Yaxing Zhang ^1* Shuangling Yang ² Jiongshan Zhang ³ Luyao Xu ^4* Yajie Guan ^5* Chun Fang ⁶ Shuhui Zheng ^7* Hongzhi Yang ^3* Haimei Liu ^1*

• ¹ Department of Physiology, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China
• ² School of Health Sciences, Guangzhou Xinhua University, Guangzhou, China
• ³ Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
• ⁴ Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China
• ⁵ Henan University of Chinese Medicine, Zhengzhou, Henan Province, China
• ⁶ Yangtze University, Jingzhou, Hubei Province, China
• ⁷ The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China

Hydrogen gas (H2), the lightest gas in the universe, can act as an important antioxidant. Supplementing exogenous H2 can improve many diseases. However, the physiological and pathological significance of endogenous H2 is not clear. In the recent issue of Cell, McCurry et al. reveal that endogenous H2 drive 21-dehydroxylation for transforming glucocorticoids to progestins, which may further influence female health, such as postpartum depression (McCurry et al., 2024). We are very interested in this milestone study that reveals the physiological effects of endogenous H2. Based on the history, here, we will discuss the process of this important finding, the uncovered questions, the current status and future of endogenous H2 in female health. The gut bacteria chemically modify glucocorticoids into progestins through 21dehydroxylationIn 1969, H. Eriksson et al. reported that the feces from germ-free (GF) rats contain corticoids but no pregnanolones, while the feces from conventional rats contain pregnanolones but no corticoids (21hydroxylated steroids), which suggested that gastrointestinal progestin formation is dependent on gut microbiome (Eriksson et al., 1969a, b). Similar to this, it has been reported that fecal slurries from rats and humans can convert glucocorticoids into progestins through reductive removal of C21 hydroxyl group, a process called 21-dehydroxylation (Morris and Brem, 2019;Morris and Ridlon, 2017).Therefore, this means the presence of 21-dehydroxylation products in the gut might depend on the gut microbiome. However, it is unclear how gut bacteria chemically modify steroids and how the resulting metabolites affect the host health.In 2024, Dr. Megan D. McCurry et al. had developed an ultra-high-performance liquid chromatography (UHPLC)-based method to quantify glucocorticoids and progestins in the biological fluids and tissues (McCurry et al., 2024). Similar to H. Eriksson's reports (Eriksson et al., 1969a, b), total levels of tetrahydroprogesterones (THPs) were significantly reduced in the feces of GF female mice compared with specific-pathogen-free (SPF) female mice (McCurry et al., 2024). Then, they further identify physiologically relevant substrates for 21-dehydroxlation through targeted corticoid profiling on human bile, and found that the average concentrations of tetrahydrodeoxycorticosterone (THDOC), e.g., 3α5αTHDOC and 3α5βTHDOC, in the abundant biliary corticoids were higher than other components (McCurry et al., 2024). This indicated that the gut bacteria are exposed to physiologically relevant concentrations of 3α5αTHDOC and 3α5βTHDOC. Because that 3α5αTHDOC levels in bile are higher in pregnant women, and its 21-dehydroxylated products have potential biological activities in the context of pregnancy and women's health (Cadeddu et al., 2020;Laatikainen and Karjalainen, 1972;Nappi et al., 2001), so, they next focused on 3α5αTHDOC, and hypothesized that 3α5αTHDOC can be 21-dehydroxylated into 3α5αTHP.To verify their hypothesis, Megan D. McCurry cultured the feces of pregnant GF and SPF mice in the presence of 3α5αTHDOC and measured the levels of progestin, and found that a large amount of 3α5αTHP and 3β5αTHP were produced from pregnant SPF mouse fecal slurries, while fecal slurries from pregnant GF mice did not produce THPs (McCurry et al., 2024). The culture pools with the human feces from healthy females and males 21-dehydroxylated 3α5αTHDOC, and moreover, the feces from pregnant human donors contained substantially higher levels of THPs than the feces from males and non-pregnant females. These indicate that both murine and human gut microbiome have the abilities to 21-dehydroxylate 3α5αTHDOC to produce progestins (McCurry et al., 2024). However, why is there more 21-dehydroxylation product of 3α5αTHDOC in the context of pregnancy? One reason is that 3α5αTHDOC levels in bile are higher in pregnant women (Laatikainen and Karjalainen, 1972).Dr What are the key mechanisms of induction of 21-dehydroxylation by E. coli? It has been reported that 21-dehydroxylation activity is inversely correlated with media redox potential (Noecker et al., 2023).According to this, Dr. Megan D. McCurry first examine the hypothesis that E. coli lowers the redox potential and thus promoting 21-dehydroxylation. Their data showed that lower redox potential does not enable 21-dehydroxylation in E. lenta 14A (McCurry et al., 2024). Second, they tested whether a 21-dehydroxylation-promoting factor was produced by EcN. The physical interactions between microbes can occur during cooperative metabolism, however, they found contact between the two species is not required to induce 21-dehydroxylation (McCurry et al., 2024). Third, they further test whether EcN release extracellular inducing factors, they found EcN syringe-filtered supernatant but not vacuum-filtered supernatants induced 21-dehydroxylation in E. lenta (McCurry et al., 2024). The basic difference between vacuum filtration and syringe filtration is that the dissolved gases are removed in the former one. They therefore hypothesized EcN may produce a gas that leads to 21dehydroxylation in E. lenta. In healthy individuals, the colonic gases are comprised of H2, carbon dioxide (CO2), methane (CH4), nitrogen (N2) and oxygen (O2) as well as several odiferous trace gases, and the former three are produced solely by colonic microbes (Carbonero et al., 2012). So, which gas or gases could be responsible for 21-dehydroxylation?It has been reported that E. coli can produce H2 during its stationary phase of growth (McDowall et al., 2014), which is when Dr. Megan D McCurry start to observe the substantial 21-dehydroxylation. Under anaerobic conditions, H2 can be used by microbes as an electron donor to drive sulfate reduction and methanogenesis (Wolf et al., 2016). Moreover, bile acids oxidation by E. lenta can be inhibited by H2, and E. lenta performs reductive metabolic reactions under high H2 partial pressure (Harris et al., 2018).Therefore, they hypothesized that H2 might provide the reducing equivalents required for this highly reductive transformation.The methylene blue (MB) is well known to react with an equimolar amount of H2 in the presence of platinum (Pt) or palladium to produce colorless reduced MB (leucomethylene blue, leucoMB), as follows: MB blue + 2H + + 2 e-→ leucoMB colorless (Seo et al., 2012) 19743 (McCurry et al., 2024). Therefore, they concluded that higher H2 produced in co-culture promote 21-dehydroxylation activity.So, which candidate genes that could be involved in 21-dehydroxylation in E. lenta? They performed comparative genomics analysis on the genome sequences of producer and non-producer strains from the Actinobacteria strain library, and identified gene cluster Elen_2451-Elen_2454 as the candidate genes for 21-dehydroxylation (McCurry et al., 2024). They co-cultured one of the top producer strains, E. lenta 14A, with EcN with or without tungstate, which is known to inhibit oxidoreductase activity of Elen_2453, and used homologously express and heterologously express the Elen_2451-2454 cluster, and confirmed that Elen_2451-2454 is responsible for 21-dehydroxylation in E. lenta (McCurry et al., 2024). The feces from pregnant subjects are enriched in Elen_2451-2454 cluster-containing bacteria, and the concentrations of THPs positively correlated with the abundance of Elen_2451-2454 homologs (McCurry et al., 2024). The feces from co-colonized female GF mice with producer strain E. lenta 14Aand EcN contained significantly more THPs than female GF controls (McCurry et al., 2024). Moreover, the co-colonized GF female mice with EcN and the cluster-containing strain E. lenta 14A, rather than the cluster-lacking strain E. lenta A2, produced higher levels of total THPs and 3α5βTHP in the feces (McCurry et al., 2024). Elen_2451 is a formate dehydrogenase family accessory protein FdhD, Elen_2452 is 4Fe-4S ferredoxin iron-sulfur binding domain protein, Elen_2453 is molybdopterin oxidoreductase, and Elen_2454 is an SPFH domain band 7 family protein (McCurry et al., 2024).However, we are still not clear about enzymatic mechanisms by which the above cluster regulate 21dehydroxylation. Discussion: There is still long way to go for endogenous H2 in female healthThe ovarian hormone (progesterone and estrogen) levels increase over 100-fold during pregnancy, and consequently, neurosteroid allopregnanolone (also as brexanolone, 3α5αTHP) is also elevated in the brain (Maguire and Mody, 2008;Mody, 2019). The GABAAR expression in the brain is reduced by neurosteroids during pregnancy to prevent sedation (Maguire and Mody, 2008;Mody, 2019;Nemeroff, 2008). Upon parturition, hormones rapidly return to pre-pregnancy levels, but the expression of GABAAR may take time to recover (Mody, 2019). Therefore, a long-lasting mismatch between neurosteroid levels and GABAAR numbers may underlie postpartum depression (Mody, 2019). The serum levels of allopregnanolone were detectable postpartum and were significantly decreased in women with maternity "blues." (Nappi et al., 2001). Brexanolone is a first Food and Drug Administration (FDA)-indicated drug for postpartum depression (Wisner et al., 2019). The gut bacteria both consume and produce H2, and the remaining net H2 in a fecal culture from donor F2 was sufficient to complete 21-dehydroxylation in co-culture and produced THPs (McCurry et al., 2024). The THPs, such as 3α5αTHP, 3α5βTHP, 3β5αTHP, and 3β5βTHP, are two orders of magnitude higher in feces from pregnant people in the third trimester compared with the feces of male and nonpregnant female subjects (McCurry et al., 2024). However, we are unclear about the changes in levels of gastrointestinal derived THPs in pregnant women with postpartum depression before and after delivery. We don't know whether progestins produced by E. lenta remain in the gastrointestinal tract or are absorbed into enterohepatic or systemic circulation (McCurry et al., 2024). Thus, it is not clear whether gut-derived THPs can affect postpartum depression (Figure 1). Supplement of exogenous H2 protected against depression in mice (Zhang et al., 2016), if THPs can be absorbed into enterohepatic or systemic circulation and have effects on depression, it is difficult to distinguish the observed effects of exogenous H2 on depression are caused directly by H2 or indirectly by produced THPs, or by the synergistic effects of H2 and THPs (Figure 1).Sex is a science of cutting edge but bathed in mystery, and it is a fundamental pleasure and quality-oflife issue (Zhang et al., 2021). H2 has been emerged as a novel bioactive molecule involved in modulating sexual organs homeostasis (Zhang et al., 2021). Supplement of exogenous H2 can improve erectile dysfunction in diabetic rats model (Fan et al., 2013), alleviate polycystic ovary syndrome (Makav et al., 2023), chemotherapy-induced ovarian injury (Meng et al., 2015), uterine inflammation (Nakano et al., 2018), mastitis (Geng et al., 2022) and breast cancer (Frajese et al., 2018;Zhang et al., 2019), and postmenopausal osteoporosis (Wu et al., 2019). It also has the effect of vasodilation, and attenuates chronic intermittent hypoxia-induced hypertension in rats (Guan et al., 2019;Zheng and Yu, 2012). However, the effects of endogenous H2 on female health, such as post-menopausal endothelial dysfunction, osteoporosis after menopause, normal breast development and mastitis, ovarian function, uterine inflammation, vaginal microbiota, and clitoral erection are unclear (Figure 1). To answer the effects of endogenous H2 on female health, we should confirm biological distributions of endogenous H2 (and its related metabolites, such as THPs) produced by microbiota, and analyze the changes of endogenous H2 (and its related metabolites, such as THPs) and microbiota in blood and feces and organs/tissues pre-and post-disease (or compare between normal individuals and patients). The animal models should be used to answer the causal relationship between endogenous H2 and female health.Moreover, the strategies to reduce endogenous H2 and increase endogenous H2 should be employed.Reducing endogenous H2 is typically achieved by systematically use of antibiotics, however, the effects of antibiotics are broad and not specific, both beneficial and harmful bacteria will be affected, therefore, this indiscriminate treatment cannot accurately answer the effects of endogenous H2. After killing gut microbial community by the antibiotics, it seems that supplementing with indicated strains can provide a relatively accurate answer to the physiological functions of an indicated bacteria (Kajiya et al., 2009).We should notice that it is not possible to have only one type of bacteria in the body, and the indicted bacteria can interact with one or more other bacteria or the host cells in multiple dimensions, thereby producing the whole physiological effects. The gut microbiota-derived endogenous H2 in female is necessary and sufficient to promote robust 21dehydroxylation, which is a key step for converting abundant biliary corticoids into progestins, e.g., allopregnanolone (3α5αTHP). The feces from pregnant human donors contained substantially higher levels of THPs than feces from males and non-pregnant females. The low serum levels of 3α5αTHP are related to symptoms of depression in late pregnancy. Allopregnanolone (3α5αTHP), also known as Brexanolone, is the first drug approved by the FDA to treat postpartum depression. 3α5αTHP, as a metabolite of gut microbiota, may also have essential impact on gut health. These are indirect effects of endogenous H2 dependent on the production of progestins, endogenous H2 may also have direct effects independent on progestins, e.g., affecting intestinal and brain health, etc. The exogenous H2 is emerged as a novel bioactive molecule involved in modulating sexual organs homeostasis and improving many reproductive diseases in animal models, including erectile dysfunction, polycystic ovary syndrome, chemotherapy-induced ovarian injury, uterine inflammation, mastitis and breast cancer, and postmenopausal osteoporosis. However, the effects of endogenous H2 on female health, such as post-menopausal endothelial dysfunction and osteoporosis, normal breast development and mastitis, ovarian function, uterine inflammation, vaginal microbiota, and clitoral erection are unclear.