease, frailty and disability? Three
hypotheses were examined. First, that menopause is age-related, but
separate from the aging process. In the Women's Health and Aging Study,
surgical menopause had no significant effect on health outcomes in women
over the age of 65, suggesting that the absence of ovaries many years
later does not affect aging variables. Second, that menopause is a
direct cause of changes in aging. The PROSALMAN study reported that in
age-matched pre- and postmenopausal women, PM women had less muscle mass
and were weaker on measures of isometric and isokinetic strength. Thus
loss of muscle strength is associated with menopause, not age. Third,
that menopause is a sentinel event leading to increased vulnerability
with age. In this hypothesis, increased inflammatory status associated
with decreased sex hormones, primarily estrogen, in turn causes
functional declines associated with aging. All three hypotheses are
plausible and all may be operative in explaining relationships between
menopause and aging.

Animal Models Discussion Session, chaired by Thomas Clarkson, Wake
Forest University School of Medicine

Patricia Hoyer (University of Arizona) presented information on various
gene knockout (KO)/transgenic rodent strains (Bax KO; Bcl-x KO or the
FSH-receptor +/-) or ovarian follicle toxicant (4-vinylcyclohexene
diepoxide)-treated rodents as models of irregular cycles associated with
the perimenopause that may be useful in exploring postmenopause health
relationships. All of these models maintain the presence of the ovary
and have altered (accelerated or reduced) rates of female reproductive
aging that allow exploration of the role of the ovary in health changes
associated with reproductive aging. Adriana Maggi (University of Milan,
Italy), described a rodent model for investigating in vivo and in real
time bioluminescent activation of the estrogen response element, either
directly via estrogen or indirectly by ligand-independent mechanisms.
Jay Kaplan (Wake Forest University School of Medicine) presented data
showing the utility of social living groups to enhance a
perimenopausal-like situation in younger monkeys rather than wait until
age 20-25 when natural perimenopause occurs, near the end of their
lifespan. Subordinate female monkeys have a high occurrence of irregular
and anovulatory cycles with lower estrogen levels; this is associated
with increased vulnerability to disease, including atherosclerosis and
lower bone density. Tom Clarkson then presented data on behalf of Erika
Honore (Southwest Foundation for Experimental Biology) associated with
irregular cycling (perimenopause) in middle-aged baboons. These animals
have reduced estrogen levels, increased FSH, increased fracture
incidence, vaginal atrophy, and atherogenic LDL:HDL ratio on a high fat
diet relative to premenopausal females.

Adipose Discussion Session, chaired by David Kelley, University of
Pittsburgh, on behalf of Lewis Kuller, University of Pittsburgh

Molly Carr (University of Washington) discussed research showing that
metabolic changes occurring across the menopause lead to increased
visceral fat mass and lowered insulin sensitivity, even with no changes
in body mass index (BMI). Epidemiologic data shows that women are at
greater risk for the metabolic syndrome following menopause than before.
David Kelley (University of Pittsburgh) reviewed the host of
inflammatory cytokines associated with adipose tissue, particularly
those cytokines secreted from large adipocytes. Weight gain and the
increased adiposity that occur with the menopause, leading to insulin
resistance, decreased adiponectin, lipoprotein changes and
atherosclerosis, are associated with a high fat diet and sedentary
lifestyle. Evan Simpson (Prince Henry's Institute of Medical Research,
Australia) presented data obtained with the estrogen synthetase
(aromatase) KO mouse he developed. In the absence of estrogen
biosynthesis, these mice show significant increases in adipose tissue,
insulin resistance and developing metabolic syndrome. He also suggested
that estrogen biosynthesis in peripheral tissues may be more important
for regulation of metabolic phenotypes than serum estrogen levels in
postmenopausal women and in men. Mary Dallman (University of California
at San Francisco) presented data on the effect of diet in a transgenic
rat model where cAMP-phosphodiesterase expression is targeted to GnRH
neurons, resulting in blunted luteinizing hormone (LH) secretion in
ovariectomized females. These animals gained significant weight via
abdominal fat depots on a high energy diet relative to wild type control
animals on the same diet. This weight gain appears to be associated with
greater food intake. They surmise that GnRH neurons regulate food intake
via GnRH secretion to observed adjacent proopiatemelanocortin (POMC)
neurons, which are known to be involved in regulation of food intake.

Immune System Discussion Session, chaired by Betty Diamond, Albert
Einstein College of Medicine

Paul Kincade (Oklahoma Medical Research Foundation) described estrogenic
effects on lymphopoiesis. Lymphoid progenitor cells in the bone marrow
have both estrogen receptors, with ERα being particularly important in B
cell lymphopoiesis. Precise mechanisms for estrogen effects on the
immune system in humans are not known and need greater research
attention. Betty Diamond (Albert Einstein College of Medicine) addressed
the issue of estrogen enhancement of autoantibody production in
autoimmune disease. Using a transgenic mouse BALB/c strain expressing
heavy chain of an anti-DNA antibody, the fraction of B cells expressing
the transgene increases in the presence of high physiologic levels of
estrogen. This leads to glomerular immunoglobin deposition and
proteinuria. In this model estrogen, presumably acting through B cell
ERα and/orERβ, decreases the B cell receptor signal that in turn causes
the survival of more autoreactive B cells. Russ Tracy (University of
Vermont) focused his talk on the relationship between inflammatory
markers and disease risk, particularly in older people where the
relationship is more likely to change with increasing age and disease
status than in younger people. The association between inflammatory
markers and components of the metabolic syndrome, particularly
adiposity, begins very early in life, and interventions as simple as
weight loss can reduce the levels of these markers. HT also can reduce
many of these markers. A mouse study suggests that the inflammatory
marker IL-6 is involved in the etiology of atherosclerosis through
modification of T helper cell populations.

Bone Discussion Session, chaired by Sundeep Khosla, Mayo Medical School

Osteoporosis, associated with high fracture risk, is a major health
problem for older men and women, currently affecting over 40 million
people and costing the healthcare system almost \$17 billion in 2001.
Thus the need for better prevention and treatment strategies is
substantial. Assessing bone density is not sufficient -- bone quality
assessment, a more difficult measure, is required. Women lose primarily
cancellous bone starting around age 50 more rapidly and to a greater
extent than men, and estrogen treatment is effective in reducing or even
preventing this bone loss.

Joel Finkelstein (Massachusetts General Hospital) described several
longitudinal studies in middle-aged women showing progressive loss of
bone density in specific bone regions across the menopausal transition.
Rates of bone loss were greater in women weighing less than 80 kg than
in women weighing over 80 kg. Stavros Manolagas (University of Arkansas
for Medical Sciences) described his work using a new therapeutic steroid
(estren) that increases bone mass and strength in ovariectomized mice
over that of the estrogen-replete state. Estren activates a
non-genotropic estrogen signaling pathway, leading to reduced osteoblast
apoptosis and increased osteoclast apoptosis, resulting in increased
bone mass. Deborah Kado (University of California at Los Angeles)
discussed how environmental risk factors and stress could affect both
the menopausal process and development of osteoporosis via activating
the hypothalamic-pituitary-adrenal (HPA) axis as well as inflammatory
pathways. Cliff Rosen (Maine Center for Osteoporosis Research and
Education) noted that rates of bone loss and BMD are independent
measures of fracture risk, where high rates of bone loss and low BMD
result in greater risk than either separately. There is also a strong
genetic component to rates of bone loss, as seen in various mouse
strains. Bone quality is another very important factor for fracture
risk. There is a need for more and better biomarkers to identify those
at risk for early and extensive bone loss.

Cardiovascular Discussion Session, chaired by Michael Mendelsohn,
Tufts-New England Medical Center

Cardiovascular tissue can both synthesize and respond to estrogen.
Estrogen directly affects vascular dilatation via genomic and rapid,
non-genomic effects (involving endothelial nitric oxide synthase
(eNOS)), probably via both ERα and ERβ in vascular tissue. Vascular
tissue also contains progesterone (PR) and androgen receptors (AR), as
do immune system cells that circulate within vascular tissue. Thus a
variety of HPO axis hormones have the potential to regulate
cardiovascular physiology and pathophysiology.

Kim Sutton-Tyrrell (University of Pittsburgh) discussed epidemiologic
factors associated with CVD and relationships with HPO hormones.
Estrogen, testosterone, and sex hormone binding globulin (SHBG) levels
are all related to carotid wall thickness, arterial stiffness, and
endothelial function, and effects may be specific to each vascular bed.
Thus it is important to extend the research on cardiovascular function
beyond estrogen alone. Cardiovascular tissue in women traversing the
menopause is affected by both age-changes as well as HPO hormonal
changes. Richard Karas (Tufts-New England Medical Center) presented
analyses showing that major epidemiologic studies leading to the
interpretation of reduced cardiovascular risk in pre- relative to
post-menopausal women or the protective effect of estrogen therapy in
surgically PM women are both questionable due to the very small number
of events in each group. Further analyses of one of these major studies
pointed out the difficulty in sorting out CVD risk by age or menopausal
status. Philip Shaul (UT Southwestern Medical Center) outlined what is
known regarding the effects of estrogen and other HPO axis hormones on
the vasculature. Of all the HPO axis hormones, most is known about
estrogen, but generally in a HT context rather than endogenous estrogen
in the serum and that synthesized locally in the vasculature via
aromatase. Very little is known about the effects of progestins,
androgen and activin. An oxysterol cholesterol metabolite
(27-hydroxycholesterol), which is at high levels in atherosclerotic
vessels, interferes with estrogen action and thus could explain the lack
of protective effect of estrogen therapy in patients with existing
vascular disease. Barbara Konkle (University of Pennsylvania) and David
Ginsburg (University of Michigan Medical School) discussed effects of
hormones and aging on blood clotting processes. Both estrogen and aging
affect the various steps in the blood coagulation cascade, and both tend
to be procoagulant. Thus changes would be expected across the menopausal
transition in middle-aged women, although this has not been carefully
studied. Risk for venous thrombosis increases with age, but again
effects of menopause are not known.

Cognition, Mood, and Brain Function Discussion Session, chaired by
Pauline Maki (University of Illinois at Chicago)

There is very limited data available on changes in cognitive test
performance across the menopause transition. Results from several
studies were presented showing increase in "forgetfulness" and "memory
declines compared with the past" across the transition, while no
differences were seen in objective cognitive test performance in most
studies. A number of factors, such as poor sleep and history of
depression, may be associated with depressive symptoms in middle-aged
women. However data from the Baltimore Longitudinal Study of Aging
Perimenopausal Study suggest menopausal symptoms and stage influence
cognition.

Session discussants brought up a number of important points. Regarding
the effects of sex hormones on brain organization and cognition, Susan
Resnick (National Institute on Aging) mentioned that young girls with
congenital adrenal hyperplasia, exposed prenatally to excess androgens,
had better spatial abilities; also females do well in verbal memory
during high estrogen phases of the normal menstrual cycle compared to
their performance during phases of the cycle when estrogen levels are
low. Neuroimaging studies showed estrogen-induced changes in cerebral
blood flow in women, but this was not seen in monkey studies. Barbara
Sherwin (McGill University) described results of her studies showing
that estrogen treatment in estrogen depleted women (oophorectomized or
Lupron-treated) enhanced verbal learning and memory, but not visual
memory or spatial abilities. Peter Schmidt (National Institute of Mental
Health) stated that perimenopause is associated with increased risk for
depression only in some women, and estrogen treatment has an
antidepressant effect only in perimenopausal, but not postmenopausal,
women. C. Dominique Toran-Allerand (Columbia University) presented the
concept that estrogen likely affects CNS function in two ways: directly
via estrogen receptors in various brain regions, and indirectly by
upregulating neurotropins and neurotropin receptors, which in turn may
regulate estrogen receptors. John Morrison (Mt. Sinai School of
Medicine) discussed estrogen-induced dendritic spines and excitatory
synapses in hippocampus and prefrontal cortex in monkeys, and the
improvement of various memory tasks following E2 treatment. Susanna Park
(New Jersey Medical School) described her pilot study showing
age-related CNS changes in LH responsiveness to estrogen treatment in
middle-aged women with regular cycles.

# A Paradigm on Staging the Menopause Transition

Sherry Sherman (NIA), Nancy Woods (University of Washington), and
Estella Parrott (NICHD) discussed the need for common definitions for
stages of the menopause transition that facilitate comparison of
findings. A workshop held in 2001, Staging Reproductive Aging Workshop,
focused on developing a paradigm for menopausal staging involving cycle
irregularity and increases in FSH secretion.

Summary and recommendations:

Several general points were made by workshop participants. First, there
is a dearth of studies on biologic effects of individual HPO axis
hormones other than estrogen on pathophysiologic processes in
non-reproductive somatic and neuronal tissues. Even for studies
involving estrogen, most utilized supraphysiologic or pharmacologic
levels, rather than physiologic levels. Participants suggested the lack
of validity for considering single hormones and target tissues in
isolation; rather investigators must begin to seriously consider studies
of interactions (or "cross-talk") of multiple hormones, cytokines and
other biologic regulators on multiple interaction organ systems. Use of
appropriate model systems is key to the success of this approach. Second
was an emphasis on studying the role of aging on tissue biology, tissue
and hormonal interactions, and tissue sensitivity and response to
hormones. Finally, better biomarkers of menopause and disease
progression are needed, as well as more sensitive hormonal assays of
serum and tissue levels of hormones.

List of Acronyms

27HC 27-hydroxycholesterol

ANGELS activators of non-genotropic estrogen-like signaling

AR androgen receptor

ArKO aromatase knockout

BMD bone-mineral density

BMI body mass index

CAH congenital adrenal hyperplasia

CHD coronary heart disease

CHS Cardiovascular Health Study

CNS central nervous system

CRF corticotropin releasing factor

CRP C-reactive protein

CVD cardiovascular disease

DHEA dehydroepiandrosterone

DHEAS dehydroepiandrosterone sulfate

DHT dihydrotestosterone

E2 estradiol-17β or generic "estrogen," as appropriate

ER estrogen receptor

ERα estrogen receptor alpha

ERβ estrogen receptor beta

ERE estrogen response element

FAS free alpha subunit

FSH follicle-stimulating hormone

FSHR follicle-stimulating hormone receptor

GnRH gonadotropin-releasing hormone

HDL high-density lipoprotein

HPA hypothalamic-pituitary-adrenal axis

HPO hypothalamic-pituitary-ovarian axis

HT hormone therapy to indicate either estrogen or estrogen plus
progestin therapy

IGF-1 insulin-like growth factor - 1

IHC immunohistochemistry

IL-1 and -6 Interleukins 1 and 6 (cytokines)

KO (gene) knockout (usually in mice)

LDL low-density lipoprotein

LH luteinizing hormone

Luc luciferase

MRI magnetic resonance imaging or images

NFκB nuclear factor κB

NO nitric oxide

NOS nitric oxide synthase

eNOS endothelial NOS

P progesterone

PAI-1 plasminogen activator inhibitor

PM postmenopause

POF premature ovarian failure

PR progesterone receptor

SERMs selective ER modulators

SHBG sex hormone-binding globulin

SWAN Study of Women's Health across the Nation

T testosterone

TNFα tumor necrosis factor-alpha

VCD 4-vinylcyclohexene diepoxide

WHAS Women's Health and Aging Study

WHI Women's Health Initiative

BIOLOGY OF THE PERIMENOPAUSE: IMPACT ON HEALTH AND AGING

Summary of an NIH Workshop

May 26--27, 2004

# Introduction

Menstrual cyclicity in women is controlled by an intricate interplay of
timed events involving the hypothalamic-pituitary-ovarian (HPO) axis.
Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH)
acts on pituitary cells (gonadotrophs) to stimulate the pulsatile
release of follicle-stimulating hormone (FSH), luteinizing hormone (LH),
and gonadotropin free alpha subunit (FAS). LH and FSH in turn stimulate
follicular maturation in the ovary, eventually culminating in ovulation
and corpus luteal formation. There is a resultant ovarian synthesis and
release of estradiol (E2) and progesterone (P) in a highly orchestrated
manner, as well as of testosterone (T) and androstenedione. Inhibin A
and inhibin B also are synthesized coordinately in the ovary during this
process. The intricate interplay leading to ovulation prepares the
uterine lining for eventual implantation of a fertilized ovum. In the
absence of implantation the uterine lining sloughs off, and a new cycle
is initiated.

There are many events, external and internal, that affect the menstrual
cycle. Disruption of the menstrual cycle is accompanied by alterations
in the level of ovarian hormones. In addition to stimulating and
maintaining reproductive organs, E2 and P bind specific receptors in
many parts of the central nervous system (CNS), act on bone, influence
adiposity, and alter cardiovascular and immune systems as well as
behavior. Each of these various end-points for steroidal hormone action
has significant implications for disease processes and quality of life
issues. Perturbations in the menstrual cycle begin by the third and
fourth decade of life, first by a disruption in the regularity of the
cycles. This is intersperced with a return of cyclicity preceding a
longer absence of cycling (perimenopause), with amenorrhea extending for
longer periods, eventually culminating in the total cessation
characterized as menopause. The age of menopause varies, but is thought
to occur in the U.S. by the median age of 52 years.

It has been noted that premenopausal women appear to be protected from
disease risk relative to men and age-matched postmenopausal women;
disease risk increases as menstrual cycles become irregular and
eventually terminate at menopause. Epidemiologic evidence suggests that
exogenous ovarian hormones provided during postmenopause (PM) preserves
health and function. However, such ideas have been challenged by recent
findings from the Women's Health Initiative (WHI) placebo-controlled
intervention study. Furthermore, little is understood of the apparent
disease protection provided to premenopausal women and the loss
conferred by aging of both the reproductive system and somatic tissue.

An advisory workshop was convened in Bethesda, MD from May 26-27, 2004,
with sponsorship from the National Institute on Aging and the NIH Office
of Research on Women's Health, to explore the biological mechanisms that
may precede and be associated with perimenopause, and the subsequent
impact of perimenopause on health and aging. The goal was to assess a
number of factors in the HPO axis that occur in the transition from
irregular cycles to menopause and to focus on underlying biological
mechanisms that may be involved in this transition. The workshop was
structured to provide background information on hormone actions, the HPO
dynamics, and aging. A series of discussion sections was then held on
animal models, adiposity, immune system, bone, cardiovascular, and
cognition. The workshop was designed to address four areas concerning
altered reproductive cycles across the menopause transition and the
connections between this alteration and increased somatic health risk in
PM:

-   What is the evidence for altered health risk across the menopausal
    transition?

-   What are the underlying biological mechanisms responsible for this
    altered health risk?

-   What are the effects of environment, behavior and lifestyle on these
    biological mechanisms?

-   What important unanswered research questions need to be addressed?

The workshop concluded with recommendations for future research
initiatives, a key objective of this advisory meeting.

# Premenopausal Protection against Chronic Diseases of Aging: Re-thinking the Paradigm

**Nanette Santoro, Albert Einstein College of Medicine**

Life expectancy of women is increasing such that those born today can
expect on average to live to age 90. Longer lifespans create concerns
about health issues that are associated with such a long period of PM
life. Thus, it is important to make linkages between aging and menopause
in ways that may be helpful.

A number of diseases are associated with the PM period, and while
cardiovascular diseases (CVD) have greater morbidity and are often
mentioned, the incidence of osteoporotic fractures are much more common.
Such fractures account for a high incidence of disability, although they
may not be fatal. A high incidence of breast and lower incidence of
cervico-endometrial-ovarian cancers also are associated with aging.

Problems with fracture are not just a bone density phenomenon, but are
age related. Data show that a woman of age 50 with a low bone density
score is far less likely to suffer a fracture than is a woman of 80 with
a similar low bone density score (Kanis et al., 2002). While bone
density measurements are reliable and excellent measures, other factors
associated with aging become more relevant to the occurrence of a
fracture (e.g., bone structure and susceptibility to falling).
Nevertheless, hormone therapy (HT) protects bone mass in PM women
regardless of whether initiated at menopause or several years later
(Christiansen et al., 1981), suggesting bone response to exogenous E2 is
somewhat independent of age. Thus, bone represents a relatively simple
model in terms of E2 effects.

More complex models are type 2 diabetes and CVD. The onset of type 2
diabetes in women is age-dependent, sharply increasing over age 45. When
looking at heart disease by age and gender, a different relationship is
seen. Rates of heart disease in women increase sharply over age 55.
Regarding gender differences, the numbers of men with heart disease
exceeds the numbers of women until age 74, but then for ages 75--84,
more women have heart disease than men. The reasons for such findings
are not clear. In older PM women HT appears to reduce the incidence of
diabetes by a small but significant effect (Margolis et al., 2004),
while not protecting women from CVD.

There is not just a simple relationship between aging or menopause and
the origin of disease. There is a need to think about shifting
paradigms---e.g., as women age and transition through the menopause, are
protective factors diminished, or are risk factors increased, in either
case altering disease risk? Are endocrine factors the sole source of
disease protection in premenopausal women, or is aging in other organ
systems involved in diminished protection against disease?

An issue not addressed in longitudinal studies is early hormonal
intervention(s), which may make a critical difference in outcomes,
especially for cardiovascular and possibly cognition effects. It is
important to consider whether there are perhaps irreversible losses that
occur that could be prevented by early exposure to exogenous hormone as
endogenous hormones decline. This is a very fertile area in need of
further exploration. The issue is whether the significant interactions
between reproductive and somatic aging have been neglected. The
endocrine-mediated event of menopause tends to mask equally important
effects of organismic aging (e.g., progressive, age-related changes
known to occur in vascular distensibility, increases in blood pressure,
and changes in lipids and lipoproteins); the menopause-related changes
in such factors are not that large.

Women with premature ovarian failure (POF) have onset of
hypogonadotropic amenorrhea prior to age 40, providing an example of
young bodies with old ovaries. Such patients do not appear to have other
age-related problems with the endocrine axes, as do those with the
polyglandular failure syndrome. In the latter the adrenal axis appears
completely normal. However, women with POF have an increase in CVD
mortality. In the Study of Women's Health across the Nation (SWAN),
these women had some history of cardiovascular failure, which preceded
the diagnosis of POF, suggesting that a weakened cardiovascular system
may cause early failure of the ovaries. Young women with functional
hypothalamic amenorrhea, a central rather than an ovarian defect, had a
high odds ratio of CVD with a 69 percent versus 29 percent prevalence of
cardiovascular artery disease (Bairey Merz et al., 2003a). Given their
slimness, youth, and activity levels, they would not be predicted to
suffer from cardiovascular arterial disease.

Other non-gonadal hormones, such as adrenal androgens for which serum
levels decline with age, may interact with the cardiovascular system in
ways not discerned until ovarian hormones decrease. Also, data from the
SWAN study suggests that high body mass index (BMI) is not only
associated with insulin resistance, which dramatically increases CVD
risk, but also with different menopausal hormonal patterns relative to
normal BMI, such as decreased serum FSH and E2 levels in the early
perimenopause.

There is a need to distinguish between chronological and endocrine
aging. This may require sophisticated experimental models. Model systems
of increasing complexity may be used to provide a framework to formulate
productive research questions that integrate aging and reproductive
hormones into a fuller picture of female health following menopause.

Discussants raised several additional considerations. One is that when
reproduction ends, a different stage of life is entered. Thus, the
premenopausal model may not be appropriate for the PM period; there may
be evolutionary adaptations for the reproductive period, but not for the
post-reproductive period. It was noted that longevity genes found in
people living beyond age 100 appear to be unrelated to reproduction.
Indeed, there appears to be no selectivity pressure for longevity, as
there may be for reproductive fitness.

Another suggested perspective was to look at gender differences, keeping
in mind that the male appears disadvantaged throughout the life cycle
with respect to mortality and CVD. Women maintain a lifetime advantage
compared to men, and such differences may offer important clues to basic
biological mechanisms. A related approach would be to consider prenatal
programming and possible relationships to later aging.

# Neurobiology of Ovarian Steroids

**Bruce McEwen, Rockefeller University**

For many years the brain has been ignored in considering what happens
after menopause; concerns have concentrated on CVD, cancer, reduced
bone-mineral density (BMD) and fractures. There is increasing evidence
that this thinking is changing, and there is an interest in how the
brain responds to gonadal hormones.

Gonadal steroids have profound effects on brain structure and function.
Berthold's (1849) studies restoring aggressive behavior after testes
were implanted in roosters and peacocks were followed 100 years later by
studies wherein T implants in hypothalamic regions of capons restored
sexual reproductive behaviors. Such studies were later extended to
rodents, where steroid implants in various CNS regions were noted to
affect behavior, gonadotropin release, and other important reproductive
activities. Subsequent studies extended such findings by hypothesizing
that dimorphic differentiation occurred in parts of the hypothalamus
signaled by neuronal density and functional differences in response to
the organizing effects of T. Other important studies looked at the
metabolism of T and its conversion to E2 and dihydrotesterone (DHT).
Early studies focused on the actions of steroid hormones via nuclear
localized receptors, and later studies focused on the non-genomic action
of such hormones.

The ventromedial hypothalamus, part of a circuit that mediates female
rat sexual response (lordosis), contains nuclear receptors for E2
(estrogen receptor, ER). E2 induces oxytocin receptors, which spread out
to areas with oxytocin innervation. Reportedly, interference with
oxytocin receptors also interferes with the lordosis response. E2 also
causes changes in numbers of progesterone receptors, induction of new
synapses, and regulation of a variety of neuropeptides and other
receptors as part of a complex network of interactions activating the
lordosis circuit.

There are other important developmental effects of estrogens in the
brain. Neurogenesis and neuronal survival during development of the
brain establishes gender differences in brain circuitry. Explants of the
hypothalamus, hippocampus, and cerebral cortex exposed to endogenous
estrogens causes an outgrowth of neuritis, presumably stimulating the
growth response that aids in forming neural connections. Exogenous E2
exerts even greater effects on outgrowth. Because the notion of
structural plasticity regulated by ovarian hormones in the adult brain
is extended throughout the lifespan to aging, a question of interest is
whether this capacity is lost or whether ovarian hormones are necessary
to maintain plasticity.

Ovarian hormones have been shown to have many effects on
non-reproductive functions, e.g., motor coordination and motor skills;
certain epileptic seizures; cyclicity-of-mood disorders, including
premenstrual dysphoria; depressive disorders; stroke risk and amount of
damage following stroke, pain mechanisms; and cognitive functions,
including risk for dementia.

Brain areas shown to be responsive to ovarian hormones are numerous: the
widely connected cholinergic, catecholaminergic and serotonergic systems
in the forebrain; hippocampus; cerebellum; cerebral cortex; and spinal
cord. In addition to the neurons, the various types of glia cells are
affected. Most of the noted areas do not have well-defined nuclear
receptor localization for E2, which binds with ER alpha (ERα) or beta
(ERβ). Localization of nuclear ERα and ERβ is concentrated mainly in the
hypothalamus and amygdala, with lower densities in cells scattered
throughout the brain. The latter brain areas may be involved in
non-genomic estrogen signaling via estrogen receptor-like proteins on
the cell membrane.

Studies on the ovarian regulation of synapses in the hippocampus,
amygdala, and prefrontal cortex are under way. The hippocampus and
prefrontal cortex appear to be very much involved in spatial, episodic,
context, and working memory, whereas the amygdala is thought to provide
emotional context. The three areas are sensitive to ovarian hormones and
may be involved in mediating verbal memory deficit affects of the GnRH
agonist Lupron, which can be restored by exogenous E2. Cyclic changes in
spine-synapse formation have been observed in CA1 neurons of the
hippocampus. These changes peak on the day of proestrus, then rapidly
decline following the LH surge in response to the secretion of P on the
day of estrus. E2 given to ovariectomized rats can induce the formation
of spine synapses much like that observed during endogenous E2 increases
between diestrus and proestrus; exogenous P causes a rapid decline in
spinal synapses. Downregulation of dendritic spine synapses has been
shown in several species to correlate with interference in
hippocampal-mediated memory. The variety of signaling pathways affected
by E2 is impressive, and the existence of E2 effects in many brain areas
may be understood in terms of the widespread presence of the
E2-regulated, extragenomic signaling pathways.

Other interesting relationships have been noted. In the rat hippocampus,
putative non-nuclear ERα is located in dendritic spines (Towart et al.,
2003), and E2 treatment increases interconnections of regions not
usually connected (Yankova et al., 2001). E2 activates putative membrane
ERα, causing it to bind to phosphatidylinositol 3-kinase, thus
activating protein kinase B and ultimately resulting in increased levels
of the dendritic spine scaffolding protein PSD-95 in a differentiated
neuron *in vitro* model system (Akama & McEwen, 2003). Finally, the
hippocampus of male rats does not respond to E2 unless brain sexual
differentiation is blocked right after birth; yet male rats do respond
to DHT, which induces synapses in the male hippocampus as robustly as E2
does in the female rat hippocampus.

Other major issues have been noted. First, selective ER modulators
(SERMs) enhance cholinergic function in the rat, but at least one SERM
blocks E2 induced synapse formation. Second, brain-derived neurotrophic
factor fluctuates during the estrous cycle and is highest during stages
dominated by E2 (Scharfman et al., 2003). Third, E2 effects on the brain
include the dopaminergic system. One way this is manifested is in the E2
regulation of symptoms related to Parkinson's syndrome and motor
coordination in general; here, E2 effects are dose related. High doses
worsen symptoms (Bedard et al., 1977), whereas low doses are
pro-dopaminergic, beneficial, and potentially neuroprotective (Becker,
1990). Fourth, besides neurons, oligodendrocytes express both ERα and
ERβ (Takao et al., 2004), and other studies show that other types of
glia cells, i.e., astrocytes and microglia, respond to E2. Fifth, the
brain, along with fat, aromatizes androgens to estrogens, and the brain
may have the capacity to convert small amounts of cholesterol all the
way to E2. This capacity becomes evident in animal models of stroke.
Knock-out of the aromatase gene makes mice extremely sensitive to
ischemia-induced brain damage, and this damage can be reduced by E2
treatment (McCullough et al., 2003). Aromatase blockers also increase
sensitivity to ischemia-induced brain damage, which raises questions
about possible side effects of the treatment of cancer patients with
aromatase inhibitors instead of SERMs.

When considering gender differences in stress effects and stress
responses that may be influenced by gonadal hormones, consideration
should be given to their interactions with cortisol, catecholamines, and
other stress mediators. Although this area needs extensive exploration,
a few findings can be highlighted from studies in rodents. First,
gonadectomy increases reactivity of the hypothalamic-pituitary-adrenal
(HPA) axis and the sympathetic nervous system in both males and females.
Second, males and females are affected differently by stressors, with
males in some situations showing enhanced performance, and females
poorer performance in conditioning tasks (Wood & Shors, 1998). Third, in
males, androgens may mediate effects of acute stress on structural
plasticity, such as synapse formation in the hippocampus, whereas stress
appears to suppress synapse formation induced by estrogens in the female
hippocampus. Fourth, developmentally programmed structural and
functional sex-based differences exist in many brain regions, and these
may influence how the male and female brain responds to stressors.

During the discussion, it was mentioned also that tamoxifen becomes an
agonist when used long term in the treatment of breast cancer. To the
best of anyone's knowledge, co-regulators that may be involved in the
transition from antagonist to agonist have not been explored, nor has
tamoxifen effects been compared in neural versus reproductive tissues.
It was mentioned that tamoxifen and other SERMs may act via non-genomic
pathways not involving co-regulators. SERMs appear to act in areas
outside the brain; perhaps it may be necessary to use E2 in the brain
and block SERM access to the brain. With respect to blocking access to
the brain, the blood-brain barrier (non-fenestrated blood vessels) was
mentioned in respect to ovarian hormones, but outside the generalization
that they do not affect vascularization. Nothing else was noted
regarding specific brain regions. It might be added that several
critical hypothalamic regions in the HPO axis probably lie outside the
blood-brain barrier.

Regarding a more important role for progesterone in female versus male
brains, it was noted that in rats female brains contain more P receptors
(PR). These receptors may therefore play a role in protecting brain
function.

Mention was made that different progestins have different effects, as do
different estrogens; there are different activities of SERMs as well. It
would be valuable to examine a panel of estrogens and progestins to
discern differing effects on the ability to induce dendritic spines.
Given the decline of dehydroepiandrosterone sulfate (DHEAS) with age,
there is a question concerning possible actions of
dehydroepiandrosterone (DHEA) or DHEAS in the brain and whether they
afford any protective affects (e.g., via aromatization). Different DHEA
metabolites may have different effects, including interference with
aromatization.

# Dynamics of the Female Reproductive System and Changes with Aging

**Janet Hall, Massachusetts General Hospital**

The dynamics of the HPO axis are such that as the gonadotropins
stimulate the ovaries, there is follicular maturation and commensurate
increases in the synthesis and release of E2. Not only does E2 stimulate
proliferation of the uterine lining preparatory to implantation, but it
importantly feeds back on the hypothalamus and pituitary to stimulate
additional LH, culminating in ovulation. Subsequent development of the
corpus luteum is associated with increased production and release of P.
As aging progresses, there is a diminution in the number of viable
follicles.

The progressive reduction in the number of follicles accelerates during
the 40--50 age range (Richardson et al., 1987). In the 35--46 age group,
there is an increase in FSH in the early follicular phase, even in the
presence of normal cycles (Welt et al., 1999). An early marker of
reduced numbers of follicles is the reduced level of ovarian inhibin B.
Inhibin B is not stimulated directly by the gonadotropins, but is a
biomarker of follicular granulosa cells. There also is a decrease in the
amount of inhibin A secreted by the mature follicle and the corpus
luteum, as well as a reduction in P; T and DHEA levels also decline with
aging. E2 levels are preserved during early phases of reproductive
aging; the reduced feedback of inhibin enables FSH levels to increase,
thus supporting the higher levels of E2 characteristic of early
perimenopause.

Changes from pre- to postmenopause is a waxing and waning process that
occurs over several years. The period of the menopausal transition is
marked by a great deal of variability in hormone levels from day to day
and from cycle to cycle. The changes in gonadal steroids and peptides
may have widespread and pronounced effects on non-reproductive
functions, but this area has received little research attention.

T levels fluctuate during the cycle in women with normal reproductive
cycles, but decrease in PM women. Data suggest that T levels are
slightly decreased in ovariectomized women compared to PM women, as is
DHEAS. DHEAS is produced primarily by the adrenal glands and shows a
decline with aging. Studies have indicated that the ovaries in PM women
lack the steroidogenic enzymes and gonadotropin receptors essential for
synthesizing androgens, suggesting an adrenal source for PM androgen
deficiency (Couzinet et al., 2001). However, the assays used for
measuring steroid levels constrain the kinds of studies that may be done
during PM, and advances in this methodology would be desirable.

PM women provide a unique model to study and examine the effects of
aging on the brain with respect to the neuroendocrine components of the
reproductive axis. The question posed is whether there are age-related
changes in components of the neuroendocrine system that may be
independent of changes in ovarian feedback characterizing the transition
to menopause. Data show that the half-life of plasma LH is lower in PM
women, compared to premenopausal women, following blockade of GnRH
(Sharpless et al., 1999); PM women have fewer of the more basic isoforms
of LH and FSH (Wide & Naessen, 1994). In premenopausal women more basic
isoforms have shorter half-lives and increased *in vitro* activity,
compared to PM women.

There is a significant decrease in FAS pulsatility in older compared to
younger PM women, indicating decreases in the activity of the GnRH pulse
generator (Hall et al., 2000). Despite the decrease in GnRH pulse
frequency, there is an increase in overall GnRH secretion in PM women.
Levels of LH, FSH, and FAS decrease with E2 and E2 plus P (Gill et al.,
2002). There is a decrease in the frequency of FAS pulses with E2 plus
P, but not with E2 alone, and an increase in the overall amount of GnRH
secreted with E2 plus P. The changes in pulse frequency and quantity of
GnRH are parallel in older and younger PM women. Taken together, the
data suggest that aging occurs at the hypothalamus and pituitary level,
as well as at the ovarian level.

In the discussion addressing questions related to hormonal pulsatility
versus continuous exposure, it was noted that pulsatile release of GnRH
is important. Studies of GnRH exposure show that continuous GnRH first
stimulates then desensitizes pituitary gonadotropin secretion. It's not
thought that pulsatile versus continuous LH or FSH has a substantial
effect on ovarian function. Periodic release of E2, as seen in different
phases of the menstrual/estrus cycle, affects neuronal plasticity, while
continuous E2 exposure reduces ER levels in most tissues, except for the
liver, where ER increases with E2 exposure.

The presentation mentioned that the size of the gonadotrophs in the
pituitary shrinks as a function of aging alone. It is not clear whether
there are similar reductions in the aging male, but the absence of
gonadal failure in the male to the extent seen in females make such
comparisons difficult.

There were several comments concerning pharmacologic versus physiologic
E2 doses.

While continuous exposure to pharmacologic E2 levels downregulate ER in
many tissues, continuous exposure to low physiologic levels, which may
be effective against CVD, osteoporosis, and loss of cognitive function,
does not downregulate ER. Even across the normal cycle, with high and
low E2 levels, different effects are seen. Cell membrane ER may be most
sensitive to continuous versus pulsatile E2 exposure.

It was noted that pulsatile release of T in studies of adult women with
Turner's syndrome or oophorectomized women appears related to the
pulsatile release of LH and may relate to LH receptors in adrenal
tissue.

There is a literature demonstrating strong aging effects on GnRH
amplitude and pulsatility, even in women with regular menstrual cycles,
suggesting a variety of different hypothalamic-pituitary changes
depending on age

# Relating Aging and Outcomes of Menopause

**Linda Fried, Johns Hopkins School of Public Health and Anne Cappola,
University of Pennsylvania School of Medicine**

Although aging and menopause are each defined differently, the
consequence of each appears to be fairly similar. Functional decrements,
including frailty, body composition changes, osteoporosis, and
cognition, are all considered outcomes of each. Both are manifested
after the success of living to old age, but the possible relationship
between the two is obscure. It is not clear whether menopausal changes
are unique occurrences or part of the aging process, or whether one
presages the other. Nor is it clear how much a role hormones play.
Hormone levels decrease during menopause, but that is not unique to
menopause, as disease and other physiological events associated with
aging may cause decreases in hormone levels as well.

The Cardiovascular Health Study (CHS) has assessed 5,201 men and women
aged 65 and older in 1989--1990 for risk factors for CVD and
consequences of CVD, including disability and mortality. Function in
high-energy physical tasks decreased over the 10 years of the study. In
the context of events prior to and after the onset of coronary heart
disease (CHD), characterized by a gradual decline in physical activity
preceding CHD and a sharp decrease in function immediately following
onset, 20 of 78 characteristics are independently predictive of 5-year
mortality. Sex is an important predictor. Males have a significantly
greater risk than females. In comparison, age is not a major
contributing variable until the mid-80s, after data were adjusted for
diseases, health habits, and sociodemographic characteristics. In the
context of frailty, it has been hypothesized that there is a cycle of
frailty wherein one antecedent precipitates a frail event, leading to a
cycle of adversity. On the basis of an accepted, standardized definition
of frailty, a higher proportion of female CHS participants than male
participants between 65 and 90 years of age are frail (Fried et al.,
2001). Men appear to die earlier from chronic disease than do women,
whereas women tend to live with chronic disease, becoming frail and
disabled.

The reasons for these gender differences are not known, but several
hypotheses have been put forward. The first is that menopause is age
related, but it is a separate event from the aging process. In the
Woman's Health and Aging Study (WHAS), no significant effect of
ovariectomy on aging outcomes, such as frailty, disease, mortality,
disability, or falls, has been observed in women over the age of 65.
Further, survival times are not lessened by ovariectomy when observed
over a 5-year period. Thus, all things being equal, these data suggest
that the absence of functional ovaries many years following menopause
does not appear to affect aging variables. There are however, a number
of uncontrolled variables, such as hormone therapy (HT) and disease,
that can confound the relationship between aging and menopause. A
separate analysis from the CHS has revealed that estrogen and
corticosteroid users have lower testosterone levels, whereas women with
increased body weight have higher levels.

A second hypothesis posits that menopause is a direct cause of changes
in aging. A study of 30 age-matched pairs of premenopausal and PM women
has examined strength, muscle mass, and functional reserve (Bandinelli
et al., 2002). Most measures of isometric strength and all measures of
isokinetic strength show PM women to be less strong, with less muscle
mass than women with cycles. Loss of functional reserve also is
associated with menopause, but not with age.

The third hypothesis is that menopause is a sentinel event that leads to
vulnerability to aging. Physiological changes are associated with
decreased E2 following menopause: increases in proinflammatory cytokines
\[i.e., the interleukins 1 and 6 (IL-1, IL-6) and tumor necrosis
factor-alpha (TNF-α)\]. Possible consequences of inflammation include
artherosclerosis and anemia. It has been noted that the more systems
involved, the greater the incidence of frailty. Data from WHAS
participants age 70--79 show that lower levels of DHEAS and insulin-like
growth factor-1 (IGF-1), along with higher levels of IL-6, are
associated with frailty (Cappola et al., 2003; Leng et al., 2004).

All three hypotheses are plausible, and all may be operative. Future
studies of the PM period into old age are worthwhile, as is
consideration of multisystem effects and their interactions.

During the discussion it was pointed out that in the CHS, depression was
associated with mortality but its possible overlap with frailty has not
been assessed, though the physiological conditions are present for such
effects.

Regarding the study of age-matched premenopausal and PM women, in which
muscle function differed between the groups, the issue of whether early
menopause is a biomarker for aging was discussed. Through early
menopause, women may be attacked in other ways (e.g., cytokine action),
and therefore physiologically older). An approach to this question would
be to replace missing hormones with exogenous ones to determine whether
(there is a gain in functions such as strength and endurance. This
approach would require the use physiological replacement levels,
however, and would be difficult to accomplish. It also could be
confounded by disease associated with early menopause.

One participant noted that because of the association of multiple
diseases with frailty, there might be endogenous, multisystem changes
with age that are not stress induced, and there may be similar changes
attendant with chronic stress associated with allostatic load. So there
could be, hypothetically, common pathways. Loss of resilience and
reserve are at the core of this question. In terms of measures, research
measures for looking at frailty potentially could be used for examining
allostatic load.

It was agreed that frailty and undereating form a condition where energy
expenditures are greater than intake, making it likely that the HPA axis
is activated. Inflammation also is considered central to the development
of frailty.

# Animal Models Discussion Session

**Session Chair: Thomas Clarkson, Wake Forest University School of
Medicine**

The use of animal models may be viewed in a continuum of investigations
from basic *in vitro* studies, to preclinical studies using animal
models, to clinical studies involving human subjects, and finally, to
clinical applications. The presentations and discussions in this session
focused on animal models used in studying the perimenopausal transition.

Such models will be useful in assessing genetic, endocrine, and cellular
pathogenetic events associated with perimenopause, particularly the role
of declining E2 production antecedent to the chronic diseases of aging.
For example, PM bone loss begins with declining E2 levels during the
perimenopausal period (Falch et al., 1987, Ettinger, 1988). In monkey
studies, small decreases in plasma E2 concentrations, comparable to
those in the early perimenopausal transition, are associated with
decreased endothelial function evidenced by paradoxical constriction to
intracoronary artery injection of acetylcholine. This endothelial
dysfunction may represent an initial stage of atherogenesis. In another
example, women's memory scores are better if HT is initiated during the
transition than if it is initiated after the first missed period.
Similar results have been observed in the ovariectomized rat model,
which shows that exogenous E2 is effective when the interval between
surgery and hormone administration is short.

## ***Rodent Models*** 

**Patricia Hoyer, University of Arizona**

Various rodent models are used for studying entry into perimenopause and
the impact of perimenopause on non-reproductive organs. These models
consist of knockout (KO) models such as Bax^-/-^ (Perez et al., 1999)
and Bcl-x^-/-^ (Rucker et al., 2000), transgenic models such as
follicle-stimulating hormone receptor (FSHR)-haploinsufficient mice
(Danilovich et al., 2002a, b), and the ovarian follicle-selective
toxicant model \[4-vinylcyclohexene diepoxide (VCD; Mayer et al.,
2004)\]. Postmenopausal models include ovariectomized rodents,
FSHR-haploinsufficient mice, and VCD.

The benefits of using the Bax^-/-^ model include maintained ovarian
function into older ages as a result of reduced follicular atresia.
These mice have more ovarian follicles and increased uterine weight than
wild type at older ages.

The Bcl-x^-/-^ model is advantageous because it has a compromised
follicle pool at up to 3 months postnatally, with an extended period of
ovarian failure. A quarter of these females can become pregnant,
although the litter sizes are small. As with the Bax^-/-^ model, the
follicular pool in Bcl-x^-/-^ animals may never be totally depleted.

In the FSHR-haploinsufficient model, ovarian failure occurs within 1
year, and fertility is compromised from 3 months to a year. At 3 months
reductions are observed in ovulation, numbers of ova, ovarian weights,
and corpora lutea. This trend continues as 7 months, with commensurate
reductions in E2 and progestins, but here FSH, LH, and T increase. At 12
months the hormonal pattern is similar, but the follicles are depleted,
with no corpora lutea, and fetuses are resorbed. Uterine pathologies
also are noted at this stage. This model is good for perimenopausal
studies from 3 months to 1 year and for PM studies at one year.

A number of benefits are evident with the VCD model (Mayer et al.,
2004). Primordial and primary follicles can be selectively destroyed in
these mice or rats, but cycling will continue until no primordial
follicles remain to be recruited. At that time, premature ovarian
failure occurs. The length of time to complete ovarian failure can be
adjusted, making the model appropriate for perimenopausal or
postmenopausal studies. Ovarian failure can be induced at any age, thus
the effects of age can be separated from ovarian failure. Also, the
animals can be used with altered or normal genotypes. Whereas there may
be nonspecific chemical effects, none have yet been observed.

## ***Mouse Models***

**Adriana Maggi, University of Milan**

The ERE-Luc reporter mouse (Maggi et al., 2004) is a unique model to use
for *in vivo* ER activity studies. The luciferase (Luc) reporter is used
because it has a rapid turnover, can be quantitatively assayed by
immunohistochemical (IHC) methods, and can be imaged *in vivo*. One can
co-localize Luc and ER, using IHC, and make parallel measurements of
endogenous E2 response genes. Bioluminescent reporter imaging in cycling
ERE-Luc mice show that E2 is highest on the proestrous day of the cycle
and stimulates liver production of a growth factor. On the basis of this
model, the mechanisms of ER transcriptional activation in fertile
females is E2-dependent in reproductive tissues and not dependent on E2
in non-reproductive tissue activation. Considering that ER activation
appears to be beneficial in non-reproductive tissue---e.g., prevention
of osteoporosis---but carcinogenic in reproductive organs, it is of
interest to identify the molecules involved in ligand-independent
activation of ER, because they might lead to an option for a safer
pharmacological treatment of the menopause.

## ***Rodent Models Discussion***

In reference to the observation of bone Luc activation within 24 hrs of
treatment with tamoxifen, but not E2, this simply could be a matter of
dosage. Tamoxifen acts as an agonist of ER in the absence of E2 and as
an antagonist in the presence of E2. This reflects the fact that
tamoxifen was given at higher dosage than E2. The laboratory is
currently looking at E2-specific gene expression patterns.

The ERE-Luc mouse model shows, for the first time *in vivo*, that ER can
be transcriptionally activated in the absence of estrogens (aromatase
inhibitors are used to block endogenous estrogen synthesis). It is
therefore a very good model to study ligand-dependent and
ligand-independent activation of ER. The non-genomic effect of estrogens
cannot be visualized with the ERE-Luc mouse. ICI 182,780 is used to
distinguish ligand-dependent from ligand-independent activation, but
ones needs to be aware that it can have effects on its own and that it
doesn't block ER in all tissues.

In the use of non-conditional KO mouse models, one should be aware that
developmental processes may be different from that in wild-type animals,
possibly leading to a misinterpretation of the data.

## ***Macaque Models***

**Jay R. Kaplan, Wake Forest University School of Medicine**

The reproductive cycles of monkeys are very similar to that of the human
female, but the length of the perimenopausal and PM periods differ
greatly. Monkeys have a long perimenopausal period and short PM, whereas
humans have short perimenopausal periods and lengthy PM. Much of what is
known concerning reproductive cycles in women is based on earlier
studies of the rhesus monkey.

Because it is impractical to wait for monkeys to reach 25 years of age
to assess monkey perimenopausal periods, investigators use group social
living to approximate what may occur during the monkey perimenopause.
Observations from these studies indicate that a hierarchy is soon
established that results in a pecking order from dominant to subordinate
animals. In such situations it has been shown that those at the top of
the order have a low risk of developing artherosclerosis, as opposed to
the more subordinate ones whose risk is much greater.

Similar effects are noted with regard to reproductive cycles and ovarian
function. In dominant animals 70 percent of their cycles are normal
compared to only 46 percent in subordinate animals. Disruption of normal
cyclicity also is associated with an increased incidence of ovarian
dysfunction in subordinate animals. For these animals, 23 percent of the
cycles are anovulatory compared to only 10 percent in dominant females.
Corpus luteal deficiency is greater in subordinate monkeys, and plasma
E2 levels are quite low. These patterns are thought to equate to what
occurs in perimenopausal women aged 30 to 45. Alternately, such patterns
as observed can be induced in monkeys less than 9 years old.

The noted results have an impact on vulnerability to disease.
Subordinate monkeys have an accelerated development of artherosclerosis
premenopausally, most likely as a result of low E2 levels (Kaplan et
al., 1995). Exogenous E2 inhibits artherosclerosis in subordinate
monkeys, and their profiles appear similar to those of dominant monkeys.
Another line of evidence supports this thesis. If dominant monkeys are
ovariectomized, they become more like subordinates, and a dominant
phenotype can be restored with exogenous E2. E2-deficient animals also
have less bone density. In animals that are skeletally mature,
subordinate animals have less bone density than dominant females, and
the decrease in bone density is probably caused by social manipulation
in adulthood. Similar to observations made in the monkey, premenopausal
E2 (birth control pills) results in less coronary artery disease after
menopause when compared to menopausal women not exposed to E2 (Bairey
Merz et al., 2003b).

## ***Peri/postmenopausal Baboon Models*** 

**Thomas B. Clarkson, Wake Forest University School of Medicine, on
behalf of Erika Honore, Southwest Foundation for Biomedical Research**

Characteristics have been collected on a large colony of about 300
baboons aged 16 to 32 years. Menstrual cycle data is collected daily and
classified as cycling, irregular (perimenopause), or acyclic (PM)
(Martin. Perimenopausal animals on a high fat diet show a more
atherogenic ratio of low-density lipoprotein (LDL) to high-density
lipoprotein (HDL) cholesterol (Chen et al., 1998), the incidence of
vertebral fractures increases in the transition of peri- to menopause
(Aufdemorte et al., 1993), and vaginal atrophy appears in animals close
to and in menopause (Hubbard et al., 1997).

Recent hormone data confirm earlier findings in aged baboon females. E2
levels are lower in perimenopausal and PM females associated with
increases in FSH levels. These findings, and the changes in bone density
and LDL in reproductive cycles suggestive of perimenopause, menopause
and PM, indicate that the baboon model is suitable for studies on the
perimenopause.

## ***Monkey Models Discussion***

It is possible to change the order of dominance within a group of
monkeys, and after doing so, changes in behavior usually precede
hormonal changes. If dominance is stable, the survival curve is much
lower in subordinate females; such differences are not noted in males.

Monkeys provide a good model for reproductive cycles, but it is unclear
whether age changes in these models are comparable to age changes in the
human. In reference to the social model of perimenopause and PM, it is
not possible to compare these observations with events that occur in the
wild. This model involves stress responses, which work similarly in all
species. Such events appear to be different from normal, menopausal
ones.

A participant mentioned the potential for a sheep model of
perimenopause; sheep exposed prenatally to T showed enhanced follicular
recruitment in the first year and, in the second year of life, became
anovulatory and developed cardiovascular problems, insulin resistance,
among other issues.

A lively and interesting discussion developed around whether it would be
possible to extend the postmenopausal lifespan in rhesus monkeys by
using better health care to extend the total lifespan. Another
participant noted that in caloric-restriction experiments, in which all
monkeys are provided with better-than-average health care, we can see,
as the study develops, whether lifespan will be extended in the ad lib
animals as a result of improved health care. The comment was made that
the ratio of time spent in perimenopause relative to PM is greater in
rhesus monkeys and baboons (1:1 perimenopause to PM ratio) relative to
women (1:5 perimenopause to PM ratio).

# Adipose Discussion Session

**Session Chairs: David E. Kelley on behalf of Lewis Kuller, University
of Pittsburgh**

Adipose tissue metabolism buffers energy balance, controlling fatty acid
flux after eating (Frayn, 2002). Small fat cells in adipose tissue are
more insulin sensitive, whereas larger fat cells tend to be insulin
resistant and have a high incidence of inflammatory products such as
TNF-α and interleukins. More recent research has indicated the endocrine
importance of fat cells, and it has been learned that they produce
enormous arrays of proteins including leptin, which provides energy
balance to the body and increases with the number of fat cells. A large
number of fat cells is associated with less circulating adiponectin,
which mediates insulin sensitivity in the liver and stimulates muscle to
oxidize fat, thus having importance in developing obesity.

Abdominal computed tomography scans and magnetic resonance images (MRI)
provide means to quantify visceral fat, which is located inside the
abdominal wall, and subcutaneous fat, which is located outside the
cavity. Visceral fat is a good marker of insulin sensitivity, because
large amounts of visceral fat show more insulin resistance. Insulin
resistance may be mediated by visceral fat-cell release of large
quantities of the identified proinflammatory products, by resistin, by
reduced levels of adiponectin, and by increased release of fatty acids.
These factors are released into the portal circulation with direct
access to the liver. A relationship between the amount of visceral fat
and a fatty liver and the development of type 2 diabetes has been
observed (Kelley et al., 2003). Subcutaneous fat generally is not as
strongly associated with insulin resistance as is visceral fat, but some
studies have found that subcutaneous fat is associated with insulin
resistance and type 2 diabetes, with one potential cause for this
variance being larger fat cell size.

## ***Estrogen Insufficiency and the Metabolic Syndrome*** 

**Evan Simpson, Prince Henry's Institute of Medical Research
(Australia)**

Models of estrogen insufficiency have revealed new and unexpected roles
for estrogens in both males and females. These models include natural
mutations in the aromatase gene, as well as knock-out (KO) mutations in
the mouse aromatase (ArKO; Fisher et al., 1998) and ER genes (ERKO).
Some of these roles, such as bone, vascular and metabolic syndrome
phenotypes, apply equally to males and females and do not relate to
reproduction.

In both male and female ArKO, there was a significant increase in the
percent of adipose tissue, though in the males it took longer to
develop, and the gain was not as great. Such animals also develop
insulin resistance at 1 year, but there are no effects on glucose
levels, suggesting that the animals have not yet developed diabetes. As
would be expected, serum leptin levels also are elevated in both males
and females, but the levels at 1 year are higher in the females.

Gonadal fat deposit per body mass was examined in the ArKO and wild-type
models following exogenous E2 administration or placebo. Reduction in
the fat ratio was notable in both groups but was very dramatic in the
ArKO model because of the significant gains in the ratio in the absence
of E2. The effects of exogenous E2 also were assessed in the male ArKO
mouse, which shows a fat pattern not observed in the female, such as
hepatic fat deposits. E2 administration clearly reduced fat deposits in
the male liver.

Studies were next done on the arcuate nucleus of the male ArKO mouse.
The arcuate was chosen because of prior studies indicating that this
area may be involved in satiety and may serve as a site of action for
leptin, adiponectin, and insulin. The data indicated a dramatic
reduction in the number of tyrosine hydoxylase cells in the male KO
model when compared to the wild-type animal.

Estrogen is important in both men and women in ways unrelated to
reproduction. Peripheral estrogen synthesis is the most important source
of estrogen influencing target tissues in PM women and men---circulating
levels reflect rather than direct estrogen action in these sites. The
absence of estrogen is associated with development of the metabolic
syndrome in mice and men (Maffei et al., 2004). There is a dimorphic
partitioning of lipid such that increases of fat in the liver are seen
in the male ArKO mouse, but not in the female, and E2 reverses such
affects. E2 thus should be considered to be another hormone synthesized
in adipose tissue, which acts to regulate lipid homeostasis along with
leptin, adiponectin, resistin, and cortisol.

## 

## ***Menopausal Changes in Body Fat Distribution and Metabolic Syndrome***

**Molly C. Carr, University of Washington**

Metabolic changes occur during menopause, resulting in changes in body
fat distribution and insulin resistance. Studies indicate that there is
an increase in intra-abdominal fat in PM women compared to those in
premenopause; such differences remain after adjusting data for age and
body fat mass (Toth et al., 2000). During this period adipocyte
cytokines, such as adiponectin, plasminogen activator inhibitor (PAI-1),
TNF-α, and IL-6, increase (Pfeilschifter et al., 2002).

The metabolic syndrome is characterized by increases in central obesity,
insulin resistance, dyslipidemia, high blood pressure, hypercoagulation,
and proinflammation. Diagnosis is based on the presence of at least
three risk factors: high blood pressure, elevated triglycerides, and low
HDL cholesterol. National Health and Nutrition Examination Survey III
(NHANES II) data show that the incidence of the metabolic syndrome
increases with age in men and women, but that women are at greater risk
following menopause (Ford et al., 2002; Park et al., 2003). Ethnicity is
a factor as well. CHD also occurs with greater frequency in those with
the metabolic syndrome and no diabetes but has a higher prevalence among
those with both the metabolic syndrome and diabetes (Alexander et al.,
2003).

In a study begun in 1997, 120 premenopausal women aged 47 to 65 years
were followed from premenopause through the transition into PM. E2
replacement was given either orally or transdermally, with a switch-over
design following a wash-out period. Preliminary data have been collected
on LDL size, fasting lipid levels, hepatic lipase, visceral adiposity,
fasting glucose and insulin levels, and a number of proinflammatory
factors (Carr et al., 2002). Initial results indicate that there are no
changes in pre- and PM body mass index (BMI), but there are significant
increases in visceral adiposity after menopause. Truncal fat also is
increased to a significant degree.

In PM intra-abdominal fat deposits, fasting lipids, and insulin and
glucose levels increase, and there is a greater prevalence of the
metabolic syndrome (Carr, 2003). Most studies on PM are problematic in
that they are predominately cross-sectional and do not have premenstrual
controls. It is difficult to control for aging and BMI in
cross-sectional studies.

## ***Obesity and Premenopausal Risk Factors***

**David Kelley on behalf of Lewis Kuller, University of Pittsburgh**

The decline in E2 production across the menopause, when combined with a
diet of high saturated fat and a sedentary lifestyle results in
significant weight gain and accrual of visceral fat. This is closely
linked to insulin resistance and decreased adiponectin, which in turn
mediate changes in lipoprotein composition and development of
atherosclerosis. Thus, it is not just simply the menopause-related
decline in E2 that accounts for an increased incidence of disease during
the PM years.

Data from the Healthy Women Study show that as visceral adipose tissue
increases, adiponectin levels decrease, with commensurate increases in
insulin levels. As weight gains increase, compared to baselines in PM
women, the levels of adiponectin decrease, and LDL cholesterol and
fasting levels of insulin increase. These changes have subsequently been
related to increases in calcium levels in coronary arteries. Weight (not
calcium) increases relate to a stiffening of arteries, which increases
the velocity of blood flow. Increased adipose mass also is related to an
increased incidence of cancer (Cottam et al., 2004).

## ***Interaction between GnRH and Diet***

**Mary Dallman, University of California at San Francisco**

The GPR-4 transgenic rat model targets expression of the cAMP-specific
phosphodiesterase to GnRH neurons (Paruthiyil et al., 2002). In joint
experiments between the Weiner and Dallman laboratories, the interaction
between GnRH and diet has been assessed in this model. These rats have
blunted GnRH responses mediated by cAMP *in vitro* and diminished LH *in
vivo*, and females possess polycystic ovaries and are anovulatory by 9
months of age. Subsequent studies on 2- to3-month-old rats show that the
GPR-4 animal gains a significant amount of weight when on a high-energy
diet, compared to control females that fail to show significant gains in
body weight. Weight is not gained on a diet of regular chow in either
group. The weight gain appears in abdominal fat deposits and is
attributed to overeating rather than hormone differences. If both groups
of animals are ovariectomized and given exogenous E2 replacement, both
gain weight and show abdominal obesity on the high-energy diet. This
result may be due to the high amounts of exogenous E2 that, after 3
weeks of treatment, probably inhibit the activity of GnRH neurons in
both GPR-4 and wild-type controls.

One hypothesis is that GnRH may provide inhibitory control of food
intake. Reduced GnRH secretion in GPR-4 rats and in ovariectomized
E2-treated females reduces stimulatory GnRH input to the cells in the
hypothalamic arcuate region. The arcuate, via
alpha-melanocyte-stimulating hormone, stimulates
corticotrophin-releasing factor (CRF) neurons in the paraventricular
hypothalamic nucleus, reducing food intake.

## ***Discussion***

Several issues were raised with respect to E2. While fat cells
synthesize E2, the amounts in premenopausal women are quite small when
compared to E2 secreted by the ovaries. However in postmenopausal women
where the ovaries cease to make estrogens, fat tissue becomes the major
estrogen source. E2 can reverse glucose intolerance. It is not clear
what the mechanisms are for E2 to alter fat distribution as well as
lipid levels. The variables are so interconnected, it would be difficult
to differentiate effects on one variable at the exclusion of all others.
Information is not available on the effects of E2 manipulation in animal
models of obesity.

The point also was made that low birth weight babies are more prone to
develop the metabolic syndrome in adulthood, and that fat storage may
differ in young and older people. The change in body fat distribution
happens over time.

# Immune System Discussion Session

**Session Chair: Betty A. Diamond, Albert Einstein College of Medicine**

A number of questions concern possible interactions of the immune system
with hormones or of hormones with the genetic background or non-genetic
modulators of the immune system. There is a higher incidence of
autoimmune disease in women than in men. It is difficult to compose an
overall statement concerning hormones and autoimmune disease, because
the mechanisms of hormonal effects, which are thought to be very
complex, are not well understood. Some autoimmune diseases occur in the
premenopause period, while others occur during PM. In some diseases such
as rheumatoid arthritis, E2 appears to be protective, whereas in other
diseases like lupus, E2 seems to be deleterious. There is a lack of
sophistication and understanding of the mechanisms underlying autoimmune
diseases and the effects of hormones on the immune system.

## ***Hormonal Effects***

**Paul W. Kincade, Oklahoma Medical Research Foundation**\
It is known that sex steroids have a major influence in replenishing the
immune system, in regulating the process of generating new lymphocytes,
and in regulating the steady state (Igarashi et al., 2001). Rare
lymphoid progenitor cells in the bone marrow are influenced by sex
steroids and contain ERα and ERβ. Stromal cells also are hormone
sensitive, although hormone influences occur only in adult life.

ERα is particularly important for the observed E2 responses in B
lymphopoiesis. ERαKO mice are not as sensitive to E2 actions as
wild-type mice. ERβ is not involved in the B-lymphocyte action. In
studies on humans it was found that lymphocytes are made in the bone
marrow throughout life, with no apparent differences according to
gender.

The precise mechanisms for hormonal regulation of lymphocyte formation
or effects of hormonal withdrawal on lymphocyte production in humans are
not known. Although hormones in the mouse model influence components of
the mature immune system, it is uncertain whether there are similar
effects in PM women.

## ***Autoreactivity in the Immune System***

**Betty Diamond, Albert Einstein College of Medicine**

As antibody molecules and B cell receptors are generated, autoreactivity
also is generated at the same rate. B cells are protective against
pathogens, but the process of eliminating autoreactive cells informs
human disease. Mice carrying a transgene of the heavy chain against the
DNA antibody form a model to understand hormonal effects. In these mice,
the heavy chain associates with a full spectrum of light chains, which
leads to several outcomes: production of antibodies with no affinity;
production of antibodies with low affinity; production of two
high-affinity antibodies, deletion of cells carrying the germ line light
chain that are immature, and anergy induction in cells with mature light
chains, rendering the cells unable to respond to antigen. If BALB/c mice
are given E2 so that serum levels are about 70 pg/ml, causing titers of
anti-DNA antibody to rise, the mice develop glomerular immunoglobulin
deposition, which is the gold standard for pathogenic anti-DNA antibody.
E2 also causes an expansion of B cells, with damage to the transgene.
BALB/c mice also show proteinuria.

A number of efforts have been made to discover what happens in the B
cells and to identify genes that are upregulated in the B cells of
E2-treated mice. The signal is transmitted to the B cells when the
antigen engages the immunoglobulin or the B cell receptor. E2-treated
animals have lower amounts of B-cell calcium flux than placebo-treated
animals do. Immature B cells that receive a strong B cell
receptor-mediated signal undergo apoptosis. Thus, for any antigen
concentration, increased concentrations of E2 decreases the B-cell
receptor signal that will cause the survival of more autoreactive B
cells than when E2 is absent. This leads to less stringency in the
negative selection of B cells; the threshold is altered by E2. Prolactin
has similar effects (anti-DNA titers increasing and glomerular
immunoglobulin deposition), but it uses an entirely different mechanism
to achieve them.

The observed E2 affects in the BALB/c mouse are absent when using the
C57BL/6 mouse. This suggests that hormonally responsive immune systems
are genetically determined. The C57 strain shows a more sensitive
uterine response to E2.

## ***Inflammatory Markers and Mechanisms***

**Russell P. Tracy, University of Vermont**

The meaning of "general inflammatory status" can be very confusing.
Inflammation status as measured by various markers is associated with
CVD risk and chronic diseases of older people. However, the strength of
the association (relative risk) for a given marker may change with age
and possibly with increased disease burden. In younger people, where
myocardial infarction(MI) is the outcome for which inflammation markers
demonstrate long predictive ability with little change over time.
However, in an unpublished study assessing cardiovascular mortality
using a composite measure of inflammatory status, the hazard ratio is
highest soon after inflammation markers are drawn and decreases greatly
over subsequent years; the risk is substantially higher in men than in
women, although the pattern of decline over time is similar. In the CHS,
almost half the deaths are cardiovascular related, but overall less than
10 percent is attributable to MI. This marks a situation that differs
from that observed in younger populations. Most cardiovascular deaths in
the elderly are due to sudden heart stoppage.

Other chronic disease outcomes associated with inflammation markers have
been noted. Inflammation markers also are associated with components of
metabolic syndrome, especially adiposity. The inflammation
marker-adiposity associations begin early in life, even as early as 2
years of age. Thus, even in early childhood, obesity is associated with
the presence of markers implicated in cardiovascular morbidity and death
later in life (Ferguson et al., 1998). Other data show that 12 weeks of
caloric restriction in obese healthy women, resulting in an average
weight loss of 7.9 kg, is associated with marked decreases in
cholesterol, LDL, HDL, triglycerides, and the C-reactive protein (CRP)
marker (Heilbronn et al., 2001), demonstrating that simple interventions
such as weight loss can have an important impact on this situation.

Medications can have a large impact on inflammatory markers. For
example, examining the effects of HT on coagulation factors and lipids
has revealed a mixed set of results, with increases and reductions in
good and bad risk factors. Most markers of inflammation decrease with
HT; only CRP increases, and then only with oral estrogen, but not with
SERM, usage (Cushman et al., 1999; Cushman et al., 2001; Walsh et al.,
2000). We do not know whether this increase has important physiological
consequences; perhaps the increase in CRP indicates an improvement in
the innate immune system function rather than increased risk for
disease.

To better understand the association of the inflammatory marker IL-6
with atherosclerosis, Dr. Tracy's laboratory has asked whether IL-6 is
just a marker or whether it is involved in the biology of
atherosclerosis. Atherosclerosis-prone mice (such as C57BL/6 on a
high-fat diet) injected with a very small but chronic dose of IL-6,
sufficient to raise the fibrinogen level by about 10 percent, have a 2-
to 5-fold increased risk for atherosclerosis (Huber et al., 1999). Dr.
Tracy's laboratory has focused on the adaptive immune response and the
ability of IL-6 to amplify T helper (TH) cell differentiation, where TH1
cells activate lipid uptake into foam cells via secreted interferon γ,
and TH2 cells produce anti-atherogenic IL-4. When C57BL/6 mice, which
normally make primarily TH1 cells, are modified genetically to make
primarily TH2 cells, atherosclerosis risk decreases substantially. In
BALB/c mice, which make primarily TH2 cells and have a lower
atherosclerosis risk, blocking IL-4 action results in substantially
increased atherosclerosis (Huber et al., 2001). An epidemiologic study
is planned to explore the association between TH1/TH2 distribution and
atherosclerosis risk in people.

## ***Discussion***

Concerning possible effects of E2 and P on lymphopoiesis, it may be that
P accentuates the effects of E2. Human mitogenic responses to P are not
great, but this field is in its infancy. E2 increases the number of B
cells, and prolactin affects follicular B cells. T cells also need to be
present. Regarding the role of androgen, testicular feminization (tfm)
mice (like ovariectomized mice) have substantially elevated number of B
cell precursors in the spleen compared with wild-type mice. Furthermore
ERαβ double KO mice still show a lymphopoietic response to E2, possibly
via the AR.

# Bone Discussion Session

**Session Chair: Sundeep Khosla, Mayo Medical School**

Osteoporosis, or low bone mass, currently affects 44 million U.S. women
and men aged 50 years and older. This number is estimated to increase to
52 million by the year 2010 and to 61 million by the year 2020. The
estimated annual cost in 2001 dollars to the U.S. health care system was
about \$17 billion. A better understanding of the pathogenesis of
osteoporosis is key to prevention, as well as to developing more
physiologically relevant treatments. Compared with a young woman's bone
structure, there is a marked disruption of the microstructure of
osteoporotic bone. Bone density does not provide information concerning
the quality of bone that is present. Thus, there is a need to go beyond
bone density to an assessment of bone quality, which is difficult to
measure.

In comparing the lifetime risk of fractures in women at 50 years of age
to that of men at 50 years of age, women's risk for hip, forearm, or
vertebral fracture is greater by 3-fold or more. The incidence of hip
fracture and vertebral fractures rise earlier and is greater in women
than in men. These differences may be attributed to men having bigger
bones and perhaps differences in quality; volumetric bone densities are
similar in men and women.

Data have been pulled together from a number of studies to provide a
perspective on menopausal effects on cortical and cancellous
(trabecular) bone. The data indicate a fairly rapid and immediate loss
of cancellous bone, with a slower but significant loss of cortical bone,
starting at 50 years of age. Losses in men are more gradual and of a
lesser amount. Other data show that relatively small doses of exogenous
E2 following ovariectomy reduces or prevents bone loss (Genant et al.,
1982). Considering the effects of age and menopausal transition status
on bone formation and turnover, increases in formation fail to keep pace
with increases in bone resorption (Garnero et al., 1996).

## ***Menopause Transition***

**Joel Finkelstein, Massachusetts General Hospital**

In a longitudinal study of the menopause transition (Slemenda et al.,
1987) based on 84 women aged 42 to 48 years, bone density studies over 3
years show lower midshaft and distal radial BMD values as menopause
progresses from early perimenopause to late PM. The SWAN study, a larger
and more definitive study, followed 2,193 women (mean age 46 years at
entry) for 4 years. The results were based on nine adjustments,
including age, weight and weight change, ethnicity, smoking, alcohol
use, calcium intake, and physical activity. Bone density change from
premenopause through PM in lumbar spine and hip shows large reductions
in bone density in late perimenopause and PM. Little bone loss is
observed in early perimenopausal women. The rates of bone loss are much
greater in women who weigh less than 80 kg than in women who weigh more
than 80 kg.

## ***Anti-Osteoporotic Effects of Sex Steroids*** 

**Stavros C. Manolagas, University of Arkansas for Medical Sciences**

The anti-osteoporotic effect of sex steroids results from the
suppression of the rate of bone remodeling, as a consequence of
inhibitory effects on osteoclastogenesis and osteoblastogenesis (De
Gregario et al., 2001), as well as from maintaining a balance between
bone resorption and formation, as a consequence of stimulating
osteoclast- and attenuating osteoblast-apoptosis (Kousteni et al.,
2003).

The effects of estrogen or androgen on bone result primarily from
extranuclear effects of the ER or androgen receptor (AR) that involve
the activation of cytoplasmic kinases and kinase-mediated control of
gene transcription, which is distinct from classical nuclear ER and
estrogen response element (ERE) interactions or protein-protein
interactions between the ER or AR and other transcription factors.
Activators of non-genotropic estrogen-like signaling (ANGELS), such as
the steroid estren, are small molecules that mimic estrogen/androgen
non-genotropic effects, but lack their genotropic actions. ANGELS are
able to activate the non-genotropic actions of the ER/AR by activating
kinase signaling cascades that increase bone mass and strength without
affecting reproductive tissues. Multiple signaling pathways involving
bone morphogenic protein and Wnts are activated by ER-stimulated kinases
and appear to be involved in bone growth.

## ***Environment, Hormones, and Bones***

**Deborah M. Kado, UCLA**

Environmental risk factors that may affect osteoporosis are diet,
physical activity, smoking and stress. Such factors may act through the
HPO, influencing early menopause and its resulting effects on bone loss
and osteoporosis. Alternately, such environmental factors may act
directly on the bone loss/production system without interacting with the
HPO. The preponderance of evidence supports the idea that risk factors
act through the HPO rather than bypassing that axis.

Stress has various definitions, but when it acts through the
hypothalamus, in simplified terms, two axes may be affected (Tsigos &
Chrousos, 2002). Reduced GnRH may influence  ovarian function leading to
lower sex steroid production and/or increased CRF may affect the adrenal
glands leading to higher cortisol production, with both avenues
eventually resulting in bone loss. A different pathway could act via
norepinephrine and the proinflammatory protein IL-6 to affect bone loss.
A number of defined stress factors appear to influence early menopause
and similarly are associated with osteoporosis.

## ***Changes in Bone Mass with Age***

**Clifford J. Rosen, Maine Center for Osteoporosis Research and
Education**

Data show that bone mass peaks at about the same age in men and women,
although there is much less mass in women. Bone loss begins in women
aged 50 to 60 years; one-third of the women lose trabecular bone at a
faster rate than the others do. It may be that E2 loss in some early PM
women may predispose them to excessive bone loss. It is clear that the
relationship of E2 and bone loss is a complicated one. Future research
should be able to identify those women prone to lose bone more rapidly;
the SWAN study is an early step in that direction.

Bone loss is important because it is an independent predictor of
fracture---bone density is a predictor of fracture risk. Bone loss,
combined with low bone density, is a very strong indicator of fracture
risk (Riis et al., 1996). Studies in four different strains of mice show
vertebral and tibia bone loss following ovariectomy, but there are
significant strain-related differences in whether the loss is greater
for vertebral or tibia bone (Myers et al., 2003). This may indicate a
genetic predisposition to lose bone. There is a need for biomarkers or
other indices to identify women predisposed to early bone loss. The
predictors may be genetic or chemical.

Understanding about bone quality, which may be very important, is just
beginning. The effects of exercise, for example, may be quite marginal
on fracture, but they may be reflected in differences in bone quality.
Ultra structure determination will be very important to bone loss and
rate of bone acquisition. There is virtually no *in vivo* longitudinal
data on women going through the menopause. More research initiatives are
needed on other predictive factors, such as vitamins, environmental
factors and/or trace metals, that interact in addition to estrogen to
effect bone quality.

## ***Discussion***

Skeletal bone is constantly changing, regenerating about 10 percent
every year. Thus, multiple pathways are involved in the constant repair
of stresses on the bone that result from daily living. E2 does not
appear to account for the differences in bone loss by body weight. BMI
may have effects because of weight bearing characteristics. There is
some uncertainty whether studies of the effects of cortisol on bone have
been done properly.

Starting out at different baselines can affect the rate of bone loss,
underscoring the need for longitudinal studies such as the SWAN study.
The relationship between IGF levels and bone density is not very clear.
There is some data that IGF-1 is strongly related to bone density. There
are high quantities of aromatase in bone that may have some impact, but
it is not clear what that impact may be. The role of genetic variations
in key genes in regulating bone loss, such as the gene encoding ER, also
merits further study.

# Cardiovascular Discussion Session

**Session Chair: Michael Mendelsohn, Tufts-New England Medical Center**

Most of the biological models used to date do not account for the timing
and loss of hormones and the timing and mode of HT influences. ERα and
perhaps ERβ mediate rapid activation of endothelial nitric oxide
synthase (eNOS). Two broad categories of effect of hormone on the
vascular wall are posed (Mendelsohn and Karas, 1999). A rapid effect is
vasodilation due to an increase in bioavailability of nitric oxide (NO)
that works through a non-genomic mechanism. The other is a recently
discovered protein mediated interaction between ERα and eNOS. ERα
appears to mediate the ability of E2 to inhibit the vascular injury
response in mouse models. If ERβ is absent, there is an abnormal
vascular contraction, with a high contractile effect and high blood
pressure.

Many cardiovascular tissues possess hormone receptors. Animal studies
have produced the following results. Myocardial cells express ERα, ERβ,
and produce aromatase. Vascular endothelial cells express the same
receptors in addition to activin. Vascular smooth muscle cells express
these proteins plus the PR and AR. Blood cells also possess the E2 and
ARs, as do T lymphocytes. Cells involved in coagulation and thrombosis
also express hormone receptors. Megakaryocytes express ERβ and AR, and
hepatocytes express ERα, AR, and aromatase.

It should be noted that there have not been studies of HT during the
perimenopause period. Although the study populations included
postmenopausal women as young as age 50, HT was initiated on average at
age 62 in the WHI study and on average at age 67 and in the Heart and
Estrogen/progestin Replacement Study.

## ***Epidemiological Factors***

**Kim Sutton-Tyrrell, University of Pittsburgh**

Several hormones and epidemiological factors have been associated with
CVD. Menopause is associated with thicker carotid walls, stiffer
arteries, and reduced endothelial function. HT is protective for
subclinical disease. Low T is correlated with thicker carotid walls. Low
sex hormone-binding globulin (SHBG) relates to cardiovascular risk
factors (Sutton-Tyrrell et al., 2005) and thicker carotid walls. E2
administration improves endothelial function and has a positive
inotropic effect.

Areas worth exploring involve looking beyond E2 to T and other
androgens, SHBG, and the relative balance between androgens and
estrogens. All vessels are not the same; thus hormones may affect
different vessels differently. Possible interactions with age or
duration of menopause are worth exploring, as is vascular remodeling.
Further studies on healthy women, such as those being supported by NHLBI
and the WHI, would be useful.

An interesting "Double Whammy" hypothesis was stipulated, which
essentially states that at menopause arteries are exposed to higher
levels of risk factors during a time of increased vulnerability to
damage because of the changes in the hormonal environment.

## ***Hormonal Effects***

**Richard Karas, Tufts-New England Medical Center**

The Framingham Heart Study has reported that premenopausal women have
lower cardiovascular risk than PM women. Incidence of CVD increases with
each age group, but this increase is greatest for PM women (Kannel et
al., 1976). In viewing these data one is struck by the small sizes of
each of the study groups. Another landmark study, the Nurses Health
Study, has compared natural with surgical menopause and never- versus
ever-users of HT (Colditz et al., 1987). Although the results of this
study lead to the conclusion that surgical menopause increases the risk
of coronary heart disease and estrogen use prevents it, the sample
numbers in this study are small.

The argument against the idea that loss of ovarian hormones leads to an
increased risk of CVD can be seen in population-based studies that show
a monotonic increase in cardiovascular death rates with age. In
contrast, breast cancer deaths show an inflection point when plotted
against age (Liu et al., 2003). If menopause increases CVD risk, one
should also see an inflection point. However, age and hormone factors
appear confounded when considering CVD risk. When the Framingham data
are re-plotted, the risk of CVD in premenopausal women increases
exponentially with age; there is no age effect once women reach
menopause. This re-analysis also shows that women who experience
menopause earlier have more CVD risk than those who have later
menopause. The risk/benefit ratio of hormones may change, and it
especially may change by disease state.

## ***Longitudinal Effects***

**Philip W. Shaul, UT Southwestern Medical Center at Dallas**

Regarding longitudinal effects of hormones on vascular health, not much
is known about the HPO axis and the increase in vascular dysfunction or
atherosclerosis that occurs during the menopausal transition. There
appear to be some E2 effects, based on observational studies and the
changes observed in CVD following ovariectomy, whereas the
interpretation of trials evaluating the impact of HT on CVD is less
straightforward. The possibility of genetic markers related to estrogen
action, which may predict atheroscelerosis and myocardial infarction,
should be examined. The possible effects of progestins, androgens, and
inhibin/activin are not clear.

There are various sites and modes of E2 action on the vasculature. These
include antioxidant effects, the increase in HDL and decrease in LDL, a
decrease in homocysteine, and important anti-inflammatory actions. In
the endothelium there is an increase in nitric oxide (NO) production
that occurs via genomic upregulation and non-genomic activation of eNOS.
Prostacyclin synthesis is also increased via genomic and nongenomic
mechanisms. In vascular smooth muscle there are increases in inducible
NO synthase expression, and there are blunted intercellular adhesion
molecule expression and nuclear factor κB (NFκB)-mediated mechanisms, to
name a few.

There are several ways in which the HPO axis may modify atherosclerosis
and vascular dysfunction. Progestins may oppose the effects of E2 on
antioxidant actions and lipoprotein metabolism, and they may
downregulate ER. Alternatively, androgens at physiologic levels decrease
flow-mediated dilation, and they are negatively correlated with carotid
intimal thickening. Activin inhibits the propagation of endothelial
cells and enhances the differentiation of monocytes into macrophages,
but it inhibits foam cell formation. Possible affects of gonadotropins
are not well studied.

This information leaves several unanswered questions with respect to CVD
and the menopausal transition:

1.  Do changes in vascular function and atherosclerosis progression in
    the perimenopause relate to hormonal changes?

2.  What are the effects of prolonged E2 deprivation?

3.  What is the contribution of systemic versus local hormone
    production?

4.  What is the role of genomic and non-genomic actions of E2?

5.  Are there genetic contributors and other factors that may affect the
    actions of hormones?

New work on cholesterol metabolites known as oxysterols \[for example,
27-hydroxycholesterol (27HC)\] suggests the possibility that they may
function as endogenous ER antagonists.

27HC at levels found in atherosclerotic lesions interfere with ER
function in an *in vitro* transcriptional assay, with ERβ inhibited to a
greater extent than ERα. Preliminary studies in a genetic model of
elevated 27HC in mice indicate that reendothelialization is attenuated.
Thus the presence of 27HC in established atherosclerotic lesions may
prevent local estrogen actions, potentially explaining a lack of benefit
of HT in patients with existing vascular disease

## ***Changes in Hemostasis with Menopause and with HT***

**David Ginsburg, Howard Hughes Medical Institute and University of
Michigan Medical School, and Barbara Konkle, University of
Pennsylvania**

Injury to the blood vessel results in platelet formation and a cascade
of processes resulting in clots formed by fibrin; too much bleeding or
thrombosis are two extremes of potential problems. This process may
differ in different blood vessels, such as large or small arteries and
various veins. In general estrogens are thought of as pro-thrombotic.

Hormones change much of the machinery responsible for platelet/clotting
actions. E2 increases some of the factors and reduces others. There is
the issue of genetic factors and their influence; menopausal events may
fit under this umbrella as well. Factor V Leiden affects many
individuals and is associated with venous thrombosis. Genetic factors
interact with hormone and age factors in key ways.

Regarding non-genetic factors of coagulation, there are numerous changes
in homeostasis with aging, such as increases in procoagulants and
natural inhibitors and local changes in endothelium. Events that change
around the time of perimenopause have not been studied, although the
SWAN study may provide information on changes that occur as a result of
aging, as opposed to those occurring during menopause.

HT affects coagulation (Post et al., 2003). There is an increase in
procoagulant factors and a decrease in natural anti-coagulants, some of
which differ from those seen in aging, and an increase in thrombin
generation. Very little is known about the role of progestins, which are
thought to have few effects on thrombosis, but recent thinking suggests
there may be some progestin effects. The risk for venous thrombosis
increases with age, but a specific effect due to menopause has not been
shown.

A number of questions remain concerning the effects of estrogens and
progestins on thrombosis: whether there are biological differences
between the effects of endogenous versus exogenous hormones; the links
among atherosclerosis, inflammation and hemostasis; and the role of
coagulation in HT-induced increases in arterial thrombosis.

## ***Discussion***

Mention has been made of the relation of SHBG and T to heart disease.
One participant cautioned that SHBG is a powerful marker of metabolic
disease, and the changes in the dynamics of free and bound T suggest
that SHBG may have little relation to heart disease.

There are issues pertaining to where damage begins within the vessels,
the corrugation of blood vessels, and the possibility that
expansion-contraction of corrugated vessels increases the production of
free radicals. It is unclear whether E2 plays a role in such processes.
There are 30 to 40 years of data suggesting that injury to the
endothelial cell is at the start of the process of atherosclerotic
disease.

In respect to the noted research questions, the need for more
perimenopausal models and data was emphasized. The rate at which women
lose hormones has an enormous effect on cardiovascular outcomes. There
are virtually no studies in animals of natural menopause; almost all
studies involve surgical menopause. When hormones are replenished in
animals, it is critical how, what, and when this is done. In known
animal studies, hormones are never administered cyclically. The cyclical
issue has been untouched and needs to be studied.

# Cognition, Mood and Brain Function Discussion Session

**Session Chair: Pauline M. Maki, University of Illinois at Chicago**

A number of studies (SWAN, the Melbourne Midlife Women's Health Study)
generally show no differences by menopausal phase in objective cognitive
test performance, including verbal memory and attention, the two areas
thought to be most affected by hormones (Meyer, et al., 2003). However,
one study using SWAN data shows significantly lower prevalence of
forgetfulness among premenopausal women compared to women at later
menopausal stages (Gold, et al., 2000), and a study of Chinese women
finds worse performance among postmenopausal women on "Trail Making Test
Part B," a test of psychomotor speed and mental flexibility, even after
controlling for the effects of age (Fuh et al., 2003). Results from the
Seattle Midlife Women's Health Study show more frequent reports of
memory declines in the early and middle transition compared to the late
menopause. Women attribute changes in memory to age, stress, and health,
but not to hormone status (Woods, et al., 2000; Mitchell and Woods,
2001).

There is not uniform agreement that depressive symptoms increase in
middle-aged women. A recent study has assessed a number of factors that
may be associated with depression in midlife women (Freeman et al.,
2004). These include a history of depression, current severe
premenstrual syndrome, or poor sleep; being African American or
unemployed; and being in the early and late transitional stages of the
menopause. FSH level, when modeled as a quadratic term, is also
associated with increased depression.

Data from the Baltimore Longitudinal Study of Aging suggest that
menopausal symptoms and menopausal stage interact to influence
cognition. For example, mood ratings in pre- and perimenopausal women do
not differ. However, mood complaints are related to poorer visuospatial
performance in perimenopausal women only. This suggests that research
should not be confined to the main effects of menopausal stage or of
mood, but that it also should consider the interaction between effects
of menopausal stage and mood.

## ***Brain Organization Effects of Hormones*** 

**Susan M. Resnick, National Institute on Aging**

Regarding the effects of sex hormones on brain organization and
cognition, studies on congenital adrenal hyperplasia (CAH) have looked
at specific cognitive abilities in girls and found specific effects on
specific cognitive functions during a critical period of development.
Girls with CAH, who are exposed to excess androgens prenatally, have
higher spatial abilities than their unaffected female relatives but have
comparable performance on other cognitive tests. In addition, studies of
menstrual cycle variation in cognition show that only some aspects of
cognition vary with the cycle. Therefore it is important to consider
different types of specific cognitive functions, because brain function
can be affected at very specific times of development, and because it
can affect some aspects of cognition and not others. In studies of the
menopausal transition or effects of postmenopausal HT, it is critical to
focus on those aspects of cognitive function that are sensitive to the
effects of hormones.

Regarding neuroimaging studies looking at the differences between monkey
and human data in terms of neural targets of estrogen, there is no
overall increase in cerebral blood flow as a result of hormones in
animal models. However, imaging in women do show increases in specific
areas of the brain, such as the hippocampal, prefrontal, and
temporal-lobe areas, when longitudinal observational data are examined.
Patterns of brain activation do differ in women as shown in different
paradigms such as MRI, blood flow, etc. There are no 5- to 10-year
studies looking at neurobiological data in monkeys.

## ***Menstrual Cycle Studies***

**Barbara Sherwin, McGill University**

Numerous investigators have examined whether changes in aspects of
cognitive performance occur during different phases of the normal
menstrual cycle. High-estrogen phases of the cycle are associated with
better performance on tests of cognitive functioning in which females
typically excel, such as tests of verbal memory , compared with
performance during cycle phases when estrogen levels are low (Hampson,
1990; Maki et al., 2002; Phillips and Sherwin, 1992a). Studies have also
examined whether low levels of estrogen are associated with better
performance on tests in which males typically excel. Sherwin's
prospective studies of premenopausal women who have had ovariectomies
for benign diseases found a specificity of E2 effects on verbal learning
and verbal memory, but not on visual memory or on spatial abilities
(Sherwin, 1988; Phillips and Sherwin, 1992b). It is important to note
that not all cognitive abilities are affected by hormones.

In a Lupron study on women with intact ovaries, there is a significant
decrease in scores on tests of verbal memory and learning following
ovarian suppression that could be reversed with E2 administration. This
supports the specificity of estrogen's protective effects on memory seen
in the menstrual cycle and in surgically menopausal studies.

## ***Mood Alterations and Depression***

**Peter Schmidt, National Institute of Mental Health**

It is not clear whether alterations in mood occur during the
perimenopause or whether changes in female reproductive hormones are
related to the development of depression during this stage of life.
Depression is one of the leading causes of disease-related disability in
the Western world, second only to heart disease. It also has been
recognized that in addition to major depression, minor depression
(defined by the presence of fewer symptoms but not necessarily by less
symptom severity or level disability compared with major depression)
contributes to a considerable amount of disease-related disability. The
presence of comorbid depression is associated with a substantially worse
outcome in several medical conditions including CVD. Indeed, a recent
study has reported that independent of several risk factors (including
past history of cardiovascular disease, diabetes, smoking,
hypertension), depression is a significant predictor of all-cause
mortality in postmenopausal women (adjusted RR=1.32).

The majority of women do not develop depression during the perimenopause
or in PM. However, in some women the perimenopause is associated with an
increased risk for developing depression. Number range CHAPTERCurrent
understanding of the role of the perimenopause in depression has
developed from several recent findings: three randomized,
placebo-controlled studies demonstrating the antidepressant efficacy of
E2 in perimenopausal (but not postmenopausal) women meeting diagnostic
criteria for depression, and two longitudinal demonstrations of the
significantly increased risk of new-onset depression during the
perimenopause (compared with the years prior to the appearance of
menstrual cycle irregularity). These data suggest an association between
perimenopause-related hormonal events and the onset of depression.
However, the mechanisms whereby alterations in ovarian function convey a
vulnerability to develop depression in some women at midlife are not
known. Additionally, analogous to the postpartum, there is no increased
prevalence of depression, but both the perimenopause and postpartum
periods are associated with an increased risk for the onset of
depression in some women. These depressions may be distinct from those
occurring at other times in a woman's life (such as
estrogen-responsivity).

## ***CNS Effects of Estrogen***

**C. Dominique Toran-Allerand, Columbia University**

Regarding the direct effects of hormones on neural substrates of
cognitive ability, it is uncertain whether E2 effects on the brain are
directly due to the stimulation of neurotrophins or to E2 itself. It is
probably both. E2 upregulates neurotrophin receptors, and the
neurotrophins in turn upregulate ERs. There is probably a significant
interaction between the two.

## ***Neural Targets of Estrogen and Underlying Mechanisms of Action***

**John H. Morrison, Mount Sinai School of Medicine**

Although estrogens appear to act on the cholinergic and serotonergic
systems, the most dramatic effect is the estrogen-induced increase in
dendritic spines. Estrogen-induced spine increases are accompanied by an
increase in excitatory synapses and have been described in hippocampus
in rodent models, and both hippocampus and the prefrontal cortex in
monkeys. Behavioral effects are more difficult to ascertain, but there
are data to suggest increased memory performance. In older monkeys that
were cyclically injected with E2, performance was moderately improved on
delayed non-matching to sample, a hippocampal dependent task, but
dramatically improved on a delayed-response task, reflective of
prefrontal cortex function. These data support the human data of Resnick
and Sherwin suggesting that the prefrontal cortex may be a more
important target of estrogen than the hippocampus. Dendritic spine
growth continues in the primate following several years of growth and
cyclical E2 treatment. These studies are interesting because of the
cyclical effects on cognition and because protective effects have been
found for aged monkeys.

## ***Altered CNS Mechanisms during Perimenopause***

**Susanna J. Park, New Jersey Medical School**

It has not been clear whether alterations in CNS mechanisms occur during
the perimenopause, especially early on, or whether these alterations are
distinct from the mechanisms underlying ovarian function. A pilot study
has found that of seven perimenopausal women with regular menstrual
cycles, only one shows a rise in LH after E2 dose. Six of the eight
controls, who were younger aged women show a rise in LH after E2.
Although the sample size is small, the difference is statistically
significant.

## ***Discussion***

In considering hormone-behavior interactions in menopausal women, issues
of cognition and depression are of clinical importance. The need for
additional studies in this area is underscored by a recent report that
the incidence of depression is increased during the perimenopause,
particularly for women with a history of depression. These findings
emphasize the importance of individual differences in cognitive and mood
changes in the perimenopause. Our understanding of the neural substrates
for these changes is based largely on data from ovariectomized, young
animals. E2 withdrawal in such situations is instantaneous rather than
gradual as in natural menopause. Our understanding of cognition in
midlife women can be furthered by the use of comprehensive
neuropsychological batteries that include tests sensitive to both age
and hormone effects. Attention to cross-cultural issues in the design of
those studies would help to ensure the applicability of findings to
minority women. Finally, not all cognitive functions and related brain
regions are equally affected by estrogen. Non-human primate studies
suggest that the prefrontal cortex and related cognitive functions may
be particularly sensitive to estrogen.

A current focus in this area of research is the so-called "critical
period hypothesis," the view that hormones might provide neuroprotection
if initiated early in the menopause but might be harmful if initiated
later in life. There is some direct evidence to support the biologic
plausibility of this hypothesis in rodents (Gibbs, 2000), though
age-related increases in silent ischemic events might be a mediator of
increases in dementia with aging.

# A Paradigm on Staging the Menopause Transition

**Sherry Sherman, NIA; Nancy Woods, University of Washington; Estella
Parrott, NICHD**

Successful cross-talk among investigators requires that researchers from
different fields speak the same language, using common definitions for
stages of the menopause transition that facilitate comparison of
findings. Toward this end, the Staging Reproductive Aging Workshop in
2001 focused on developing a paradigm for menopausal staging (Soules et
al., 2001). Very dynamic physiological changes are occurring during the
menopause transition that would make a staging paradigm very useful. In
the late reproductive period, cycles begin to lengthen, and FSH
secretion increases. Stages of the early and late menopause transition
are variable in length. The early menopausal transition is marked by
cycle lengths greater than 7 days different from normal but with mostly
regular menstrual cycles and FSH levels continuing to increase. The late
menopausal transition is defined by missing more than two cycles, with
amenorrhea of 60 or more days, and is associated with the greatest
reporting of vasomotor symptoms. FSH increases further during this
period as well as during PM. Early PM is defined as within 4 years of
menopause, and late PM from the end of the 4 years until the death of
the woman. All stages are of variable lengths except for the two in the
PM period.

# Wrap Up Session

**Session Co-Chairs: Nanette Santoro and Bruce McEwen**

Several themes have arisen from the meeting. First, context is
everything. Researchers are increasingly seeing beyond a single hormone
and understand that the problems are much more complicated. It is time
to build on the WHI results and to begin to address the key questions.
Patient-oriented research is emphasized, and the human is clearly the
animal of interest and one of few animals to experience menopause.

Second, there is an increasing convergence of biological and
epidemiological approaches, in particular mechanistic and probabilistic
approaches. Biology shows that there are clear-cut effects of hormones.
For example, E2 action can be characterized in tissues and in whole
organisms, and specific pathways are getting clearer in brain, bone, and
the cardiovascular system. We need to take into account how the liver,
adipose tissue, and immune system are influenced by hormones and aging.
We can no longer afford to consider hormones in isolation, but we must
consider multiple hormones and biologic effectors acting on specific
tissues. Modeling at various levels is key to moving concepts learned
from underlying biological mechanistic studies to the bedside.
Biological models include *in vitro* studies of tissues, cells, etc. to
work out cellular pathways; small-animal studies to extrapolate
mechanisms in physiological settings; large-animal studies to explore
applicability to humans and provide "proof of concept" for
interventions; and experiments of nature to provide "proof of concept"
for human interventions.

Epidemiologic studies document weight gain among the U.S. population as
a major problem that needs to be addressed; we need to link
epidemiological issues with biological approaches to understand
causation and underlying mechanisms. We also need to develop risk models
as a basis for testing new interventions and for examining nonspecific
interactions with aging. An advantage of the epidemiological approach is
the forced emphasis on context, including social order as is seen in
monkeys. There are nonspecific interactions with aging. Because most
"risk factors" are small and not completely consistent, it is important
to figure out how to integrate them. Epidemiology provides directive
clues for biology and supports therapeutic studies by facilitating the
generation of physiological hypotheses to test in biological systems.
Nuances exist in the transition from normal reproductive cycles to the
perimenopausal period. Little is understood concerning the hormone and
biological events operating in this period. Better ways are needed to
assess hormone levels with greater sensitivity and specificity, and
better biomarkers need to be developed.

Third, collaborative cross-cutting research is strongly needed to
address some of the key themes of this workshop, and grant award
mechanisms such as the program project grant and interactive projects
will be especially appropriate. Four cross-cutting issues have been
identified. Three of these relate to treatment issues and one to the
underlying biology. However, even those related to treatment questions,
which was not a main focus of this workshop, relate ultimately to
biological questions both during the menopausal transition and in
hormonal responses and continuity of hormonal exposure in the latter
stages of the transition.

1)  Are there are similar or different critical time windows for HT or
    E2 treatment for protecting different systems (e.g., cognition,
    cardiovascular, bone, adiposity, immune)? Existence of such windows
    around the time of the menopause implies that there are ongoing
    processes related to aging that are not yet understood. Moreover, it
    remains to be seen whether E2 treatment during such critical windows
    should be followed by treatment later in life. Conversely, if E2
    treatment is NOT given during the critical window, does it become
    ineffective or deleterious? Results from WHI would suggest that this
    might be the case for some outcomes, but WHI results are very much
    dependent on the HT regimen used and the physiological
    characteristics of the subjects recruited into the study.

2)  What is the importance of discontinuous or episodic versus
    continuous administration of hormones, and how important is the
    timing of their administration in HT studies? For example, evidence
    suggests that enhancement of cholinergic function in the basal
    forebrain may be lost when E2 treatment is continuous as opposed to
    episodic.

3)  The importance of evaluating the effects of progesterone vs.
    synthetic progestins and the timing and dose of such treatment in
    relation to E2. It was noted that progesterone has neuroprotective
    effects of its own and yet is known to reverse or antagonize some of
    the CNS actions of E2, such as the activation of female sexual
    behavior in the rat. This biphasic action of P is also true for
    other systems in the body.

4)  How can we recognize and understand multiple interacting systems
    throughout the body? The network of mediators that are involved in
    adaptation (such as stress hormones, cytokines, the autonomic
    nervous system, and other hormones regulated via the hypothalamus
    and pituitary) operates in a non-linear fashion, with each mediator
    exerting effects on other mediators and receiving reciprocal
    influences that may be biphasic, depending on the dose and presence
    of other mediators. Gonadal hormones interact with these systems in
    non-linear ways. The same may be said of the myriad of signaling
    pathways. Although biomedical science still operates as if these
    systems communicate in linear pathways and will continue to operate
    in this way for the forseeable future, it is important to recognize
    that this non-linear complexity exists. What this means is that
    short, linear pathways of cause and effect will be very much
    dependent on "context," or the activity of mediators that lie
    outside of the linear sequence under study.

**Research Agenda Items presented by Discussion Session Chairs**

## ***Adipose*** 

Fasting lipids, body fat distribution, insulin and glucose levels are
all affected by menopause. Genetic and environmental factors contribute
to adiposity, including body-fat distribution and shift in body fat
levels. The mechanism(s) by which altered hormonal profiles during the
menopause transition and hormonal treatments in postmenopausal HT may
affect adiposity requires more research.

## ***Animal Models***

Research using rodent models has made more progress than larger animal
model systems. Although the monkey is a good model because of its
similarities to the human, there are substantial problems with the use
of young, ovariectomized monkeys. Such models show E2 depletion: less
than 5 pg/ml, in contrast to the E2 concentrations of postmenopausal
women (10 to 30 pg/ml). Additionally, ovariectomized monkeys have plasma
testosterone concentrations of about 5 to 10 ng/dl, in contrast to
postmenopausal women, who have about 35 ng/dl. Ovariectomized mice have
undetectable androgen levels because rodent adrenals do not make C19
steroids. A major area of research interest concerns the effects of PM
estrogen and progestogen replacement on the breast and on markers of
breast cancer. Because testosterone is an important modulator of the
breast's response to estrogens and progestins, postmenopausal
testosterone concentrations become important characteristics of the
animal model. The VCD model appears to offer the possibility of a
perimenopausal to PM transition, with estrogens and androgens more
comparable to naturally perimenopausal and PM women. There is a need to
extend the VCD-treated rodent studies to a suitable large-animal model.

## ***Immune System***

It is known that the immune system is responsive to hormones and that
the immune system interacts with other organ systems in complex ways.
Relationships of inflammation to a number of factors, such as heart
disease, adiposity, diabetes, may include cognitive function. More
research is needed to understand the relationship between HPO hormones,
individually and in combination, and the immune system. Understanding
the effects of aging on hormonal-immune system interactions are crucial.
Biomarkers of immune function that correlate with menopause and disease
burden also need to be identified.

## ***Bone***

Bone loss may result from risk factors other than just loss of E2 across
the menopause transition. Bone density needs to be considered because
fracture risk increases with age, not just with the increased number of
falls. We need to find better, noninvasive ways to relate bone density
to fracture risk and to find better ways to assess bone quality. The
mechanisms of sex steroid regulation of specific pathways in bone
metabolism need to be further defined.

# *Cognition, Mood and Brain Function* 

Recent epidemiological studies suggest a need for a better understanding
of individual differences in vulnerability to cognitive and mood
disturbance and interaction between cognition and mood during the
menopausal transition. Studies of ER polymorphisms might help to explain
mechanisms underlying individual differences in CNS function in midlife
women. The critical period hypothesis suggests a need to distinguish
between proximal and distal effects of the menopause on CNS function
early in the menopause. The WHI Memory Study is a critically important
study addressing hormones and brain function, particularly in older
women, but its relevance to midlife women is untested. Midlife women
will continue to use HT to treat symptoms. Neuroimaging studies of
microvascular changes may be important in understanding the risks and
benefits of HT on specific aspects of cognitive function.

## ***Cardiovascular***

Cross-talk and --fertilization among various research areas and organ
systems is crucial for a better understanding of the pathobiology of
vascular and endothelial dysfunction across the menopause transition.
For example, understanding the full impact of the metabolic syndrome is
extremely important. Also a better understanding of the role of
genetics, as can be obtained from the SWAN and other longitudinal
studies of middle-aged women, is extremely important. Model systems have
varying strengths and weaknesses. The power of using the mouse model is
the ability to ask single gene questions, which in turn has provided a
much needed stimulus to research.

# 

# Research Agenda Items from Workshop Participants (presented either at the workshop or contributed following the workshop)

**Geriatrician perspective**: Research studies in this area would
benefit from inclusion of a geriatrician's, as well as multidisciplinary
research, perspective. We need a better understanding of the physiology
of reproductive hormones on functional and disease outcomes
non-reproductive tissues during the peri- and postmenopausal life
periods, as well as better hormonal assays for the lower levels commonly
found in older people. Long-term follow-up is crucial in animal or human
studies.

**Genetics and genomics**: In the next generation of large epidemiologic
studies, more thought should be given to harnessing the incredible power
of genetics and genomics to open up new avenues of research. These
approaches to large multi-ethnic longitudinal studies across the
menopause transition (such as the Study of Women's Health Across the
Nation (SWAN) Study
([www.edc.gsph.pitt.edu/swan/public](http://www.edc.gsph.pitt.edu/swan/public))
and associated biospecimen repository with available DNA specimens
([www.swanrepository.org](http://www.swanrepository.org/))) with
long-term outcomes could be especially fruitful. Use of sib pairs in
this type of research would be very powerful.

**Menopause affect on aging**: Because human female mortality rates
indicate very little acceleration around menopause, effects of menopause
on health and disease independent of aging will be subtle if they exist
at all. Virtually all studies confound aging and menopause. Even
stratifying studies by menopause status keeping age constant may not
cleanly separate these effects because age-at-menopause seems to be
associated with future longevity.

**Model systems**: good model systems may be available to separate aging
and menopause-related effects on health and disease. One approach would
be to modify the age at "natural" menopause to young, middle-aged, and
old ages so that HPO axis hormones change while the non-reproductive
tissues vary from young to old ages. Menopause occurs in humans at
middle-age when mortality rate is approximately 20-fold higher than just
before puberty, but 4-fold lower than it will be by age 65, and 16-fold
lower that it will be at age 80. A major problem with rodent models is
that they exhibit female reproductive aging too young -- at an age when
mortality rates have hardly begun to increase at all (from limited
information on age-specific mortality in mice and rats). A particularly
informative model might be one in which menopause (or female
reproductive aging) normally occurs late in life, but can be manipulated
to terminate reproductive cycles at a range of physiologic ages in the
presence of the ovary.

**CNS clocks regulating endocrine aging**: There has been a lot of focus
on the role of the ovary in the menopausal process in the human female
HPO axis, but not as much attention has been paid to the role of the
CNS, and even less to central clocks regulating releasing hormone
pulsatility. Given the rise of cortisol and decline of DHEA with age,
might there be an overarching timekeeper orchestrating endocrine aging?

**Role of dietary phytoestrogens vis a vis endogenous estrogens**: Women
are increasing their consumption of foods and supplements containing soy
isoflavone phytoestrogens. What are the health consequences of this
exposure on estrogen reproductive and non-reproductive target tissues.
Effects of phytoestrogens may depend on the endogenous estrogen levels
in these women, so effects may potentially be greater in older women
with lower endogenous estrogen levels, and the tissue functional effects
in older peri- or postmenopausal women may differ from that in estrogen
replete younger women.

**Potential neuroprotective effects on the brain's immune system**:
While what little is know of estrogen effects on the somatic immune
system were discussed at this workshop, no attention was paid to the
effect of estrogen on the brain's immune system as one of the ways
estrogen may be neuroprotective. This area should be explored in a
future workshop.

**Sex hormones on inflammation**: Knowledge of the effects of sex
hormones on inflammation may be key to understanding the apparent
protection young cycling women have against cardiovascular and
neurodegenerative diseases, as well as osteoporosis and perhaps
diabetes. There should be more attention paid to this potentially
fruitful research area.

**Non-human primate models of menopause**: Is the postmenopausal life
span, which is considerably longer in humans relative to non-human
primates (e.g., rhesus macaques) and for which some think may contribute
to the survival of children and grandchildren, subject to natural
selection? Does the postmenopausal life span represent the same
phenomenon in humans and in non-human primates (e.g., rhesus macaques)?

**Rodent model of perimenopause**: Preliminary data suggests that the
GPR-4 transgenic rat model (Paruthiyil et al., 2002) described in the
Adipose Discussion Session report above become anovulatory at 9 months
of age, when only about 25% of wild type animals are anovulatory. This
may allow exploration of the role of the intact HPO axis on
pathophysiologic processes in non-reproductive tissues associated with
postmenopausal health problems in women. Further work needs to be done
to confirm the suitability of this model.

**Role of prenatal programming in adult disease**: The role of prenatal
programming in affecting susceptibility to diseases and health
conditions associated with the menopause, or possibly to the age at
menopause or hormonal changes associated with the menopause, needs to be
considered.

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SUBJECT INDEX (NOTE: page designations were assigned prior to inserting
a number of additional references in the text. Therefore, some of the
indexed terms may be found on the page following the designations below)

A

Activators of non-genotropic estrogen-like signaling (ANGELS), 21

Adiponectin, 14, 15, 16

Adipose

and menopause, 31

metabolism of, 14

Adiposity, see also Fat deposits

and HT, 31

during menopause, vi, 31

Aging

in animal models, 13

and blood clotting processes, viii, 25

and bone mass, 21--22

chronological *vs.* endocrine, 3

diseases associated with, 2

endocrine, 33

female reproductive system and, 7--8

and GnRH pulsatility, iv, 8

and menopause, 3, 8--10, 33

and tissue biology, ix

Androgen

deficiency, 7

effects on bone of, 21

Animal models, 10

baboon, 12--14

bone loss in, 10

caloric-restriction experiments in, 14

effects of social behavior in, v, 12

estrogen response in, v, 10, 12, 13

irregular cycling in, v, 13

limitations of, 33

macaque, 12--14

monkey, v, 13, 28, 31--32

mouse, 11--12, 32--33

non-human primates, 34

rat, 5, 16

reproductive aging in, v, 12

rodent, 11, 12, 28, 31, 33, 34

Aromatase KO (ArKO) models, 14--15

Atherosclerosis

in animal models, 13

and HPO axis, 24

inflammatory markers and, 19

and obesity, 16

Autoimmune diseases, 17

Autonomic nervous system, 31

B

BALB/c mice, 18, 19

Baltimore Longitudinal Study of Aging Perimenopausal Study, viii, 26

Birth control pills, in animal models, 13

Blood clotting processes, viii, 25

Body fat distribution, menopausal changes in, 9, 15, 31

Body mass index (BMI), vi, 3, 16

Bone, menopausal effects on, 20

Bone density

and aging, 2

and fracture risk, 21

studies, 20

Bone loss

in animal models, 10

genetics of, 22

in menopausal transition, vii, 20

rates of, 20

risk factors for, 32

timing of, 21

Brain. *See also* Central nervous system

during perimenopause, 32

response to ovarian hormones of, 5

C

Caloric-restriction experiments, in animal models, 14

Cardiovascular disease (CVD)

gender differences in, 9

incidence of, iii, 9

and inflammation, 18--19

and menopausal transition, 24

risk of, viii, 4, 23

Cardiovascular Health Study (CHS), v, 9

Cardiovascular tissues, hormone receptors in, 22

Central nervous system (CNS)

effects of estrogen on, ix, 28

and endocrine aging, 33

in midlife women, 32

during perimenopause, 28--29

Cholesterol, and inflammation, 19

Cognition

cyclical effects on, 28

during menstrual cycle, 27

during perimenopause, 9, 26--29, 32

Congenital adrenal hyperplasia (CAH), viii, 26--27

Coronary heart disease (CHD)

in animal models, 13

gender differences in, 9

and metabolic syndrome, 16

C-reactive protein (CRP), and inflammation, 19

Critical period hypothesis, 29, 32

Cross-cultural issues, 29

Cytokines, 31

D

Dehydroepiandrosterone sulfate (DHEAS), 7

Depression

and frailty, 10

in midlife women, viii, 26

and perimenopause, 27--28, 29

Diabetes, type 2, 34

aging and, 2

incidence of, iii, 2

subcutaneous fat in, 14

Diet, and GnRH, 16--17

Disease

autoimmune, 17

and frailty, 10

impact on menopause of, 9

E

Endothelial dysfunction, 32

Endothelial nitric oxide synthase (eNOS), vii, 22

Estren, vii, 21

Estrogen (E2),

in animal models, 10, 12, 13

antidepressant efficacy of, 28

and brain\'s immune system, 34

breast\'s response to, 32

and CNS function, ix, 28

critical time windows for, 30--31

effects on bone of, 21

effects on brain of, iv, 4

neural targets of, 27, 28

new roles for, 14

non-reproductive role of, 15

phytoestrogens *vs.* endogenous, 34

and thrombosis, 25

and vasculature, 24

Estrogen insufficiency, in animal models, 14

Estrogen receptor (ER),

> and atherosclerotic lesions, 24
>
> in brain tissue, 4, 5

Estrogen response element (ERE) interactions, 21

> ERE-Luc reporter mouse, 11, 12, 13

Ethnicity, and metabolic syndrome, 16

F

Fat cells, endocrine importance of, 14

Fat deposits. *See also* Adiposity

in male liver, 15

visceral, 14, 16

Fat distribution, during menopause, 15, 31

Follicle-stimulating hormone (FSH)

in animal models, 13

effects of aging on, 8

during menopausal transition, 29

Follicle-stimulating hormone receptor (FSHR)-haploinsufficient mice, 11

Food intake, and GnRH neurons, vi, 16--17

Forgetfulness

among premenopausal women, 26

and menopausal transition, viii, 7

Fractures, osteoporotic

and bone loss, 32

gender differences in, 20

incidence of, iii, 2

risk for, vii, 21

Frailty

cycle of, 6

disease and, 10

and menopause, 9

Framingham Heart Study, 23

Free alpha subunit (FAS)

and aging, 8

Functional hypothalamic amenorrhea (FHA), iii

Functional reserve, and aging, 9

G

Gender differences

in CVD risk, 4, 9

in fracture risk, 20

in stress effects, 6

Genetics, 33

of hormonally responsive immune systems, 18

and pathobiology, 32

Genomics, 33

Geriatrics, and menopausal transition, 33

Glucose, and menopause, 31

Glucose intolerance, and estrogen, 17

Gonadal hormones, 31

Gonadal steroids

and aging, 7

effects on brain structure of, 4

Gonadotropin-releasing hormone (GnRH)

and aging, iv, 8

and diet, 16--17

GPR-4 transgenic rat, 34

H

Healthy Women Study, 16

Heart and Estrogen/progestin Replacement Study, 23

Heart disease, and aging, 2. *See also* Cardiovascular disease; Coronary
heart disease

Hemostasis, in menopause, 25

High-density lipoprotein (HDL), in animal models, 13

Hormones,

and aging process, 9

biology of, 30

and blood clotting processes, viii, 25

and brain function, 26--27, 32

HPO, iii, viii, ix, 32

and immune system, 17

risk/benefit ratio of, 23

Hormone therapy (HT), 23

beneficial effects of, 2--3

critical time windows for, 30--31

and diabetes, iii

and hemostasis, 25

impact of, 9

postmenopausal, 27

timing of, 31

and vascular changes, 32

27-hydroxycholesterol (27HC), 24--25

Hypothalamic-pituitary-adrenal (HPA) axis, vii, 6

Hypothalamic-pituitary-ovarian (HPO) axis

in animal models, 34

and atherosclerosis, 24

in middle-aged women, iv, 32

and osteoporosis, 21

Hypothalamic-pituitary-ovarian (HPO) axis hormones, iii, 32

and cardiovascular physiology, viii

studies on, ix

Hypothalamus, 31

I

Immune system

autoreactivity in, 18

brain\'s, 34

hormones in, 17

interactions of, 32

Inflammation

and CVD risk, 18--19

and frailty, 10

sex hormones and, 34

Inflammatory markers, and disease risk, vii, 18--19

Inhibin, vi, 7, 24

Insulin, and menopause, 31

Insulin resistance, 15

and CVD risk, 3

and estrogen, vi

and visceral fat, 16

Insulin sensitivity, and visceral fat, 14, 16

K

Knockout (KO) rodent models, 11

L

Learning, and estrogen treatment, ix, 32. *See also* Cognition

Life expectancy, of women, 2

Lipids, and menopause, 31. *See also* Adiposity

Lordosis response, 4

Low-density lipoprotein (LDL), in animal models, 13

Lupron study, 5, 27

Lutenizing hormone (LH)

effects of aging on, 8

Lymphopoiesis, vi, 18, 19

M

Medications, and inflammatory markers, 19

Melbourne Midlife Women\'s Health Study, 26

Memory

and estrogen treatment, ix

and menopausal transition, viii

and menstrual cycle, 27

and perimenopause, 26

Menopausal transition

longitudinal study of, 20

staging, ix, 29

vascular dysfunction during, 24

Menopause

and aging, v, 8--10, 33

and biomarkers, ix

and CVD, 23

early, 10

metabolic changes in, vi, 9

non-human primate models of, 34

Menstrual cycle studies, 27

Metabolic syndrome, vi, vii

in animal models, 14

characteristics of, 15

impact of, 32

inflammation markers associated with, 19

Mood alterations, during perimenopause, 26, 27--28, 29, 32. *See also*
Depression

Mortality, depression and, 27

Muscle mass, and aging, 9

Muscle strength, and menopause, v

N

National Health and Nutrition Examination Survey III (NHANES III), 15

Nurses Health Study, 23

O

Obesity

epidemiology of, 30

and inflammation markers, 19

and premenopausal risk factors, 16

Osteoblastogenesis, 21

Osteoclastogenesis, 21

Osteoporosis, vii, 9, 34. *See also* Fractures, osteoporotic

environmental risk factors for, 21

incidence of, 20

Ovarian follicle-selective toxicant models, 11

Ovarian follicles, effect of aging on, 7

Ovarian function

and aging, 8

in animal models, 13

Ovarian steroids, neurobiology of, 4--8. *See also* sex steroids

Oxysterols, 24

P

Perimenopause

altered CNS mechanisms during, 28--29

in animal models, 12, 14

coagulation and, 25

and depression, 27--28

rodent model of, 34

Phytoestrogens, effects of, 34

Pituitary gland

gonadotrophs in, 8

and stress, 31

Platelet formation, 25

Polyglandular failure syndrome, and CVD, 3

Premature ovarian failure (POF), and CVD, iii, 3

Prenatal programming, 34

Progesterone

effects of, 31

in female *vs.* male brains, 6

Progestins

breast\'s response to, 32

synthetic, 31

and thrombosis, 25

Proopiatemelanocortin (POMC) neurons, and food intake, vi

PROSALMAN study, v

R

Reproductive axis, neuroendocrine components of, 7--8

Reproductive cycles, in animal models, 13

Reproductive system (female)

and effects of aging, 7--8

Resilience, loss of, 10

Rheumatoid arthritis, 17

S

Seattle Midlife Women\'s Health Study, 26

Selective ER modulators (SERMs), 5--7, 19

Sex hormone-binding globulin (SHBG)

and cardiovascular risk, 23

and heart disease, 25

and vasculature, viii, 25

Sex hormones, and inflammation, 34

Skeletal bone, 22. *See also* Bone loss

Staging Reproductive Aging Workshop, 29

Strength, and aging, 9

Stress, and early menopause, 21

Stress hormones, 31

Study of Women\'s Health Across Nation (SWAN), 3, 20, 21, 22, 25, 26,
32, 33

Systemic lupus erythematosus, 17

T

Tamoxifen

in animal models, 12

effects of, 6

Testosterone

levels and aging, 7

postmenopausal, 32

Thrombosis

cells involved in, 22

risk of, 25

Triglycerides, and inflammation, 19

Turner\'s syndrome, 8

Tyrosine hydoxylase cells, 15

U

Undereating, 10

V

Vasculature

effect of hormones on, viii, 22

during menopausal transition, 24

and menopause, 3

Vasomotor symptoms, during menopausal transition, 29

VCD-treated rodent model, 12, 32

Visceral adiposity, after menopause, 16

Visceral fat, 14, 16

W

Weight gain. *See also* Obesity

among U.S. population, 30

during menopause, vi

Women\'s Health and Aging Study (WHAS), v, 9, 10

Women\'s Health Initiative (WHI), 23, 30

Women\'s Health Initiative (WHI) Memory Study, 32

AUTHOR INDEX

C

Carr, Molly, vi, 15

Clarkson, Thomas, v, 10, 13

D

Dallman, Mary, vi, 16

Diamond, Betty, vi, 17, 18

F

Finkelstein, Joel, vii, 20

Fried, Linda, v, 8

G

Ginsburg, David, viii, 25

H

Hall, Janet, iv, 7

Honore, Erika, v

Hoyer, Patricia, v, 11

K

Kado, Deborah M., vii, 21

Kaplan, Jay, v, 12

Karas, Richard, viii, 23

Kelley, David, vi, 14, 16

Khosla, Sundeep, vii, 20

Kincade, Paul, vi, 17

Konkle, Barbara, viii, 25

Kuller, Lewis, vi, 14, 16

M

Maggi, Adriana, v

Maki, Pauline M., viii, 26

Manolagas, Stavros C., vii, 21

McEwen, Bruce, iv, 5, 29

Mendelsohn, Michael, vii, 22

Morrison, John H., iv, 28

P

Park, Susanna J., ix, 28

Parrott, Estella, ix, 29

R

Resnick, Susan M., viii, 26, 27, 28

Rosen, Clifford J., vii, 21

S

Santoro, Nanette, iii, 2, 29

Schmidt, Peter, ix, 27

Shaul, Philip W., viii, 24

Sherman, Sherry, ix, 29

Sherwin, Barbara B., ix, 27, 28

Simpson, Evan, vi, 14

Sutton-Tyrrell, Kim, viii, 23

T

Toran-Allerand, C. Dominique, ix, 28

Tracy, Russell P., vi--vii, 18, 19

W

Woods, Nancy F., ix, 26, 29