The Heterogeneity of Post-Menopausal Disease Risk: Could the Basis for Why Only Subsets of Females Are Affected Be Due to a Reversible Epigenetic Modification System Associated with Puberty, Menstrual Cycles, Pregnancy and Lactation, and, Ultimately, Menopause?

For much of human evolution, the average lifespan was <40 years, due in part to disease, infant mortality, predators, food insecurity, and, for females, complications of childbirth. Thus, for much of evolution, many females did not reach the age of menopause (45–50 years of age) and it is mainly in the past several hundred years that the lifespan has been extended to >75 years, primarily due to public health advances, medical interventions, antibiotics, and nutrition. Therefore, the underlying biological mechanisms responsible for disease risk following menopause must have evolved during the complex processes leading to Homo sapiens to serve functions in the pre-menopausal state. Furthermore, as a primary function for the survival of the species is effective reproduction, it is likely that most of the advantages of having such post-menopausal risks relate to reproduction and the ability to address environmental stresses. This opinion/perspective will be discussed in the context of how such post-menopausal risks could enhance reproduction, with improved survival of offspring, and perhaps why such risks are preserved. Not all post-menopausal females exhibit risk for this set of diseases, and those who do develop such diseases do not have all of the conditions. The diseases of the post-menopausal state do not operate as a unified complex, but as independent variables, with the potential for some overlap. The how and why there would be such heterogeneity if the risk factors serve essential functions during the reproductive years is also discussed and the concept of sets of reversible epigenetic changes associated with puberty, pregnancy, and lactation is offered to explain the observations regarding the distribution of post-menopausal conditions and their potential roles in reproduction. While the involvement of an epigenetic system with a dynamic “modification-demodification-remodification” paradigm contributing to disease risk is a hypothesis at this point, validation of it could lead to a better understanding of post-menopausal disease risk in the context of reproduction with commonalities may also lead to future improved interventions to control such risk after menopause.


Purpose: Discuss the Potential Role of Variables Leading to Disease Risk in the Post-Menopausal State Having Important Roles during the Reproductive Stage of the Life Cycle, and Then Focus on Potential Mechanisms That Are Involved
Most articles regarding diseases arising in the post-menopausal phase of a female's life cycle focus on individual diseases (i.e., osteoporosis, cardiovascular disease, obesity, dementia, osteoarthritis).They usually do not view them in an integrated manner, nor do they ask the questions "why these conditions?", "why only a subset of females?", or "why do individual females not have all of these conditions?".This review article attempts to take the position that these are not just diseases, but the underlying mechanisms are advantageous to the reproduction and survival of the species down through evolution unknown, but it could relate in part, to the development of obesity as individuals with obesity can develop metabolic OA (discussed in [10]).While there may be overlap between the various conditions associated with disease risk in the post-menopausal state, only subsets of post-menopausal females are clinically affected.That is, only ~30% of females exhibit OP [15], and in that subset, the extent or progression of the condition is quite variable (discussed in [1]).Thus, females with diagnosed OP may lose bone rapidly or slowly.Therefore, even those with OP are quite heterogenous and the progression of OP is complex, likely involving a set of genes, with some involved in the initiation of the condition and others involved in the rate of disease progression.The genes involved in the latter may not be directly related to the post-menopausal state, as astronauts, mostly males thus far, also lose bone in a heterogenous manner while in microgravity (discussed in [11,16]).

Variables and Factors Influencing Post-Menopause Diseases and Conditions
A number of variables such as early menopause [17] and reproductive parity [18][19][20] have been reported to influence some post-menopausal conditions such as OP, but not all studies are in agreement [21].Early menopause can lead to OP and a high parity number can also lead to OP.The latter may be the result of repeated pregnancy and lactation bone loss without ample time to recover.However, the studies by Seo et al. [19] and Yang et al. [20] were performed with Asian populations while that of de Bakker et al. [21] was with a Canadian population that was likely mainly Caucasian, so there could potentially be racial/ethnic differences in this regard.Early menopause, defined as menopause onset < 40 years of age, has been reported to increase the risk of developing dementia and cognitive decline (reviewed in [22][23][24]).
Co-morbidities such as HIV [25] and rheumatoid arthritis [26] have been reported to lead to higher incidence of OP or fragility fractures, respectively.Other co-morbidities such as COPD [27], kidney disease [28], and periodontitis [29] have also been reported to influence OP.While not apparently influencing the incidence of OP but aggravating the condition, the basis may be related to the inflammation associated with such conditions, as inflammation can also affect bone (reviewed [30]).Some of these inflammatory influences may be associated with an age-related low level of chronic inflammation (inflammaging) [31].
Other factors that may also contribute to OP risk and progression include periostin levels associated with endocrine disease [32], the gut microbiota [33,34], anti-Mullerian hormone [35,36], and genetic factors [37], reviewed in [38,39], and mediators such as irisin [40].Vascular dysfunctions such as varicose veins have also been implicated in the development of OP [41].Therefore, there are numerous risk factors for the elaboration of OP and its progression, but the basis for why a specific subset of women (~30%) develops clinically defined OP remains unknown.
Osteoporosis after menopause onset is not the only condition that appears to be influenced by inflammatory processes.Dementia, including Alzheimer's disease (AD), is also more common in post-menopausal women than in men (discussed in [42]).Similar to OP, AD also appears to be influenced by inflammatory processes [42][43][44].Some of this influence may be at the level of the vascular component of the system as sex hormones contribute to cerebrovascular function [9,45].In support of this concept, it has been reported that adiponectin, an anti-inflammatory cytokine produced mainly by adipose tissue may contribute to a positive effect on post-menopausal cognitive decline [46].However, it remains to be determined whether inflammatory processes primarily influence disease progression rather than trigger the disease itself.Interestingly, in mouse models, vitamin D deficiency can exacerbate AD-like disease by reducing antioxidant capacity in the brain [47], again supporting a role for inflammation-like processes in disease progression but not initiation.
As some people have elevated tangles and tau protein levels but no loss of cognition, and others have cognitive decline but without elevated tau [48], there may be multiple steps in the loss of cognition and disease development, one based on some primary initiating step followed by a secondary step, potentially involving inflammation.As there also appears to be a subset of post-menopausal patients who have a vascular form of the disease, the first step(s) in disease induction may involve different components and there are multiple ways to develop the condition.However, much of mainstream research has focused on amyloid dysfunction in AD, with recent drugs designed to assist in the removal of such proteins [49], but the effects of the drugs are quite modest.Therefore, the molecular basis for the development of dementia in a subset of the post-menopausal population remains largely unknown.
Prior to menopause, women and men appear to be equally affected by OA, but after menopause, the incidence in women exceeds that in men (discussed in [1,2,10,50]).Whether the development of OA, particularly in the knee, in the post-menopausal state is a unique subset of OA patients is unknown, but the possibility has been raised [10].However, it is clear that OA is an inflammatory disease (discussed in [51][52][53][54]) and in early post-traumatic knee injuries leading to OA in preclinical models, treatment to inhibit inflammation ameliorates the development of the condition [55][56][57].Furthermore, metabolic osteoarthritis has an inflammatory component [58][59][60] and this may relate to post-menopausal women with obesity.
Before menopause, women are reported to be somewhat protected from cardiovascular diseases (CVD), but after menopause, there is an increased risk for such conditions (discussed in [61][62][63][64]).Some of this increase may be linked to the co-development of increases in body fat (discussed in [64]).As with some of the conditions discussed above, aspects of the CVD risk after menopause may be again linked to inflammatory processes [65].This may be related to calcium levels [66], as calcium is a key regulator and may be a link between OP and heart disease.Interestingly, genes involved in some inflammatory pathways have been reported to be biomarkers for coronary heart disease in post-menopausal Thai women [67].This may relate to the development of hypertension in the post-menopausal state for some women [68,69].
A subset of women with rheumatoid arthritis (RA), an autoimmune inflammatory disease, are also prone to developing cardiovascular disease [70][71][72][73].Of interest is the fact that ~70% of female RA patients undergo a remission of their disease when pregnant and disease activity is also influenced by the menstrual cycle (reviewed in [74]).Why only ~70% of female RA patients undergo remission is unknown, but certainly, pregnancy is known to lead to a downregulation of inflammatory responses, possibly to better carry an allogeneic fetus.Whatever the mechanism(s) that are involved, such findings do indicate that the regulation of inflammatory processes is different in males and females, and inflammatory processes developing in the post-menopausal state may be indirectly related to reproductive functions [75,76].Thus, future considerations should investigate the potential efficacy of anti-inflammatory therapies in the onset and progression of several of the post-menopausal onset conditions and diseases.

Summary of Points Potentially Relevant to Post-Menopausal Conditions
From the above discussion, a number of salient points regarding post-menopausal conditions and diseases have to be reconciled in order to understand how they arise, and their potential role after puberty and prior to menopause.These points include the following: A.
Not all women experience these conditions (i.e., osteoporosis, osteoarthritis, dementia, obesity, cardiovascular disease) after menopause, and those that do usually only experience a subset of them.B.
For some of the conditions, such as OP and others, there appears to be a genetic component to disease risk, as it "runs in families".C.
Most if not all of the post-menopausal conditions can be linked directly or indirectly to reproductive activities such as pregnancy and lactation.D.
Early menopause and elevated parity contribute to OP risk and low bone density, respectively.Early menopause is also linked to other post-menopausal conditions [77].
E. Inflammation appears to be a "catalyst" for post-menopausal disease progression but possibly not an inducer of the conditions.
Additional potentially relevant points include the following: AA. Epigenetic modifications of the genome via direct methylation or indirectly via modification of histones, as well as modification of RNA can occur under a variety of circumstances (discussed in [78][79][80][81][82]).Many of these alterations are reversible modifications that allow for modulation and regulation of gene expression and function.
Several of such alterations have been implicated in diseases or conditions relevant to menopause, such as osteoporosis and calcium signaling [83][84][85], cardiovascular disease [86,87], osteoarthritis [88][89][90], the nervous system and the brain [91][92][93][94], obesity [95,96], and vascular inflammation [97].BB.The onset of puberty is associated with epigenetic alterations to a variety of genes (discussed in [1,2]).Epigenetic modifications also occur in the brain during development and aging [98,99].CC.Epigenetic modifications occur with the onset of menopause [1,100] and in those with post-menopausal conditions such as OP (discussed in [1,11]), cardiovascular conditions [101], and others [98,102].However, in many of these conditions, their relationship to disease induction versus arising as a consequence of the diseases/conditions is not known.DD.Treatment of individuals undergoing gender transitioning with hormones such as estrogen leads to epigenetic modification of the genome of blood cells [103].EE.Hormonal changes associated with pregnancy involve several molecules in addition to estrogen (discussed in [104][105][106][107][108]).FF.Epigenetic modifications occurring in the white blood cells of astronauts while in space are reversible following a return to Earth [109], as discussed in [16].GG.Young women are heterogeneous with regard to menstrual cycle-associated alterations.Approximately 20% do not exhibit alterations to knee joint laxity across the menstrual cycle in spite of similar changes in estrogen levels to the 80% that do exhibit changes [110,111].HH.Pregnancy is associated with a downregulation of inflammation in a majority (~70%) of those with inflammatory autoimmune diseases such as rheumatoid arthritis, and disease activity is influenced by menstrual cycle variations in hormone levels [74].II.Pregnancy is associated with cognitive changes [104].JJ.Endothelial cells of the vasculature can differentiate to form paracrine systems with other cells in a local environment and thus form unique paracrine systems with distinctive characteristics (reviewed in [112]; discussed in [113,114]).

Involvement of Epigenetic Modification across the Lifespan of Females
Epigenetics, the modification of DNA and DNA activity, can occur across the lifespan [115,116] and can occur during aging [117], often in response to environmental stressors [118].Such changes can be reversible [16,109,119], and thus, such a system is dynamic and not unidirectional.Some of the complexity of epigenetic modifications occurring in females across the lifespan are detailed below.

During Puberty and Gender Transitioning
After a period of growth and maturation, females undergo puberty at ~11-13 years of age.Puberty is accompanied by a number of epigenetic modifications in a variety of tissues in preparation for reproduction.The affected tissues can be in the brain [120], specifically the hypothalamus [121][122][123], peripheral blood leukocytes [124], adipocytes [102], and mammary glands [125], to name several.Additional genetic and epigenetic factors have been associated with precocious puberty [126][127][128].Using saliva, Stueve et al. [126] reported that epigenetic modification of specific genes such as CYP19A1 may be a biomarker for pubertal timing.Thus, a variety of tissues are affected primarily and secondarily during puberty onset.Interestingly, using hormonal therapy for gender transitioning can also lead to epigenetic modifications of a variety of cells [103] and is being investigated in tissues [129].Therefore, the onset of puberty leads to a variety of epigenetic modifications to both prepare for reproductive activities and also the growth and maturation that occurs during the post-puberty adolescent period.

During Pregnancy and Lactation
A large number of adaptations occur in a female when she becomes pregnant.Not only is she carrying a fetus that is a histoincompatible allograft (e.g., a "foreign graft") that must be implanted, but also requires adaptations that assist in nurturing its growth and maturation, as well as accommodating stresses on a variety of the mother's organs [130,131].Some of these adaptations appear to be manifested by epigenetic modifications [130][131][132][133][134][135].However, some long-term epigenetic modifications may also contribute to adverse risks of pregnancy leading to pathological responses [136,137], but others appear to be transient and reversed during the postpartum period [131].
Subsequently, post-birth, the induction of lactation appears to be associated with epigenetic modifications to cells in the mammary glands, leading to the generation of milk [125,[138][139][140]. Evolutionarily, this of course also required the release of calcium from bones to contribute to milk formation [141][142][143].In a subset of women, this can lead to overt OP [143].
Many of the studies reported above for humans used white blood cells to assess the epigenetic changes, so it remains to be largely determined as to whether there are unique epigenetic modifications that are tissue-specific and which ones may be transient and reversible after pregnancy and lactation, and which may be more permanent.In addition, the influence of parity on the extent and permanence of epigenetic modifications occurring during pregnancy and lactation has not been well studied.

During Menopause and during Post-Menopausal Interventions
Aging in females can lead to DNA methylation alterations at the level of the ovaries, impacting fertility [100,144].The relationship of such changes to menopause onset remains undefined.However, with the advent of menopause, a variety of conditions/diseases can arise that impact a diverse set of tissues (e.g., bone, brain, heart, adipose, muscles, and tissues of the musculoskeletal system).Accompanying some of these diseases or risk for diseases are epigenetic modifications, usually detected with white blood cells in human females.For example, hypomethylation of Alu elements in the DNA has been reported for females with osteoporosis [145], DNA methylation alterations were reported for post-menopausal females associated with metabolic and immunological systems [146], and identification of an epigenetic "signature" for CVD in post-menopausal females has been reported [147].Whether similar epigenetic modifications are also evident in specific target tissues remains largely unknown.
However, efforts to overcome the loss of sex hormones at menopause with hormone replacement therapy (HRT) have had a "checkered" history, with the pros and cons debated regarding the risk factors (discussed in [148][149][150][151]), including cancer risk [149,152,153].Over the ensuing years, lessons have been learned regarding dosage, composition, when to initiate HRT, and for how long to mitigate some of the risks in these subsets of females, and this has led to more females taking HRT for treatment of post-menopausal conditions.
In menopausal females, HRT is reported to inhibit/prevent OP [154,155], metabolic syndromes [156], and loss of intervertebral disc height [157], as well as not posing a risk for breast cancer [158], but evidence for a role in protection at the level of OA [159], CVD [160,161] and dementia/Alzheimer's disease [162] is still controversial via mixed results of studies over the past 25 years.
Using nucleated blood cells, a few reports have indicated that HRT use can lead to epigenetic modifications such as DNA methylation [163,164].While interesting, these studies do not address the issue of tissue-specific epigenetic modifications.
Finally, non-drug protocols that instead use exercise protocols, which have been shown to decrease risk for a variety of post-menopausal diseases (reviewed in [1,2,11]), have been reported to lead to alterations in epigenetic modifications in blood cells of post-menopausal females with risk for diabetes [165] or in a population of older females without Alzheimer's disease [166].In the latter report, some specific genes became hypomethylated while others became hypermethylated, so the changes were not unidirectional.
Based on the above discussion, females undergo a series of epigenetic modifications at distinct times during their lifespan.However, the inter-relationships between those modifications (e.g., those occurring at puberty impacting those during pregnancy, those occurring during pregnancy impacting those occurring at menopause) remain to be determined.Furthermore, in human populations, much of the literature has focused on epigenetic changes associated with white blood cells, so tissue-specific effects regarding the genes and molecules affected largely remain to be elucidated.

The Epigenetic Modification/Demodification/Remodification Hypothesis to Address the Development of Post-Menopausal Diseases and Disease Risk
From the above discussion, females undergo a number of epigenetic modifications during their life span, with many associated with events related to sex hormones and reproductive functions (e.g., puberty, pregnancy, lactation, and menopause).Furthermore, some of these event-dependent modifications appear to be transient and reversible, such as those following pregnancy and lactation [131].Such reversals of epigenetic modifications can also occur in other circumstances so are not unique to pregnancy.
Following menopause, subsets of women develop an increased risk of developing unique diseases or conditions that relate directly or indirectly to reproductive functioning (e.g., osteoporosis, dementia, obesity, and cardiovascular disease).Not all females are affected, and some females may have more than one condition, but evidence that a subset has all of the conditions could not be found.Nearly all of the conditions/diseases are usually treated as independent conditions, in part because of the way medicine is organized and specialties are focused.Therefore, these conditions are studied and treated as if they do not have any common mechanisms or underlying basis.However, that may not be the case and the concept that the various post-menopausal conditions are all related to reproductive functioning and thus may have commonalities in mechanisms has been advanced [1].
Building on that commonality concept and the discussion contained earlier in this review, the hypothesis that the commonality [1] for post-menopausal conditions/diseases may be based on a system of reversible epigenetic signatures that are influenced by sex hormones at puberty and then during pregnancy and lactation, and, finally, by menopause is advanced.As outlined in Figure 1, during the early pre-puberty years, many genes associated with the relevant tissues are either not epigenetically modified to accommodate initial growth and maturation or are modified in a specific manner.However, at puberty, many of them are now epigenetically modified to be in a suppressed or quiescent state in preparation for reproductive functioning, and potentially some of them being active may not be compatible with the adolescent growth and maturation sequences.In this scenario, the targeted genes are maintained in an epigenetically impacted state by the recurrent monthly menstrual cycles until skeletal maturity is obtained and thereafter.However, at the time of pregnancy, these epigenetically repressed genes are likely required for a successful pregnancy and, thus, the epigenetic repression is reversed by mechanisms and enzymes that override the influence of estrogen/progesterone, leading to the involvement of systems (e.g., calcium mobilization from bone, adapting to stresses on the cardiovascular system, metabolic needs for nutrition, and brain center involvement) that benefit fetal growth and the health of the mother.This hypothetical process is outlined in Figure 2 (Panel A).In this scheme, epigenetically repressed genes required for a successful pregnancy that were epigenetically modified as a result of puberty become reactivated for pregnancy, with additional changes potentially reversed for lactation.Once pregnancy and lactation are completed, the re-institution of menstrual cycles leads to epigenetic re-repression of the involved genes.Such cycles of epigenetic repression and then de-repression would occur with each pregnancy, creating an equilibrium between the two scenarios (Figure 2, Panel B).Multiple pregnancies without sufficient time between cycles may lead to incomplete re-repression resulting in risks elaborated following early menopause.Some reports have indicated that parity is associated with increased risk for CVD [167][168][169], dementia [170], metabolic disease [171], and knee OA [172,173], but not fracture risk due to OP [21].Therefore, with increased parity, there may be incomplete re-establishment of a repressed state by menstrual cycles.However, at the time of pregnancy, these epigenetically repressed genes are likely required for a successful pregnancy and, thus, the epigenetic repression is reversed by mechanisms and enzymes that override the influence of estrogen/progesterone, leading to the involvement of systems (e.g., calcium mobilization from bone, adapting to stresses on the cardiovascular system, metabolic needs for nutrition, and brain center involvement) that benefit fetal growth and the health of the mother.This hypothetical process is outlined in Figure 2 (Panel A).In this scheme, epigenetically repressed genes required for a successful pregnancy that were epigenetically modified as a result of puberty become reactivated for pregnancy, with additional changes potentially reversed for lactation.Once pregnancy and lactation are completed, the re-institution of menstrual cycles leads to epigenetic re-repression of the involved genes.Such cycles of epigenetic repression and then de-repression would occur with each pregnancy, creating an equilibrium between the two scenarios (Figure 2, Panel B).Multiple pregnancies without sufficient time between cycles may lead to incomplete re-repression resulting in risks elaborated following early menopause.Some reports have indicated that parity is associated with increased risk for CVD [167][168][169], dementia [170], metabolic disease [171], and knee OA [172,173], but not fracture risk due to OP [21].Therefore, with increased parity, there may be incomplete re-establishment of a repressed state by menstrual cycles.Pregnancy leads to the re-activation of genes influenced by puberty-associated epigenetic modifications allowing for essential activities leading to a successful pregnancy followed by lactation.(A) Pregnancy-related factors either negate the effects of estrogen (and potentially other sex hormones like progesterone) to maintain the epigenetic modifications, or pregnancy-related factors override the effects of estrogen via enzymes that impact the epigenetic modifications.(B) Following cessation of pregnancy and lactation, menstrual cycles re-establish the epigenetic modifications that again lead to repression of the pregnancy-associated genes until the next pregnancy.With frequent pregnancies, the ability of menstrual cycles to re-establish a homeostatic state with regard to these affected genes may be compromised.
Following menopause, which is a process that can take years to complete and is not an acute event, some tissues may de-repress the estrogen-driven epigenetic modifications in the absence of pregnancy due to genetic factors or environmental factors [Figure 3].In this scenario, different tissues involved in the pregnancy/lactation-associated processes may become active by removing some of the epigenetic modifications, leading to chronic and unregulated expression of genes and gene products contributing to post-menopausal diseases and disease risks.Not all females would be at equal risk for such conditions, and not all affected females would have all of the conditions/diseases, as the modifications would be tissue-specific and not general in this construct [Figure 3].Pregnancy leads to the re-activation of genes influenced by puberty-associated epigenetic modifications allowing for essential activities leading to a successful pregnancy followed by lactation.(A) Pregnancy-related factors either negate the effects of estrogen (and potentially other sex hormones like progesterone) to maintain the epigenetic modifications, or pregnancy-related factors override the effects of estrogen via enzymes that impact the epigenetic modifications.(B) Following cessation of pregnancy and lactation, menstrual cycles re-establish the epigenetic modifications that again lead to repression of the pregnancy-associated genes until the next pregnancy.With frequent pregnancies, the ability of menstrual cycles to re-establish a homeostatic state with regard to these affected genes may be compromised.
Following menopause, which is a process that can take years to complete and is not an acute event, some tissues may de-repress the estrogen-driven epigenetic modifications in the absence of pregnancy due to genetic factors or environmental factors [Figure 3].In this scenario, different tissues involved in the pregnancy/lactation-associated processes may become active by removing some of the epigenetic modifications, leading to chronic and unregulated expression of genes and gene products contributing to post-menopausal diseases and disease risks.Not all females would be at equal risk for such conditions, and not all affected females would have all of the conditions/diseases, as the modifications would be tissue-specific and not general in this construct [Figure 3].
The investigation of this construct to explain the spectrum of post-menopausal diseases and conditions could remove it from the study of each condition/disease individually, focus research on the commonalities of the mechanisms involved, and address the spectrum of conditions as components of a system rather than a collection of disparate diseases.

Figure 3.
Possible role of tissue-specific reversal of puberty-associated genes to become re-activated following menopause.Following menopause and the loss of estrogen to maintain the epigenetically repressed genes, through either genetic variables or other unknown factors, the pregnancy-associated genes become re-activated by tissue-specific endogenous enzymes leading to the spectrum of menopause-associated conditions/diseases.Not all females would have all of the disease risks.Once initiated, the impact of the de-repression could be aggravated by ongoing inflammatory processes.The cells central to this proposed scheme may be the endothelial cells of the microvasculature in a tissue-specific manner (discussed in more detail in Section 7 of this review).
The investigation of this construct to explain the spectrum of post-menopausal diseases and conditions could remove it from the study of each condition/disease individually, focus research on the commonalities of the mechanisms involved, and address the spectrum of conditions as components of a system rather than a collection of disparate diseases.

What Cells May Be Involved in the Relevant Epigenetic Modifications Outlined in Section 6?
The conditions/diseases associated with the post-menopausal state involve a variety of tissues ranging from the brain (dementia), bone (osteoporosis), adipose tissue (obesity), the cardiovascular system (heart and vascular disease risk), and joint tissues (osteoarthritis).Thus, there is a spectrum of mechanisms that could be involved, and these include the following: (1) the cells unique to each tissue are the primary target for the epigenetic modifications; (2) as all of the tissues involved are vascularized, the primary target could be the endothelial cells in the microvasculature of each tissue; and (3) as all of the tissues involved are also innervated (or are brain centers), the primary effectors relate to the neuronal cells that then either regulate the endothelial cells (neurovascular regulation) or the target cells directly.For the latter, this may involve the brain centers affecting each tissue.The first option, that tissue-specific target cells are the targets of the epigenetically modified gene expression profile, is the most difficult to envision, as bone is widely distributed in the body and exists in a variety of mechanical and biological environments, conditions that may make it hard to target all of the cells equally or completely.Adipose tissue is also Possible role of tissue-specific reversal of puberty-associated genes to become re-activated following menopause.Following menopause and the loss of estrogen to maintain the epigenetically repressed genes, through either genetic variables or other unknown factors, the pregnancyassociated genes become re-activated by tissue-specific endogenous enzymes leading to the spectrum of menopause-associated conditions/diseases.Not all females would have all of the disease risks.Once initiated, the impact of the de-repression could be aggravated by ongoing inflammatory processes.The cells central to this proposed scheme may be the endothelial cells of the microvasculature in a tissue-specific manner (discussed in more detail in Section 7 of this review).

What Cells May Be Involved in the Relevant Epigenetic Modifications Outlined in Section 6?
The conditions/diseases associated with the post-menopausal state involve a variety of tissues ranging from the brain (dementia), bone (osteoporosis), adipose tissue (obesity), the cardiovascular system (heart and vascular disease risk), and joint tissues (osteoarthritis).Thus, there is a spectrum of mechanisms that could be involved, and these include the following: (1) the cells unique to each tissue are the primary target for the epigenetic modifications; (2) as all of the tissues involved are vascularized, the primary target could be the endothelial cells in the microvasculature of each tissue; and (3) as all of the tissues involved are also innervated (or are brain centers), the primary effectors relate to the neuronal cells that then either regulate the endothelial cells (neurovascular regulation) or the target cells directly.For the latter, this may involve the brain centers affecting each tissue.The first option, that tissue-specific target cells are the targets of the epigenetically modified gene expression profile, is the most difficult to envision, as bone is widely distributed in the body and exists in a variety of mechanical and biological environments, conditions that may make it hard to target all of the cells equally or completely.Adipose tissue is also widely distributed, but some tissues could be affected more than others leading to a selective influence on specific adipose tissues.Regarding dementia, specific areas of the brain could be involved, and this post-menopausal condition could result from epigenetic modification of cells localized to that specific type of tissue.Thus, "option 1" could be in play regarding the post-menopausal conditions that are epigenetically dependent, but if this option were the dominant mechanism, there may be challenges to implementing this mechanism given the diversity of some of the tissues.
All of the tissues involved in the post-menopausal conditions/diseases are vascularized except articular cartilage in OA and that condition is likely due to alterations in the other tissues that are involved in joint functioning and the cartilage is only the "weak link" in the disease process (discussed in [10]).While all of the relevant tissues are vascularized, it is likely that the endothelial cells in the different tissues have differentiated to effectively function in those tissues in a paracrine manner (reviewed in [112,[174][175][176]; discussed in [113,114]).Such tissue-specific characteristics appear to be lost when endothelial cells are cultured in vitro [177] but also appear to utilize epigenetic modifications in the regulation of their function in specific environments [178].Thus, a commonality in post-menopausal conditions could be the targeting of endothelial cells in the different relevant tissue environments to alter their functioning via epigenetic modification, leading to tissue-specific disease risk after menopause by disrupting the integrity of the paracrine system involving the endothelial cells and their target cells.As it is known that microvascular endothelial cells in some tissues are functionally altered during pregnancy in preclinical models [179,180], there is precedent for endothelial cells to be altered by this condition.However, as rabbits are induced ovulators, they do not have menstrual cycles as do humans, so the models have some limitations regarding the further exploration of the concept discussed.
Further evidence for the potential role of endothelial cells of the microvasculature in postmenopausal conditions can be derived from studies indicating that exercise can prevent or alleviate the progression of bone loss and conditions such as osteoporosis [181][182][183][184], can prevent the loss of cardiovascular integrity [185][186][187][188], osteoarthritis [189], and dementia [190][191][192][193], as well as the prevention of aging-related endothelial cell senescence [194][195][196][197].In contrast, physical inactivity can contribute to brain-associated changes and such changes are mediated in part by epigenetic alterations [198].Physical activity has also been reported to potentially prevent loss of cognition and function via enhanced hippocampus neurogenesis in a variety of species [199].Furthermore, some of the age-related risk for diseases is mediated by epigenetic modifications [200], and these can be influenced by exercise in systems such as the cardiovascular system [185,201].Also of relevance to this discussion are reviews that address the issue of endothelial cell senescence on the development and progression of dementias such as Alzheimer's disease [202].Finally, and relevant to the present discussion, are reports that exercise can impact the cognitive function of older females more than males, and this appears to be related to parity in females [203,204].
Based on the above discussion, the question then arises as to how exercise (e.g., mechanical loading) could influence endothelial cell epigenetics directly.Endothelial cells express the sensor of mechanical loading piezo1 [205] and this senses fluid flow [206].Piezo1-dependent regulation of brain vascular development has been reported [207,208].Relevant to the current discussion of the role of endothelial cells and epigenetics in postmenopausal conditions/diseases, mechanical stimulation of endothelial cells leads to epigenetic modifications [209], changes that could influence the interaction of the endothelial cells in specific tissues.However, it has not yet been reported as to whether mechanical loading of endothelial cells from different vascular and tissue environments responds similarly or differently.
Alternatively, some vascular functioning can be mediated by the associated neuroelements that often parallel the endothelial cells in the microcirculation.As mentioned previously, aspects of this relationship appear to be modified during pregnancy in the rabbit model [179,180].Thus, neuro-regulation of differentiated endothelial cells in specific tissues could exert tissue-specific responses and potentially contribute to endothelial responses occurring after the decline in sex hormone expression following menopause.Thus, both neuro-regulation and exercise appear to impact vaso-regulation, likely in part through the regulation of endothelial cells.Quite possibly this is not an "either or" situation and mechanical modulation of endothelial cells and neuro-regulation of the cells could work together or exert different levels of influence in different tissues.Some of these possibilities will need to be addressed by future research.

Conclusions and the Way Forward
While the rationale for the bone-associated variables being linked to pregnancy and lactation and then the development of osteoporosis is fairly well developed, how the dementia risk in the post-menopausal state relates to involvement in reproductive activities still remains to be developed.Why only a subset of post-menopausal females develop dementia and why specific areas of the brain may be affected versus others, also remains to be determined.However, in both OP and dementia, inflammatory processes appear to be involved in exacerbating the disease progression.
Given the influence of parity and early menopause on development of post-menopausal diseases and conditions, one could postulate that epigenetic processes are involved and the reason that only a subset of post-menopausal females are affected by conditions such as OP and AD is due to a combination of genetic and epigenetic variables, with the epigenetic modifications requiring longer exposure to menstrual cycles following pregnancy/lactationassociated intervals to maintain the relevant cells in a repressed state via those genes activated during pregnancy and lactation.As Alzheimer's disease usually develops long after the menopause transition in most individuals (i.e., >20 years), it is challenging to comprehend how such a time course after menopause could translate to an impact on the at-risk females during a 9-month pregnancy and perhaps a 1-year lactation.
While it is still not yet clear how the various conditions, disease risks, and diseases arising after menopause occur at the mechanistic level, the concept that it involves the microvasculature and tissue-specific endothelial cells was advanced and should be the focus of future investigations.The possible use in the future of drugs or interventions capable of modifying epigenetic alterations contributing to post-menopausal conditions/diseases in a controlled manner (e.g., removal, addition) may be a feasible avenue for research going forward.Such drugs have been proposed to be applied in conditions affecting bone [210], cancer [211,212], neurodegenerative disorders [213,214], cardiovascular conditions [215,216], and healthy aging [217].The development of such "epi-drugs" is not without challenges [218], particularly since such interventions will have to be personalized for post-menopausal conditions that arise in different tissues, may not result from a single phenotypic pattern, and may occur in conjunction with multiple co-morbidities as the individual ages.
As the epi-drug approach may involve tissue-specific epigenetic modifications and their reversal/addition via a variety of mechanisms (e.g., DNA modification, histone modification, and others), this will be challenging to perform with heterogeneous human/patient populations.In the meantime, perhaps one of the most effective non-drug interventions that are beneficial for human functioning across the lifespan is exercise protocols [219].While likely effective across the lifespan, they may be particularly effective in mid-life and beyond when mobility issues and co-morbidities may inhibit motivation for exercise in post-menopausal females.
Finally, another non-drug approach could be the use of specific diets to interfere with or deter the development of post-menopausal conditions.Of particular interest has been the study of Mediterranean diets or diets rich in soy isoflavonoids/flavones [220][221][222][223] and their effects on CVD, dementia, and metabolic disease in post-menopausal females.One component of such diets is genistein, a soybean isoflavone [224], and a molecule that can exert its influence via ER-beta, as well as has effects on endothelial cells [225][226][227][228][229]. A clinical trial of genistein alone has been reported to have a beneficial effect on postmenopausal females with metabolic syndrome [230].Relevant to the present discussion are also reports that flavonoids, including genistein, can exert epigenetic effects [231][232][233][234][235]. Therefore, several aspects of these diets and natural products are very compatible with the proposed epigenetic-based mechanism for post-menopausal conditions.
While many of the diet-related benefits in post-menopausal females are modest [221,222], going forward, a focus of research should be the combination of diets such as the Mediterranean diet or those containing soy-derived flavonoids and related molecules with validated exercise programs.The combination of these two "natural" interventions could lead to addi-tive or synergistic effects on the development and progression of post-menopausal conditions without the need for synthetic pharmaceuticals.Such a combination of interventions could also be initiated prior to the onset of menopause in a proactive manner and thus be preventative.

Figure 1 .
Figure 1.Sequence of epigenetic modifications associated with onset of puberty and progression to skeletal and physiological maturity without intervening pregnancy.Epigenetic modifications repress genes that are associated with pregnancy and lactation which allows for post-puberty growth and maturation.Epigenetic repression maintained by menstrual cycles.

Figure 1 .
Figure 1.Sequence of epigenetic modifications associated with onset of puberty and progression to skeletal and physiological maturity without intervening pregnancy.Epigenetic modifications repress genes that are associated with pregnancy and lactation which allows for post-puberty growth and maturation.Epigenetic repression maintained by menstrual cycles.

Figure 2 .
Figure 2.Pregnancy leads to the re-activation of genes influenced by puberty-associated epigenetic modifications allowing for essential activities leading to a successful pregnancy followed by lactation.(A) Pregnancy-related factors either negate the effects of estrogen (and potentially other sex hormones like progesterone) to maintain the epigenetic modifications, or pregnancy-related factors override the effects of estrogen via enzymes that impact the epigenetic modifications.(B) Following cessation of pregnancy and lactation, menstrual cycles re-establish the epigenetic modifications that again lead to repression of the pregnancy-associated genes until the next pregnancy.With frequent pregnancies, the ability of menstrual cycles to re-establish a homeostatic state with regard to these affected genes may be compromised.

Figure 2 .
Figure 2.Pregnancy leads to the re-activation of genes influenced by puberty-associated epigenetic modifications allowing for essential activities leading to a successful pregnancy followed by lactation.(A) Pregnancy-related factors either negate the effects of estrogen (and potentially other sex hormones like progesterone) to maintain the epigenetic modifications, or pregnancy-related factors override the effects of estrogen via enzymes that impact the epigenetic modifications.(B) Following cessation of pregnancy and lactation, menstrual cycles re-establish the epigenetic modifications that again lead to repression of the pregnancy-associated genes until the next pregnancy.With frequent pregnancies, the ability of menstrual cycles to re-establish a homeostatic state with regard to these affected genes may be compromised.

Figure 3 .
Figure 3.Possible role of tissue-specific reversal of puberty-associated genes to become re-activated following menopause.Following menopause and the loss of estrogen to maintain the epigenetically repressed genes, through either genetic variables or other unknown factors, the pregnancyassociated genes become re-activated by tissue-specific endogenous enzymes leading to the spectrum of menopause-associated conditions/diseases.Not all females would have all of the disease risks.Once initiated, the impact of the de-repression could be aggravated by ongoing inflammatory processes.The cells central to this proposed scheme may be the endothelial cells of the microvasculature in a tissue-specific manner (discussed in more detail in Section 7 of this review).