Progestins are a class of molecules composed of the naturally occurring progesterone [discovered (1930) and isolated (1933) by Willard Allen and George Corner] and synthesized steroids initially designed to block the proliferative effects of estrogen in reproductive tissues, specifically in the uterus. Several generations of clinical progestins have been synthesized and marketed for contraception and hormone therapy.

While the target of progestins used in hormone therapy is primarily the uterus, progestin therapy affects every major organ system including the brain, the cardiovascular system, the immune system and hematopoietic system.(1)(2)(3)(4)(5)(6)(7)(8) As in other systems, progestins exert unique profiles of outcomes in the brain, which ultimately could impact the long-term neurological health of users.

In contraceptive preparations, progestins are used to prevent ovulation and pregnancy and are often combined with estrogen to obtain better menstrual cycle control and more effective inhibition of follicle maturation and ovulation. The majority of contraceptive drugs currently on the market are estrogen and progestin combined oral formulations. Other formulations, including parenteral administration, implants, vaginal rings, transdermal gels and sprays have also been developed and commercialized.(9) Hormone therapy is often provided to surgically menopausal, perimenopausal and naturally postmenopausal women to alleviate the clinical symptoms caused by decline in ovarian hormones. In hormone therapy preparations, progestins are required for women with intact uterus to counter the estrogen-induced endometrial hyperplasia. The variety of hormone therapy formulations has followed that of contraceptive options (see Table 1).

Increasing evidence indicates that progestins differentially regulate the regenerative and bioenergetic capacity of the brain alone or in combination with estrogen.(4)(5)(6)(7) Findings from these preclinical studies indicate that the type and regimen of progestin used in hormone therapy/contraception and the timing of HT intervention during the peri- to post-menopausal years could dramatically impact neurological health and cognitive function.(16)(17)(18)(19)(20)(21)(22)(23) The focus of this Cyberounds^® is progestin action on the brain, specifically the impact of progestins on the regenerative and bioenergetic capacity of the brain, as well as their effects on cognition.

Progesterone and Progestin Receptors In The Brain

The brain is a primary target for both contraceptive and hormone therapy where these therapies regulate the reproductive cycle and hot flushes. Progestins interact with progesterone receptors that are present both in the nucleus and on the cell membrane.(24)(25)(26) Further, depending upon the chemical structure of these molecules, progestins will also interact with estrogen, androgen, glucocorticoid and mineralocorticoid receptors. (9)(27) Receptors for progesterone and other steroid hormones are abundant in the hypothalamus as well as other brain regions not directly involved in reproductive function.(25) Specifically, these molecules regulate mitochondrial function, synaptogenesis, neurogenesis and regeneration, myelination, inflammation, mood and cognition.(4)(5)(6)(7)(28)(29)(30)(31)(32)(33)

Synthetic progestins were originally developed to overcome the short biological half-life and high production cost of progesterone.(34)(35) They are structurally derived from either progesterone or testosterone (36) and are categorized into “generations”: the first generation (medroxyprogesterone acetate, norethindrone, etc.), second generation (levonorgestrel, norgestrel, etc.), third generation (gestodene, desogestrel, norgestimate, etc.) and new generation (drospirenone).(37) Compared to third and new generations, the earlier generations of progestins often have lower progestational activity and greater interaction with other steroid receptors, i.e., the androgen, estrogen, glucocorticoid and mineralocorticoid receptors.(9)(27)

The interaction between progestins and these receptors is a major source of the undesired side effects seen in hormonal contraception and hormone therapy, of which the most disturbing are the increased risks of breast cancer and cardiovascular diseases.(1)(9)(38)(39) Newer progestins were often designed to better mimic the physiological function of progesterone and minimize the side effects by reducing the interaction between progestins and other steroid hormone receptors.(38) Systematic evaluation of efficacy and safety of these newer progestins in contraceptive and hormone replacement therapy preparations is needed to determine their long-term impact on women’s health.

Emerging data indicate that progesterone has multiple non-reproductive functions in the central nervous system -- regulation of mitochondrial function, synaptogenesis, neurogenesis and regeneration, myelination, inflammation, mood and cognition.(4)(5)(6)(7)(8)(25)(31)(32)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50) Progesterone-regulated neural responses are mediated by an array of progesterone receptors that include the classic nuclear progesterone receptor A and progesterone receptor B and splice variants of each, the seven transmembrane domain 7TMPRb and the membrane-associated 25-Dx progesterone receptor PGRMC1.(25)(51)(52)(53) These progesterone receptors induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. Remarkably, progesterone receptors are broadly expressed throughout the brain and can be detected in every neural cell type. The distribution of progesterone receptors beyond hypothalamic borders provides the foundation for a much broader role of progesterone in regulating neural function.(25)

Progestin Regulation of Regeneration In The Brain

Progesterone regulation of cellular proliferative activities was first recognized in reproductive tissues, most notably in the breast(54)(55) and the uterus. In breast tissue, progesterone can promote proliferation, whereas in the uterus it has both inhibitory and stimulatory effects, depending on cell type, the regimen of treatment, whether progesterone receptor A or progesterone receptor B is expressed and the dose of estrogen and progesterone.(25)

In the adult brain, proliferation of neural cells occurs in two proliferative zones, the subgranular zone of the hippocampus and the subventricular zone of the cerebral ventricles.(56)(57) The generation of new neurons (neurogenesis) occurs in both zones. As in the uterus, progesterone regulation of mitosis in the nervous system is complex. Results from multiple laboratories have indicated that progesterone regulates neural cell proliferation in both the peripheral and central nervous system.(58)(59) Preclinical analyses indicate that progesterone can act as a neurogenic agent to promote hippocampal neural progenitor cell proliferation.(6) Further, progesterone significantly increased the expression of genes required for progression through the cell cycle and inhibited expression of genes involved in repression of the cell cycle.(6) Progesterone-induced neurogenesis was a direct effect of progesterone and did not require conversion to its metabolites.

The direct effect of progesterone on neural progenitor cell proliferation was demonstrated by inhibiting the conversion of progesterone to its metabolites 5-dihydroprogesterone and allopregnanolone by finasteride.(6) Blocking the conversion of progesterone to its metabolism had no effect on progesterone-induced neural progenitor cell proliferation, demonstrating the direct effect of progesterone. Interestingly, progesterone-induced neural progenitor cell proliferation was mediated by the membrane progesterone receptor PGMRC1. The requirement of PGMRC1 was demonstrated by RNA interference (siRNA) knock-down of PGMRC1 receptor expression in rat neural progenitor cells which abolished the proliferative effect of progesterone.

Progesterone increased proliferation of neural progenitor cell from female adult rat hippocampus with maximal proliferative efficacy within the picomolar/nanomolar range, which was reversed at the higher micromolar range.(6) In the adult female rat, progesterone plasma levels range from 1 to 200 nM, and the concentration of progesterone in the brain is thought to closely follow circulating plasma levels.(60) The mitotic efficacy of progesterone was comparable to the growth factor bFGF.(62) Importantly, progesterone-induced DNA synthesis did not persist beyond 24 hours, indicating that progesterone-responsive rat neural progenitor cells traversed the cell cycle but did not remain in a proliferative state and thus progesterone, in vitro, did not induce prolonged or uncontrolled proliferation. Other groups have reported that progesterone promotes cell proliferation in both peripheral and central nervous system at nanomolar/micromolar ranges.(58)(61)

Clinical hormone therapies are often administered as a continuous combined regimen of an estrogen and progestin. Translationally, it was important to investigate the impact of the combined exposure of estrogen and progesterone on the regenerative capacity of the brain. Estrogen alone is neurogenic, as is progesterone. When progesterone was administered simultaneously with estrogen, the neurogenic effect of estrogen was antagonized.(63)

In the aged brain, both the pool of neural stem cells and their proliferative potential are markedly diminished.(64)(65)(66) The discovery that progesterone could promote adult rat neural progenitor cell proliferation highlights the potential use of progesterone as a regenerative agent in adult brain. Indeed, several studies reported that administration of progesterone could improve cognitive performance in the aging mouse when compared with control groups.(67)(68)(69) Together with our present data, these findings suggest a promising strategy for promoting neurogenesis in the adult brain. Furthermore, the finding that the estrogen receptor and PGRMC1 were mutually exclusive in breast tumor sections, and that PGRMC1 labeling increased in hypoxic areas of the tumor,(70)(71) suggests that targeting PGMRC1 may be a novel approach to promote proliferation in the brain while avoiding tumorogenic effects in estrogen receptor-positive cells.

Clinical Progestin Regulation of Regeneration in the Brain

Our group investigated the efficacy of seven clinically relevant progestins (i.e., nestorone, medroxyprogesterone, levonorgestrel, norgestimate, norethindrone, norethindrone acetate and norethynodrel) alone or in combination with estrogen on adult rat neural progenitor cell proliferation and hippocampal cell viability, a predictive indicator of the health of newly generated and existing cells in vitro and in vivo. In vitro analyses indicated that progesterone, norgestimate, nestorone, norethynodrel, norethindrone and levonorgestrel significantly increased rat neural progenitor cell BrdU (a synthetic nucleoside) incorporation, a marker for DNA synthesis and cell proliferation, while norethindrone acetate was without effect, and medroxyprogesterone acetate decreased BrdU incorporation.(5)

Since estrogen is used together with progestin in most contraceptive and hormone therapy preparations, it was critical to investigate the impact of exposure to estrogen + progestin on neurogenesis and neuroprotection. Results of these analyses showed that estrogen significantly increased neural proliferation at a magnitude comparable with that of progesterone alone. No significant differences were observed between estrogen alone, estrogen + progesterone and estrogen + nestorone groups. Both levonorgestrel and medroxyprogesterone increased BrdU+ cell count when administered in combination with estrogen.(5) However, in parallel to the increase in BrdU positive cells, levonorgestrel and medroxyprogesterone significantly increased the number of apoptotic cells(5) indicating these cells were undergoing programmed cell death, which is consistent with the proapoptotic effects of levonorgestrel and medroxyprogesterone in the uterus.(72)

Results of these studies indicate that acute exposure to clinically relevant progestins and estrogen + progestin combinations differentially regulated neurogenic and neuroprotective responses in brain. Clinical progestins exerted a range of effects that span promotion of neurogenesis and neuroprotection to inducing apoptosis and reduced cell viability. The effects of progestins were tested at clinically relevant concentrations.(5)

The differential proliferative effects of the progestins were potentially due to progestins binding to multiple steroid receptors. Indeed, levonorgestrel and norgestimate exert androgenic effects by binding to and activating the nuclear androgen receptor. However, norgestimate, in contrast to levonorgestrel, inhibits nuclear translocation of the androgen receptor, revealing an antiandrogenic property.(73) Also, norethynodrel induces estrogenic activity through aromatization in vivo. Although nestorone can bind to the nuclear glucocorticoid receptor, it showed no glucocorticoid activity in vivo.(74)(75) While these preclinical studies were limited in scope, they pointed to differential effects on the regenerative system in brain that could have profound clinical implications for neurological health in women.

Progestin Regulation of White Matter Regeneration

The subventricular zone neurogenic niche also gives rise to oligodendrocytes, which are capable of migrating into sites of demyelination under disease or injury conditions in an attempt to repair the disrupted myelin sheath.(76) Progesterone facilitated oligodendrocyte precursor cell proliferation and differentiation, with acute treatment favoring proliferation whereas chronic administration favored differentiation.(77)(78) Oligodendrocyte death and white matter lesion is a well-characterized feature of Alzheimer’s disease.(79)(80) While progesterone was an effective remyelinating molecule under multiple disease and injury conditions(28)(81)(82)(83)(84), its potential to alleviate white matter abnormalities present in Alzheimer’s disease remains to be tested.

Progestin Regulation of Brain Metabolism

Recent data indicate that progesterone also promotes brain metabolism. Mitochondria are the primary energy producers of the cell that convert nutrients into ATP for energy through cellular respiration via the electron transport chain. Our group investigated the impact of progesterone on key mitochondrial functions, oxidative respiration and free radical generation in the central nervous system. We found that both estrogen and progesterone significantly increased mitochondrial respiration 24 hours after a single in vivo exposure.(7) Neither progesterone nor estrogen nor their combination induced evidence for mitochondrial biogenesis, indicating these effects were due to progesterone and estrogen regulation of mitochondrion function rather than an increase in mitochondrial number.(7) Together, these preclinical data revealed that the gonadal hormones progesterone and estrogen were potent regulators of mitochondrial function to increase both the magnitude and efficiency of mitochondrial respiration in brain.

Further, both estrogen and progesterone reduced free radical leak, indicating greater efficiency of electron transport. Consistent with reduced generation of free radicals, progesterone and estrogen induced a significant reduction in mitochondrial lipid peroxidation. Oxidative damage to mitochondria is posited to play a major role in aging and in neuronal populations may underlie cognitive declines associated with aging.(85)(86)(87) As electrons pass through the mitochondrial electron transport chain, some electrons leak out to molecular oxygen (O2) to form O2•−, which is dismutated (simultaneous reduction and oxidation creating two different compounds) by manganese superoxide dismutase (MnSOD) to form hydrogen peroxide (H2O2). The hydrogen peroxide can in turn be reduced to water by peroxidases such as glutathione peroxidase or peroxiredoxins.

A lack of sufficient peroxidase activity results in the peroxidation of lipids and proteins. Lipid peroxidation, the nonspecific oxidation of polyunsaturated fatty acids in cellular membranes, is a radical-mediated pathway and generates a number of harmful degradation products besides drastically altering the structure and function of the membrane. Our results demonstrated that ovarian hormone treatment reduced lipid peroxidation of whole-brain mitochondria. This result is consistent with previous reports demonstrating that estrogen(88)(89)(90) and progesterone(91) could individually reduce oxidative stress in the brain.

Respiratory control is one facet of an interlocking network regulating metabolic activity and energy production in which mitochondrial redox potential is coupled with cytosolic signaling. This tightly coupled network integrates various signals to determine cell fate based upon ATP levels, extracellular signals and energetic demands. In addition to the defensive effects of estrogen and progesterone, including regulation of the Bcl-2 family proteins,(92) the increased respiratory control efficiency and decreased oxidative load demonstrated in this study could establish a buffer, an antioxidant defense against neuronal functional decline associated with aging and neurodegenerative diseases.

Surprising to us was the lack of synergy when progesterone and estrogen were administered in combination. On all outcome measures, the combination of progesterone and estrogen resulted in a substantial decrement in response magnitude. Another point of note is that the effects of the combined treatment were not consistent across all outcome measures. In some outcomes, as in mitochondrial respiration, there was no effect of the combined treatment relative to control, and in others, as in cytochrome c oxidase activity and expression, the combined treatment significantly altered the response relative to control.(7) Thus, the neuroprotective abilities of combined estrogen and progesterone do not involve all aspects of mitochondrial bioenergetics, and the two steroids probably act through two different sets of overlapping sets of mechanisms. These distinct mechanisms may act synergistically or antagonistically resulting in the mixed profile of outcomes.

Clinical Progestin Regulation of Brain Metabolism

The impact of clinical progestins used in contraception and hormone therapies on the metabolic capacity of the brain has long-term implications for neurological health in pre- and postmenopausal women.

In preclinical models, ovariectomized female rats were treated with medroxyprogesterone, estrogen, estrogen + medroxyprogesterone or placebo, with ovary-intact rats serving as a positive control. Medroxyprogesterone alone and medroxyprogesterone + estrogen resulted in diminished mitochondrial protein levels for pyruvate dehydrogenase, cytochrome oxidase, ATP synthase, manganese-superoxide dismutase and peroxiredoxin V.(4) Medroxyprogesterone alone did not rescue the ovariectomy-induced decrease in mitochondrial bioenergetic function, whereas the co-administration of estrogen and medroxyprogesterone exhibited moderate efficacy.(4) However, the co-administration of medroxyprogesterone was detrimental to antioxidant defense, including manganese-superoxide dismutase activity/expression and peroxiredoxin V expression.(4)

As mentioned above, estrogen, progesterone, or co-administration reduced electron leak, indicating greater efficiency of electron transport.(7) Consistent with reduced generation of free radicals, estrogen completely prevented mitochondrial lipid peroxidation. In contrast, medroxyprogesterone or estrogen + medroxyprogesterone combination did not prevent ovariectomy-induced lipid peroxidation. Furthermore, medroxyprogesterone abolished estrogen-induced enhancement of mitochondrial respiration in primary cultures of the hippocampal neurons and glia (4). In vivo analyses of multiple clinically relevant progestins confirmed that progesterone, nestorone, and levonorgestrel induced a significant rise in mitochondrial CVα expression, a marker of cell viability, whereas medroxyprogesterone showed no effect, indicating the neuroprotective effects of the progestins are closely related to their ability in regulating mitochondria function.

Collectively these findings demonstrate that the effects of medroxyprogesterone differ significantly from the bioenergetic profile induced by progesterone and that, overall, medroxyprogesterone induced a decline in glycolytic and oxidative phosphorylation protein and activity. On all outcome measures except respiration, the combination of medroxyprogesterone and estrogen was not synergistic and when administered in combination led to a decreased response relative to estrogen alone. Findings from this study provide insights into progestin regulation of mitochondrial respiration and the impact of clinical regimens of hormone therapy on mitochondrial function in the brain. A major challenge now is to identify a selective progestin that is best suited for hormone therapy outcomes in brain, including sustaining cognitive function with age.

Progestin Regulation of Cognitive Function

While there have been an abundance of studies investigating the cognitive outcomes of combined estrogen + progestin hormone therapy/contraceptive use in women, the exact effects of progestins on cognition remain elusive. The Women’s Health Initiative Memory Study (WHIMS), the largest randomized, controlled study of the kind, found that the conjugated equine estrogen + metroxyprogesterone treated group of women had higher risk of probable, all-cause dementia compared to the conjugated equine estrogen only group and the placebo group.(14)(15) Estrogen has been shown to have multiple beneficial effects on brain metabolism and cognition, capable of increasing dendritic spine density and enhancing synaptic plasticity in the hippocampus, elevating brain mitochondrial efficiency and preventing metabolic decline in postmenopausal women, notably in brain regions most susceptible to Alzheimer’s disease, and protecting against oxidative stress as well as β-amyloid-induced neuronal apoptosis.(10)(92)(93)(94)(95) The discrepancies between these studies and findings from WHIMS led to the speculation that progestins may affect aspects of cognition negatively, attenuating or opposing the beneficial effects of estrogen.

Several important issues, however, were not addressed in the WHIMS study and they must be considered to better understand the true impact of progestins on cognition. First, most researchers investigating cognitive outcomes of combined hormone therapy/contraceptive used estrogen + medroxyprogesterone preparations.(20)(96) Medroxyprogesterone is reported in many preclinical and clinical studies to have detrimental biological properties. Its strong androgenic effect is associated with increased risk of breast cancer in hormone therapy users.(39)(97) In the brain, medroxyprogesterone antagonizes the estrogen up-regulation of mitochondrial function and is bioenergetically inhibitory by itself.(4) Long term weekly treatment with medroxyprogesterone causes working memory impairment.(50)

Because of its widespread use medroxyprogesterone has been extensively studied but much less is known about the variety of progestins used in the hormone therapy/contraceptive preparations listed in Table 1. Indeed, several studies reported favorable effects on cognition by alternative progestins. Post-menopausal women receiving conjugated equine estrogen + micronized progesterone for 12 weeks performed significantly better on a working memory task than the conjugated equine estrogen + medroxyprogesterone and the conjugated equine estrogen + placebo groups.(32) The preclinical data are consistent with the clinical data. Rats treated with progesterone, alone or in conjunction with estrogen, had improved cognitive behavior.(67)(68)(69) Importantly, women who used contraceptive drugs with new generation progestin performed better on mental rotation (a task in which subjects are presented with two objects, often each rotated a specific amount of degrees, and must decide whether the two objects are identical or mirror images) and verbal fluency than women using contraceptive drugs with third generation progestins,(98) further supporting the postulate that progestins modulate cognition differentially.

In preclinical studies, the regimen of progestin administration, in particular the continuous vs. cyclic paradigm, can affect the cognitive outcome. In an ovariectomized female mouse model of Alzheimer’s disease, estrogen reduced amyloid pathology and improved working memory. A hormone regimen of cyclic progesterone + estrogen enhanced the beneficial effects of estrogen including reducing amyloid pathology.(99) In contrast, continuous progesterone treatment antagonized the beneficial effects of estrogen.(99) These preclinical data comparing the impact of cyclic vs. continuous exposure to progesterone have potential clinical implications, as the majority of hormone therapies use a continuous combined regimen of treatment (see Table 1).

Discussion

Clinical progestins are an integral constituent of contraceptive and hormone therapy preparations. Progestin-containing contraceptive drugs account for an increasing proportion of modern contraceptive formulations used by women around the world. In the United Kingdom, the commercially popular progestins are 19-nortestosterone derivatives (norethindrone acetateA, norgestrel and levonorgestrel). In France, micronized progesterone and 19-norprogesterone (such as promegestone and nomegestrol acetate) are commonly prescribed, while medroxyprogesterone is the most prescribed progestin in the United States(5) and the progestin used in commonly randomized controlled hormone therapy trials including the Women’s Health Initiative(100) and Women’s Health Initiative Memory Study.(15)(19) Typically, clinical progestins are chronically administered, extending over many years to decades. For example, depomedroxyprogesterone acetate, a long-acting formulation of medroxyprogesterone, is extensively prescribed for adolescent females,(101) and Norplant implant delivers constant infusion of levonorgestrel for 5–7 years.(102)

Despite the global clinical use of progestins, little is known regarding the impact of chronic exposure to these molecules in the brains of women during and following their reproductive years. Preclinical research from our laboratory and others strongly suggest that these molecules have profound effects on neurological functions that range from regeneration in the brain to cognition. These effects could have implications for protection against or vulnerability to neurodegenerative diseases and warrant further intensive investigation.

Summary

Progestins are a class of molecules composed of the naturally occurring progesterone and synthesized steroids initially designed to block the proliferative effects of estrogen in reproductive tissues, specifically in the uterus. In addition to regulating the reproductive system, progestins impact multiple major organ systems including the brain. We and others have found that progestins have differential effects on brain regeneration, brain metabolism and cognitive function when administered alone or in combination with estrogen. Progesterone has a favorable profile of action in the brain, promoting regeneration and mitochondrial function. Under certain conditions, progesterone can improve cognitive performance. In contrast, synthesized progestins have a mixed profile of outcomes in the brain that span from beneficial to detrimental. Both preclinical and clinical data raise concern on the type and regimen of progestins currently used in hormone therapy and contraceptive preparations, and partially explain the variability observed in outcomes in brain at both the preclinical and clinical levels of analyses. Given the wide use of hormone and contraceptive therapies worldwide, understanding progestin regulation of brain function and cognition has the potential to profoundly impact women's neurological health.

Acknowledgement

The works reviewed here were supported by National Institute on Aging Grant 1 PO1 AG026572 (to R.D.B.).