Ovarian hormones such as estrogens and progesterone fluctuate across the female lifespan with natural declines occurring at menopause, typically between the ages of 45 and 55. The cessation of ovarian function during the menopausal transition has been linked to an array of neural changes (Brinton et al., 2015), including a decline in brain glucose metabolism (Ding et al., 2013), reductions in gray matter (GM) and white matter (WM) volume (Fjell et al., 2009; Goto et al., 2011; Mosconi et al., 2021; Mosconi et al., 2017), and increased amyloid-beta deposition (Mosconi et al., 2018) as well as WM lesions (Wen et al., 2009). In combination with other risk factors, these neural changes might foster the emergence of neurodegenerative diseases such as late-onset Alzheimer’s disease (AD), which is more often diagnosed in females relative to similarly aged males, with greater cognitive decline and neuropathological burden (Laws et al., 2018; Laws et al., 2016).

MHT is commonly prescribed to minimize vasomotor symptoms occurring during the menopausal transition and is generally thought to be neuroprotective, with a propensity to reduce the risk for AD (Simpkins et al., 2009; Hogervorst et al., 2000; Zandi et al., 2002; Kim et al., 2021) and improve cognition later in life (Maki et al., 2011). However, study results are equivocal (Mills et al., 2023), reporting both positive (Schelbaum et al., 2021; Erickson et al., 2005; Ha et al., 2007) and negative outcomes (Kantarci et al., 2016; Lange et al., 2020; Pourhadi et al., 2023). A 2021 study reported that reproductive history events signaling more estrogens exposure, including MHT use, were associated with greater gray matter volume in middle-aged females (Schelbaum et al., 2021), in line with other neuroimaging studies suggesting a protective effect of MHT on GM, WM, and ventricle size (Erickson et al., 2005; Ha et al., 2007). Conversely, MHT use has also been associated with greater atrophy (Resnick et al., 2009) and higher rates of ventricular expansion (Kantarci et al., 2016) in menopausal females. Similarly, in our previous UK Biobank study of ~16,000 females (Lange et al., 2020), we found positive associations between MHT use and older GM brain age, albeit with small effect sizes.

Besides mixed results in observational studies, randomized controlled trials (RCTs) such as the Women’s Health Initiative Memory Study (WHIMS) suggest an increased risk of dementia and cognitive decline with MHT use. In detail, WHIMS found negative effects of prolonged oral administration of both conjugated equine estrogen (CEE) alone (Shumaker et al., 2004) or in combination with medroxyprogesterone acetate (MPA, synthetic progestin) (Rapp et al., 2003; Shumaker et al., 2003) among females aged 65 y or older. Similarly, the Heart and Estrogen/Progestin Replacement Study (HERS) showed an association between 4 y of CEE +MPA treatment and lower cognitive performance in older postmenopausal females (71±6 y) (Grady et al., 2002). In contrast, administering oral CEE or transdermal estradiol plus micronized progesterone in recently postmenopausal females did not alter cognition in the Kronos Early Estrogen Prevention Study (KEEPS) (Gleason et al., 2015). These mixed findings raise the question of whether a combination of timing, formulation, and route of administration might play a crucial role in the effectiveness of MHT.

According to the ‘critical window hypothesis,’ MHT might be neuroprotective if it is initiated close to menopause (Maki, 2013). For example, MHT initiation during perimenopause has been associated with improved memory and hippocampal function later in life (Maki et al., 2011). Although emerging evidence supports this hypothesis (MacLennan et al., 2006; Gibbs and Gabor, 2003), oral CEE use in combination with MPA has been found to increase the risk for memory decline regardless of timing (Shumaker et al., 2003; Maki, 2013; Maki et al., 2007). Similarly, systemic MHT (i.e. oral and transdermal use) has been associated with a 9–17% increased risk of AD in a Finish nationwide case-control study, independent of MHT formulation and timing (Savolainen-Peltonen et al., 2019). However, vaginal estradiol use did not show such risk, indicating differential effects of route of administration (Savolainen-Peltonen et al., 2019). Vaginal as well as transdermal MHT formulations are mainly composed of estradiol, and CEE is a blend of compounds, mostly estrone (Wharton et al., 2013). Both estrogens have different affinities to bind to estrogen receptors (ER): relative to estradiol, estrone is approximately 2/3 the affinity to ER-alpha, and about 1/3 to ER-beta (Kuiper et al., 1997), and estrone is present at higher levels post-menopause than estradiol. Furthermore, contrary to oral use, vaginal and transdermal MHT formulations bypass hepatic metabolism, resulting in a steady-state concentration of estradiol (estrone:estradiol ratio of 1:1) (Wharton et al., 2013). Hence, vaginal, or transdermal estradiol-based MHT formulations might be more effective than oral estrone-based types. For instance, Gleason and colleagues found that females exposed to oral CEE exhibited poorer memory performance than either MHT-naïve or estradiol-exposed individuals (Gleason et al., 2006). In addition to estrogens, progestins are commonly added in non-hysterectomized females, and, like estrogens-based MHT, progestins can be administered in different forms, e.g., norethisterone acetate (synthetic progestin), micronized progesterone (bioidentical), or MPA (synthetic progestin). These progestin forms have been linked to different side-effect profiles (de Lignières, 1999) and can antagonize the effects of estrogens in MHT (Nilsen and Brinton, 2002).

In addition to the impact of MHT timing, formulation, and route of administration, effects of MHT on the female brain might be modulated by apolipoprotein ε type 4 (APOE ε4) genotype. Carried by 14% of the world’s population, the APOE ε4 allele is a known dose-dependent risk factor for late-onset AD. Yaffe and colleagues found that among non-carriers, current MHT use lowered the risk of cognitive impairment by almost half compared to never-users, while there was no such effect among carriers (Yaffe et al., 2000). Results from the Nurses’ Health Study found that MHT use was associated with worse rates of decline in general cognition, especially among females with an APOE ε4 allele (Kim et al., 2019). Conversely, we found no significant interactions between APOE ε4 genotype and MHT use on cognition in a 2023 UK Biobank study (Lindseth et al., 2023). We did, however, observe that earlier MHT initiation was linked to younger GM brain age, albeit weakly, only in APOE ε4 carriers (Lange et al., 2020). Moreover, a 2024 UK Biobank study showed smaller brain volumes in MHT users compared to never-users, specifically among females with the APOE ε4/ε4 genotype (Ambikairajah et al., 2024). Yet, the potential of APOE ε4 to modulate effects of MHT dosage, administration, and formulation on the female brain is understudied.

In summary, emerging evidence suggests differential effects of MHT formulation, age at initiation, route of administration, and genotype on the female brain and cognition. However, studies aiming to disentangle the effects of different MHT regimes are largely missing (Kim and Brinton, 2021). In this observational study, we investigated associations between MHT variables, different MHT regimes, APOE ε4 status, and brain measures in middle to older-aged females from the UK Biobank cohort. MHT variables included user status (i.e. current users, past users, never users), age at initiation, dosage and duration, formulation, route of administration, and type (i.e. bioidentical vs synthetic) as well as active ingredient (e.g. estradiol hemihydrate). MHT regimes based on prescription data were extracted from primary care records (general practice). Brain measures included GM and WM brain age gap based on brain age prediction, hippocampal volumes, and total WM hyperintensity volume as a proxy of vascular disease. These measures were chosen as they have been linked to both chronological and endocrine aging as well as MHT use in our studies (Lange et al., 2020; Ambikairajah et al., 2024; Schindler et al., 2023; Subramaniapillai et al., 2022).

This study assessed detailed MHT data, APOE ε4 genotype, and brain characteristics in a large, population-based sample of females in the UK. The results showed significantly higher GM and WM BAG (older brain age relative to chronological age) as well as smaller left and right hippocampus volumes in current MHT users, but not in past users, compared to never-users. Among MHT users, we found no significant associations between age at MHT initiation, alone and in relation to age at menopause, and brain measures. However, older age at last use after age at menopause was associated with higher GM and WM BAG, higher WMH volume, and lower left and right hippocampal volumes. Similar associations were found for longer duration of MHT use. MHT users with a history of hysterectomy ± bilateral oophorectomy showed lower GM BAG relative to MHT users without such history. In the sub-sample with prescription data, we found no significant associations between detailed MHT variables, such as MHT formulation, route of administration, type, active ingredients, and dosage, and brain measures, after FDR correction. Lastly, although we found lower right hippocampus volumes in carriers of two APOE ε4 alleles relative to non-carriers, we found no interactions with MHT variables after FDR correction.

Current MHT users showed higher GM and WM BAG as well as lower left and right hippocampus volumes relative to never-users. The effects were robust but relatively modest in magnitude, with the largest effect size indicating a group difference of 0.77 y (~9 mo) for GM BAG (standardized β = 0.218, Supplementary file 4). However, we found no significant differences in brain measures in past MHT users relative to never-users. Current MHT users were significantly younger than past- and never-users, and around 67% were menopausal relative to over 80% in the past- and never-user groups. The unequal distribution of age and menopausal status across groups may have influenced the observed findings. For instance, a larger proportion of the current users might be in the perimenopausal phase, which is often associated with debilitating neurological and vasomotor symptoms (Brinton et al., 2015). MHT is commonly prescribed to minimize such symptoms. Although MHT initiation during perimenopause has been associated with improved memory and hippocampal function, as well as lower AD risk later in life (Maki et al., 2011), the need for MHT might in itself be an indicator of neurological changes (Kantarci and Manson, 2023) here potentially reflected in higher BAG and lower hippocampal volumes. After the transition to menopause, symptoms might subside and some perimenopausal brain changes might revert or stabilize in the postmenopausal phase (Mosconi et al., 2021). Although the UK Biobank lacks detailed information on menopausal symptoms and perimenopausal staging, our results might be capturing subtle disturbances during perimenopause that later stabilize. This could explain why the largely postmenopausal groups of past MHT users and never-users present with lower GM and WM BAG than the current user group. Considering the critical window hypothesis emphasizing perimenopause as a key phase for MHT action (Maki, 2013; Kim and Brinton, 2021), future longitudinal studies are crucial to clarify the interplay between neurological changes and MHT use across the menopause transition.

Besides age-related differences, the current user group also showed a significantly unhealthier lifestyle, and higher rates of bilateral oophorectomy compared to the never-user group and the past-user group, respectively. These findings are contrary to the healthy user bias hypothesis (i.e. equating MHT use with healthy user status, Gleason et al., 2012), and might indicate that the current user group could be exposed to neurological changes prior to MHT use. According to the healthy cell bias hypothesis of estrogen action (Brinton, 2008), MHT use might be detrimental for brain health when initiated after cells are exposed to neurological degeneration. Although MHT use might have exacerbated adverse brain changes in the unhealthier group of current users, higher BAG was also linked to longer duration of MHT use and older age at last use post-menopause. Although the effect sizes were modest (Supplementary file 4), these findings might reflect subtle yet unfavorable effects of MHT on brain health in our sample, particularly when used continuously after natural menopause with uterus and ovaries still intact (i.e. all females with hysterectomy ± bilateral oophorectomy were excluded from this analysis).

On the contrary, MHT users with a history of hysterectomy ± bilateral oophorectomy showed lower GM BAG relative to MHT users without such history, and we found larger hippocampal volumes in MHT users with a hysterectomy without a bilateral oophorectomy. Previous work associates these surgical interventions with accelerated cognitive decline (Rocca et al., 2007), increased risk of dementia (Phung et al., 2010), morphological changes in regions of the medial temporal lobe (Zeydan et al., 2019), and accumulation of Alzheimer’s disease pathology (Georgakis et al., 2019). However, some of these studies relied on comparing surgery to no surgery (Phung et al., 2010; Rocca et al., 2007) without taking MHT use into account (Georgakis et al., 2016), or investigated the effect of surgery with MHT relative to surgery without MHT. For instance, a 2023 study highlighted that estrogen-only use in females with hysterectomy was associated with increased dementia rates relative to females with hysterectomy who never used MHT (Pourhadi et al., 2024). In the current study, we specifically compared the effect of MHT use on brain measures among females with and without surgical history and found lower GM BAG in the surgical MHT user group. These findings might be explained by differences in indication of MHT use for females with and without surgery, MHT formulation, and age at surgery. Without surgery, MHT use (i.e. often combined MHT formulation) is prescribed in females with intact uterus and ovaries to minimize menopausal symptoms that are largely neurological in nature, such as vasomotor symptoms as well as mood, cognitive, and sleep disturbances (Brinton et al., 2015). After hysterectomy, estrogen-only MHT might be prescribed but is not strictly needed as ovaries are still intact. Whereas after bilateral oophorectomy (=surgical menopause), combined or estrogen- only MHT is indicated to compensate for the acute and chronic deficiency of hormones normally produced by the ovaries. These distinctions might entail important differences in risk and side-effect profiles of estrogen-only or combined MHT in females with and without endogenous sex hormone production. It is also possible that the timing between MHT use and surgery is more tightly controlled and, therefore, more beneficial for brain aging (Kim and Brinton, 2021). For instance, studies suggest that MHT may mitigate the potential long-term adverse effects of bilateral oophorectomy before natural menopause on bone mineral density as well as cardiovascular, cognitive, and mental health (Blümel et al., 2022; Kaunitz et al., 2021; Stuursma et al., 2022). In addition, a 2024 UK Biobank study found that ever used MHT was associated with decreased odds of Alzheimer’s disease in women with bilateral oophorectomy (Calvo et al., 2024).

However, our study also showed group differences in several demographic and MHT-related variables which might influence the results. For instance, the hysterectomy MHT user group was significantly older than the non-surgery and bilateral oophorectomy group, started MHT at a younger age than the non-surgery group, and had surgery at a younger age than the bilateral oophorectomy group. More research is needed to disentangle this complex interplay of MHT effects in females with and without intact uteri and ovaries.

No significant associations were observed between brain measures and MHT regimes based on prescription data. However, given the relatively small sample size and the large number of comparisons, it is possible that we were unable to detect subtle effects of factors such as MHT formulation, route of administration, type, active ingredients, and dosage. Before FDR correction, we found higher WM BAG in estrogens +progestin users, in CEE + MPA users, and in mixed active ingredients users relative to never-users. In addition, WMH volume was higher among oral MHT users and lower with longer duration of estrogens use in estrogens-only users relative to never-users. These uncorrected results are partly in line with previous findings. For instance, the 2003 Women’s Health Initiative Memory Study reported that prolonged oral use of both CEE alone (Shumaker et al., 2004), or combined with MPA (Shumaker et al., 2003), increased the risk of dementia and cognitive decline among females aged 65 y and older. Contrarily, prolonged use of estrogen-only MHT has been linked to reduced white matter loss in aging (n=10) (Ha et al., 2007). Similar to our findings, Ha and colleagues did not find an association between gray matter volumes and estrogen use (Ha et al., 2007). Although uncorrected results must be interpreted with caution, our findings might indicate an unfavorable effect of mixed active ingredient use, including CEE ± MPA and oral administration, on white matter brain aging. This effect might be due to the higher estrone concentrations associated with such oral estrone-based MHT types (Longcope et al., 1985; Slater et al., 2001; Van Erpecum et al., 1991). In addition, over 50% of combined MHT users had mixed MHT use which suggests that it might be beneficial to examine the reasons for switching medication. In sum, these findings highlight the need for personalized MHT regimes, and longitudinal RCTs are needed to establish how different MHT regimes, and their respective hormonal profiles are causally linked to brain aging.

In the current study, we found significantly smaller right hippocampus volumes in carriers of two APOE ε4 alleles relative to non-carriers, which is in line with previous work linking the APOE ε4 genotype to greater rates of hippocampal atrophy in non-demented and Alzheimer’s disease samples (Saeed et al., 2021; Gorbach et al., 2020). However, after FDR correction, we did not find any interactions between APOE ε4 carrier status and MHT variables in relation to the brain measures. This finding was unexpected, as previous work has highlighted the APOE ε4 genotype as a crucial determinant of MHT effects on the female brain (Barth et al., 2023). For instance, one study associated MHT use with improved memory performance and larger entorhinal and amygdala volumes in female ε4 carriers versus non-carriers (Saleh et al., 2023). A 2023 prospective longitudinal study showed that MHT was associated with smaller changes towards Alzheimer’s disease pathophysiology than non-therapy and that APOE ε4 carrier status was linked to an amplified treatment outcome (Depypere et al., 2023). However, contrary results have also been reported. For instance, Yaffe and colleagues found lower risk of cognitive decline with estrogen-only MHT use among female APOE ε4 non-carriers, but there was no such effect among carriers (Yaffe et al., 2000). To understand these discrepancies, more research on MHT by genotype interactions is needed.

The current work represents the most comprehensive study of detailed MHT data, APOE ε4 genotype, and several brain measures in a large population-based cohort to date. Overall, our findings do not unequivocally support general neuroprotective effects of MHT, nor do they indicate severe adverse effects of MHT use on the female brain. The results suggest subtle yet complex relationships between MHT’s and brain health, highlighting the necessity for a personalized approach to MHT use. Importantly, our analyses provide a broad view of population-based associations and are not designed to guide individual-level decisions regarding the benefits versus risks of MHT use. Furthermore, several study limitations should be acknowledged. The presented data does not enable causal inference, and observational studies are subject to different sources of heterogeneity such as switching between MHT regimes (e.g. due to side effects or availability) and variable MHT formulation and dose. In addition, utilizing prescription registry data comes with its own set of challenges. In the UK, a national system for collecting or sharing primary care data is currently missing, and the availability, completeness, and level of detail in the data might vary between systems, suppliers, and over time. The primary care data used in the current study was drawn from an interim release, including data on approximately 231,000 participants. Hence, our analyses conducted on these participants’ data and the observed results might not generalize to the entire cohort. Furthermore, although we extracted prescription MHT data, medications that were prescribed were not necessarily dispensed or used. Moving forward, research utilizing unified prescription registry data is needed to overcome some of these hurdles. In addition, previous studies highlight that UK Biobank participants are considered healthier than the general population based on several lifestyle and health-related factors (Golomb et al., 2012; Bradley and Nichols, 2022). This healthy volunteer bias increases with age, likely resulting in a disproportionate number of healthier older adults. Together with the imbalance in age distributions across groups, this might explain the less apparent brain aging in the older MHT user groups. We have previously highlighted that age is negatively associated with the number of APOE ε4 carriers in the UK Biobank (Lange et al., 2020), which is indicative of survivor bias. In addition to these inherent biases in aging cohorts, the ethnic background of the sample is homogeneous (>96% white), further reducing the generalizability of the results. Lastly, although the UK Biobank has a wealth of female-specific variables, the acquired data relies on self-reports, which might not be reliable and data recording does not always align with best practice standards. For example, menopausal status in the UK Biobank is recorded based on whether the menstrual period has generally stopped, not whether it has been absent for at least 12 mo, in line with the STRAW criteria (Harlow et al., 2012).

In conclusion, our findings suggest that associations between MHT use and female brain health might vary depending on duration of use and past surgical history. Although the effect sizes were generally modest, future longitudinal studies and RCTs, particularly focused on the perimenopausal transition window, are warranted to fully understand how MHT use influences female brain health. Importantly, considering risks and benefits, decisions regarding MHT use should be made within the clinical context unique to each individual.