Neurodegenerative diseases (NDs) are a group of chronic, multivariate, progressive diseases characterized by neuronal degeneration and necrosis. Common NDs include Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS), which have been regarded as persistent and difficult to treat because of their unknown etiology, and poor prognosis. Therefore, early detection and prevention of these diseases have become a hot research topic in recent years. Epidemiologic studies suggest that there is a significant gender difference in the incidence of NDs. For example, studies have shown that two-thirds of patients with AD in the United States are women and that the prevalence of AD is twice as high in women as men [1]. Among patients with ALS, the prevalence is twice as high in men as in women [2]. For patients with PD, the prevalence is 1.5 times higher in men than in women [3]. These gender differences in disease prevalence suggest that the disease development may be related to sex hormone levels.

Sex hormones have been shown to correlate with NDs, however, the underlying biological mechanisms of these correlations are not clear [4]. Studies have shown that estrogen can reduce beta amyloid (A$\beta$) aggregation [5], which is at odds with estimates suggesting that AD is twice as prevalent in women as men. As men age, androgen levels decrease [6] and testosterone levels are lower in patients with AD than in normal individuals [7, 8]. This seems to imply the protective effect of androgens against AD. It has also been shown that estrogen reduces dopamine (DA) depletion in female rats [9, 10].

To date, the role of androgens in patients with PD is not clear [4]. Similarly, little research has been done on the role of sex hormones in ALS. Studies have shown that estrogen can counteract neuroinflammation [11] and that estradiol increases motor neuron survival in male ALS mice [12]. Sex hormone-binding globulin (SHBG) plays a crucial role in the biological action of sex hormones. SHBG is a protein synthesized and secreted by the liver, which, when it reaches the bloodstream, binds directly to sex hormones and regulates their biological activity. A biologically active sex hormone is defined as a sex hormone that is not bound to SHBG.

Although studies have shown a close association between sex hormones and NDs, these studies are not systematic and comprehensive, and due to the limitations of the experimental conditions, they often produce biased results or contradictory conclusions. This may lead to biased conclusions due to the inability of the study design to exclude reverse causality and confounding factors. In addition, traditional randomized controlled trial (RCT) experiments require a large amount of human, material, and financial resources, making it impractical to conduct RCT experiments on this issue.

Mendelian randomization (MR) has been increasingly used to infer risk factors and disease causality. Based on the stochastic nature of genetic variation during meiosis, MR uses environmentally relevant genetic variants as instrumental variables (single nucleotide polymorphisms: SNPs) to assess causality between exposure and outcome. As these genetic variants were randomly assigned at conception before disease onset, MR analyses can effectively exclude confounding factors and identify determinants of a given outcome. In the present study, we systematically evaluated the causal relationship between sex hormones and NDs by using the MR method.

Our study systematically assessed the causal relationship between sex hormones and the risk of developing common NDs, which greatly enriches the existing research findings. Our study suggests that the level of BT is negatively associated with the risk of developing ALS, whereas EL and SHBG are not associated with the risk of developing NDs. Previous studies have shown that in patients with ALS, those with higher testosterone levels have the condition progress at a faster rate and involve the respiratory muscles [19]. At the same time, it has been shown that castration in an ALS mouse model prolongs survival, while androgen supplementation exacerbates motor neuron loss and decreases survival rates [20, 21, 22]. In contrast, blocking the androgen receptor instead leads to accelerated disease onset [23]. Our study suggests that androgens reduce the risk of ALS. The beneficial impact of BT on ALS might be attributed to its neuroprotective properties. Testosterone crosses the blood-brain barrier and exerts its direct impact on the central nervous system by activating androgen receptors or by being converted into other steroid hormones involved in improving human neurons and astrocyte survival [24, 25]. Studies also reported that testosterone could act directly on mitochondrial membranes to inhibit the generation of reactive oxygen species and increase the sirtuin-1 expression [26].

The vast majority of studies suggest that estrogen has neuroprotective effects on ALS, for example, estrogen can directly target neurons and act as an anti-apoptotic agent [27, 28]. This anti-apoptotic effect also applies to motor neurons [29]. However, in our study, estrogen was not associated with the risk of developing ALS. High estrogen levels are negatively associated with the severity of PD symptoms in women, and menopausal women are at a higher risk of developing PD [30, 31, 32]. Previous studies have reported that estrogen replacement therapy during the early postmenopausal period could mitigate the risk of PD and improve prognosis [33, 34]. Estrogen is therefore thought to have neuroprotective effects by slowing down the onset and progression of the disease [35]. However, the current study did not detect the effect of estrogen on PD.

Exactly what role androgens play in the pathogenesis of PD is not well understood so far. Some studies suggest that testosterone deficiency may be associated with PD and that testosterone therapy may improve specific symptoms in patients with PD, including improving motor function and increasing general well-being [36, 37, 38]. However, some studies also provide evidence that total testosterone levels correlate with biomarkers of PD severity, suggesting that testosterone might play a role in male vulnerability to PD [39]. Therefore, the role of testosterone in PD remains equivocal. Our study suggests that testosterone is not associated with the risk of developing PD.

AD, the most common ND, has a high prevalence and mortality rate, and there is a lack of effective clinical treatments. Previous studies have shown that both BT and estrogen are effective in reducing A$\beta$ production and aggregation [5, 40]. Additionally, estrogen therapy has been shown to have a beneficial effect on slowing the development of AD [41, 42]. However, these studies are not reproducible [43, 44]. In contrast, our study aptly demonstrates that estrogen is not associated with the risk of developing AD. In men, circulating testosterone levels decrease progressively with age. In patients with AD, testosterone levels have been reported to be lower than in controls [7, 8]. In addition, androgens are naturally neuroprotective and support neuronal growth [45, 46]. However, it has also been shown that testosterone has no beneficial effects on cognitive function in men [47]. Our study also demonstrates that testosterone does not correlate with the risk of developing AD. Some studies have shown that SHBG is associated with the risk of developing AD [48], however, there are also studies showing that SHBG is associated with the risk of developing ALS, but not AD [49]. Therefore, it is currently difficult to reach a consensus on the causal relationship between SHBG and the risk of developing NDs, and our data suggest that there is no causal relationship between SHBG and the risk of developing NDs. Taken together, the current study failed to detect the effect of SHBG or estradiol on NDs or the effect of BT on NDs except for ALS, which was inconsistent with a majority of previous studies.

Some key points should be noted. Previous epidemiological studies are based on traditional observational design, which is susceptible to confounds or reverse causality, and thus the observed associations might be biased. Another point to note is that the current study used genome-wide association studies (GWAS) summary statistics and thus was unable to conduct sex stratification analysis, which could impact the MR results. Considering the controversial results, more studies are warranted to aid more relevant evidence.

To the best of our knowledge, this is the first systematic MR study conducted to study the relationship between sex hormones and the risk of NDs. MR studies have more advantages than traditional observational studies. For example, it can overcome the effects of confounding and reverse causation [50]. Thus, we were able to provide new insights that may contribute to a better understanding of the causal relationship between sex hormones and NDs. In addition, we used various MR analysis methods to obtain more accurate results. We used recently published GWAS data and performed multiple sensitivity analyses to assess the robustness of our findings.

Although we used MR analyses, the present study has some limitations: (1) as mentioned above, we are unsure of the proportion of false questionnaire reports in the GWAS data; (2) the population included in the present study was predominantly European. Since the results of causal association analysis may also be influenced by ethnicity, further MR studies in other ethnicities should be conducted to validate the present findings; (3) there may be population sharing between the two European sample sets, leading to overuse of genetic information; (4) only publicly available GWAS data were used in our study, thus making sex stratification analysis unavailable. Indeed, the beneficial effects of estradiol in women with PD have been extensively demonstrated in previous studies [51]. The E3N study has reported that postmenopausal hormone therapy might decrease PD incidence [52]. A recent MR study also reported that a later age at menopause was associated with a decreased risk of PD in women, using the Parkinson’s Environment and Gene (PEG) and the Parkinson’s Disease in Denmark (PASIDA) datasets stratified by sex [53]. These studies highlighted the important role of sex in PD. Thereby, further investigation is warranted when future GWAS data stratified by sex is publicly available.

Our study first confirmed a causal relationship between BT and the risk of ALS. However, there no causal relationship between SHBG, EL, and NDs was detected. Therefore, the detection of BT levels may be beneficial for early prevention of ALS. Further studies on the biological mechanism of this causal relationship are needed.

All the data used in this work are publicly available in the IEU database (https://gwas.mrcieu.ac.uk/). The data generated in this work are provided in the main text or supplementary materials.

JHG and QH designed the research. QH and QL were responsible for the completeness of the data analysis and the accuracy of the data analysis, wrote the manuscript, and performed the data analysis. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Only publicly available summary-level GWAS data were used in this work, and thus ethics approval or consent to participate was not required.

We are grateful for the IEU database for making GWAS data publicly available.

This research was supported by Science and Technology Innovation 2030 – Major program of “Brain Science and Brain-like Research” (2021ZD0201801).

The authors declare no conflict of interest.