Zi Chong granules improve hydroxyurea-induced decrease in ovarian reserve function

Zi Chong granules improve hydroxyurea-induced decrease in ovarian reserve function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Zi Chong granules improve hydroxyurea-induced decrease in ovarian reserve function Wenran Dong, Xinyu Guo, Hua Lu, Zhibin Liu, Lan Xie, Yi Liu, Qian Wan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3869320/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hydroxyurea (HU) is an antitumor drug. However, HU exposure is associated with diminished ovarian reserve (DOR). Zi Chong granules, a Chinese Medicine, can protect against DOR, but little is known regarding its underlying mechanisms of DOR treatment, and thus the target of the present study. Female KM mice were randomly divided into three groups: the control group (Con), the hydroxyurea group (HU), and the Zi Chong group (ZC). The ovaries and uterus of mice were examined histologically by H&E. The levels of anti-Mullerian hormone (AMH), estradiol (E 2 ), and progesterone (P) were quantified using ELISA kits. The number and quality of oocytes were assessed, and endometrial receptivity was evaluated by immunohistochemistry. 16S rDNA gene sequencing was used to analyze the composition and abundance of gut microbiome in feces, and non-targeted metabolomics was performed to detect serum metabolite profiles. Correlation analysis was performed to explore the relationships between different gut microbiota and differential metabolites. The results showed that ZC granules increased the number of primordial follicles in the ovaries, reduced excessive follicular atresia, restored low AMH, upregulated estrogen and progesterone secretion, and increased the number of mature oocytes after ovulation promotion. It also increased thickness of uterine endometrium and the number of glands, resulting in increased endometrial microvessel density (MVD), enhanced endometrial blood supply, reduced CD138 expression levels and endometrial inflammation. HU decreased the abundance of Lactobacillus spp. in mouse intestines and decreased arachidonic acid metabolism, tryptophan metabolism, spermidine and spermine biosynthesis, steroidogenesis and nicotinate and nicotinamide metabolism. Correlation analysis revealed that HU exerted its side effects by altering the gut microbiome and bacteria-derived metabolites, while ZC granules could reverse DOR partly depends on regulating gut microbiota and metabolites. Together, ZC granules may be a potential therapy for alleviating HU-induced DOR. Biological sciences/Drug discovery/Pharmacology Health sciences/Diseases/Reproductive disorders/Infertility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hydroxyurea (HU) is an oral chemotherapeutic agent that is widely used to treat various diseases, including sickle cell disease, chronic granulocytic leukemia, and other cancers. However, its clinical use can result in side effects, such as bone marrow suppression and anemia, as well as toxic effects on the human reproductive system, including diminished ovarian reserve (DOR) in women. A multicenter study of patients with sickle cell disease treated with HU and followed for 10 years found that its administration was strongly associated with low anti-Müllerian hormone (AMH) levels, leading to premature decline of ovarian reserve [ 1 ] . Another study found a significant decrease in the antral follicle counts (AFC) in the ovaries of women exposed to HU compared with controls [ 2 ] . HU also caused reduced ovarian reserve function, as well as causing embryonic cell death and embryonic malformations, in experimental animals [ 3 ] . However, although HU has been shown to decrease ovarian reserve function, the mechanism responsible for its toxic effects on the reproductive system remain unclear. Current treatments for DOR-related infertility caused by HU include pretreatment with drugs such as dehydroepiandrosterone and coenzyme Q10, controlled ovarian hyperstimulation, in vitro fertilization and embryo transfer, sex hormone therapy, bone marrow stem cell transplantation and ovarian injections of platelet-rich plasma to improve fertility, but evidence for the efficacies of these approaches is currently lacking and patient conception rates are generally < 40% [ 4 ] .There are thus no recognized drugs that can effectively mitigate HU reproductive toxicity, and strategies for improving HU-induced DOR are urgently required. Recent evidence suggests that the intestinal flora may be an important environmental factor contributing to abnormal reproductive system function. Fecal sex hormone concentrations in germ-free (GF) mice were lower than in specific pathogen-free (SPF) mice, However, after colonizing GF mice with the microbiota of SPF mice, the fecal hormone levels of the GF mice increased [ 5 ] . Multiple mechanistic studies have identified a possible correlation with β-glucuronidase production by gut microbes [ 6 ] .In addition, the gut microbiome and its metabolites, such as short-chain fatty acids, have been implicated in inflammation and immunity, and may likewise play a key role in reproductive disorders [ 7 ] . Elgart et al. found that Drosophila gut bacteria could inhibit egg formation, possibly associated with deficiency of intestinal acetate [ 8 ] . Zi Chong (ZC) granules traditional Chinese medicine formula consists of Rehmannia glutinosa (Gaertn.) DC., Dioscorea oppositifolia L. and Cornus officinalis Siebold & Zucc. , have been used in clinical practice for over 20 years and have shown efficacy in patients with DOR, mostly due to ovarian insufficiency caused by a decrease in the number or quality of oocytes, accompanied by reduced levels of sex hormones and AMH and a decrease in the number of sinus follicles (AFC). In the past ten years, our research group has continued to clarify the efficacy of Zi Chong granules in ovarian reserve function through clinical observations. Some studies have included infertile patients with premature ovarian failure, premature ovarian insufficiency, and endometriosis follicular development disorders. The controlled pre-post study was conducted. Compared with pre-treatment, Zi Chong granules promoted the growth and development of antral follicles and endometrial hyperplasia in patients. The improvement of ovarian dysfunction may be related to the improvement of blood supply to the ovaries and uterus [ 9 ] . In addition, for patients with premature ovarian insufficiency, Zi Chong granules can increase AMH, reduce FSH, and increase the number of antral follicles, suggesting a tendency to improve ovarian function [ 10 ] . The above studies all provide scientific basis for Zi Chong granules to improve ovarian function. A previous study found that ZC granules could promote estrogen secretion from granulosa cells and follicle development in mice [ 11 ] . ZC granules also induced the differentiation of human embryonic stem cells to granulosa cells, and the promotion of granulosa cell differentiation and development by this compound reinforced its positive regulatory effect on follicular development [ 12 ] . In addition, intestinal infusion of ZC granules improved follicular development in young rats and increased estradiol (E 2 ) levels in ovariectomized mice, showing estrogen-like effects [ 13 ] . These results suggest that ZC may improve reproductive function by regulating the intestinal microenvironment. However, it is not clear if ZC granules can mitigate HU-induced reproductive toxicity to restore fertility in DOR mice, and its potential mechanism of action via the intestinal bacteria also remains unclear. This study therefore aimed to observe the effects of HU gavage on the reproductive system and investigate the pharmacodynamic effects of ZC granules on reproductive function in mice. We also used 16s rRNA and targeted metabolomics techniques to detect the intestinal bacteria and blood metabolites in mice before and after HU gavage, and the differential intestinal bacteria and metabolic pathways regulated by ZC granules, in order to explore the mechanism of HU reproductive toxicity and the restoration of fertility by ZC granules following DOR. Results Chemical Compositions of Zi Chong granules Phytochemical analysis of the ZC granules was conducted using an UHPLC. The chemical compound peaks of ZC granules were identified by standards based on the specific retention time of each compound. Six main compounds were identified (Fig. 1 ), including Morroniside, Chlorogenic acid, Loganin, Hyperoside, Isoquercitrin, Acteoside. ZC reversed ovarian reserve function in mice exposed to HU According to our preliminary research, given the better treatment effect achieved with high-dose of ZC granules, mice in the high-dose ZC granules were selected for subsequent research (Supplementary Fig. 1). We investigated the effect of HU on ovarian reserve function in mice and the ability of ZC granules to reverse the effect. Oocytes in the CON and HU + ZC groups mostly comprised a single layer of flattened follicular cells in the periphery, i.e., primordial follicles, while oocytes in the HU group mostly had indistinct or absent structures and the zona pellucida was crinkled and depressed, suggesting that they were mostly atretic follicles. These observations suggested that HU accelerated abnormal follicular apoptosis in mice, while ZC granules prevented excessive follicular atresia and hindered the depletion of primordial follicles by HU (Fig. 2 a). The low AMH levels in the HU group compared with the CON group further indicated that HU reduced ovarian reserve, impaired ovarian function, and reduced oocyte quality. Compared with the HU group, AMH levels were increased in the HU + ZC group (Fig. 2 b), indicating that ZC granules restored ovarian reserve function. We also examined estrogen and progesterone levels to assess ovarian function. Serum E 2 levels were 34.66 ± 10.48 pmol/L in the CON group, but were decreased by about 37.19% in the HU group compared with the CON group, and increased about 6.71-fold in the HU + ZC group compared with the HU group. Progesterone levels showed a similar trend (Fig. 2 c and d). In addition, staging and counting of expelled oocytes after COH showed significant decreases in total oocytes (p < 0.05) and stage MII oocytes (p < 0.01) and a significant increase in abnormal oocytes (p < 0.05) in the HU group compared with the CON group, all of which effects were reversed in mice treated with ZC granules (p < 0.01, p < 0.05, p < 0.01, respectively) (Fig. 2 e-g). ZC improved endometrial receptivity in mice exposed to HU The endometrium is regulated by ovarian hormone levels, and low ovarian reserve function can lead to poor endometrial tolerance. We therefore examined the endometrium by HE staining. Compared with the HU group, the endometrium was thicker in the CON group (343.00 ± 70.09 µm), with more glands and dilated ducts and visible protein secretion, suggesting that HU resulted in a thinner endometrium. Compared with the HU group, mice in the HU + ZC group had a thicker endometrium with more glands but no significant dilation (Fig. 3 a), suggesting that ZC granules could partly restore endometrial thickness. The endometrial thickness and glandular areas were quantified, and the results were consistent with the trends indicated in the images, with a significant difference between the groups (p < 0.01) (Fig. 3 b and d). We further assessed the endometrial blood supply by determining the MVD, manifested as brownish-yellow granules in the cytoplasm of vascular endothelial cells. The granules were regular in morphology and uniformly distributed in the CON group, but were concentrated and decreased in density in the HU group. However, the granules showed normal morphology and increased density in the HU + ZC group compared with the HU group. Endothelial ERα levels detected by immunohistochemistry showed similar trends to MVD. We also carried out CD138 staining to assess the inflammatory status of the endometrium. CD138 was localized in the cytoplasm and cell membrane, with low expression in the endometrial mesenchyme in the CON group suggesting few plasma cells, compared with high expression in the HU group, suggesting more plasma cells, with reduced expression in the HU + ZC group compared with the HU group (Fig. 3 c). Zi Chong granules regulated the dysbiosis of gut microbiome species caused by hydroxyurea We investigated if the contribution of HU to the symptoms of DOR in mice and the improvement in symptoms following ZC treatment were related to the gut microbiota. We performed 16S rRNA sequencing of fecal samples from mice in the HU, HU + ZC, and CON groups to characterize the differences in their gut microbiomes. Species stacking plots for the gut microbiome at the phylum level (Fig. 4 a) showed that Bacteroidetes was the most common phylum in all groups. HU significantly decreased the proportion of thick-walled phyla compared with the CON group, and this change was reversed in the HU + ZC group. In addition, the proportion of Proteobacteria increased in the HU group relative to the CON group. At the family level (Fig. 4 b), the proportion of Lactobacillaceae was significantly decreased while Prevotellaceae were significantly increased in the HU group compared with the CON group, with no significant differences in proportions between the HU + ZC and CON groups. The most significant changes were in the abundance of Lactobacillaceae , and both filtered and log-transformed counts showed that ZC granules improved the HU-impaired levels of intestinal microorganisms in mice (Fig. 4 c). Overall, these results suggest that the addition of HU altered the composition of the gut microbiome in mice and that ZC granules reversed this effect. Zi Chong granules restored the structure of gut microbiome in DOR mice β-Diversity analysis of the gut microbiota based on OTU abundance was used to visualize the clustering of the microbial communities in each group. The box line plot of β-diversity (Fig. 5 a) showed that the HU + ZC group was more similar to the CON group than the HU group was to the CON group. The PCA plot (Fig. 5 b) showed that axis 1 (PCA1) explained 39.9% of the variability and axis 2 (PCA2) explained 17.2% of the variability. The PCA results showed that the gut microbial species differed significantly between the HU group and the CON group (r = 0.73, p < 0.01), with almost complete separation between the samples. The ANOSIM test results (Table 1 ) showed that the between-group differences were greater than the within-group differences, but the HU + ZC and CON groups had smaller R-values than the remaining two comparisons and showed a tighter aggregation. Table 1 ANOSIM results for β-diversity of bacterial communities in mice treated with HU, HU + ZC, and CON, respectively. HU/CON HU/HU + ZC HU + ZC/CON R-value 0.944 0.798 0.566 P-value 0.003 0.002 0.003 ZC granules restored the metabolome in DOR mice We determined the effect of ZC granules at the metabolite level by examining the serum metabolomes in each group of mice by PCA analysis (Fig. 6 a). The metabolome in the HU group was significantly separate from that in the CON group, while the metabolome in the HU + ZC group showed significant aggregation with the CON group(Table 2 ). This indicated that the CON group was more similar to the HU + ZC group at the metabolite level, suggesting that HU significantly altered the serum metabolome and that ZC granules had a significant restorative effect on the serum metabolome. A heatmap (Fig. 6 b) showed that HU significantly decreased the levels of 40 metabolites and increased the concentrations of about 10 metabolites, while treatment with ZC granules significantly restored the levels of these metabolites, these metabolites can also be viewed in volcano plots (Supplementary Fig. 2a, b). KEGG enrichment analysis confirmed that HU gavage significantly altered many metabolic pathways (Fig. 5 c), and that ZC granules restored these pathways, similar to levels in the CON group (Fig. 5 d). Table 2 ANOSIM results for PCA analysis of metabolome in mice treated with HU, HU + ZC, and CON, respectively. HU/CON HU/HU + ZC HU + ZC/CON R-value 0.537 0.687 0.13 P-value 0.004 0.004 0.016 Correlation between gut microbiomes and follicle counts To determine if HU-induced changes in gut microbes were responsible for DOR in mice, we examined the correlations of the different strains with the total numbers of oocytes, MII oocytes, and abnormal oocytes in the ovaries using Pearson’s correlation coefficient, and found that most of the significantly changed gut microbes were significantly correlated with the numbers of MII and abnormal oocytes (Fig. 7 ). Correlation between gut microbiomes and metabolome In order to determine which gut microorganisms were altered by HU and may thus affect metabolite levels, we examined the correlations between different microorganisms and serum metabolites using Pearson’s correlation coefficients, to further investigate the specific mechanism by which HU decreased ovarian reserve function. Notably, Lactobacillaceae showed a strong positive correlation with most metabolites (Fig. 8 ). Discussion The results of this study demonstrated significant differences between HU-treated and untreated mice, with HU reducing ovarian reserve function, lowering sex hormone levels, and inducing endometrial hypotolerance. We further investigated the mechanism responsible for the HU-induced decrease in ovarian reserve function in mice by analyzing the gut microbiome and serum metabolome. ZC granules effectively improved the HU-induced low ovarian reserve function in mice, and this improvement was closely related to alterations in metabolic pathways caused by improvements in intestinal microbiology. DOR is caused by a decrease in the number and/or quality of oocytes, leading to inadequate ovarian function and resulting in reduced fertility, accompanied by decreased AMH levels and a decreased AFC [ 14 ] . In this study, HE staining showed a decrease in the number of primordial follicles and increase in the number of atretic follicles in the ovary in HU-treated mice, as well as decreases in mature oocytes obtained by COH and an increase in abnormal oocytes, indicating a decrease in the number and quality of oocytes in mice. E 2 is a steroid that is produced from androstenedione in the follicular membrane in follicular cells through the metabolism of ovarian granulosa cells hormones [ 15 ] , while progesterone is an endogenous steroid secreted by ovarian luteal cells formed by follicles that have expelled oocytes after ovulation in the female ovary. The low serum E 2 and progesterone levels in HU-treated mice suggest that HU induced ovarian functional impairment in DOR mice. Overall, these results suggest that HU caused reproductive toxicity and DOR in mice. AMH is a member of the transforming growth factor-β family. It is expressed in follicular granulosa cells in the ovaries of reproductive-age women and plays an important role in follicular growth and development by controlling the formation of primary follicles, via inhibiting the recruitment of excess follicles by follicle-stimulating hormone [ 16 ] . Changes in AMH precede changes in follicle-stimulating hormone and E 2 , and changes in AFC can reflect the numbers of sinus and antral follicles in the ovary and indicate ovarian reserve function [ 17 – 18 ] . The decrease in serum AMH levels in mice after HU intervention corroborates the decline in ovarian reserve function induced by HU in mice. The uterus is a direct target organ for estrogen and progesterone. Estrogen and its receptor bind to the endometrium and subsequently activate various protein factors in the nucleus to initiate mRNA transcription, activate cell mitosis, and regulate endometrial thickness and function. The ER is the main driver of estrogen action, and ER-deficient mice were shown to have a dysplastic and infertile uterus [ 19 ] . Mice with epithelial-specific deletion of ER-α exhibited abnormal expression of estrogen-responsive genes and failure of implantation [ 20 ] . A prospective clinical study also found that women with low endometrial thickness exhibited abnormal ER expression patterns and differential expression of genes that bind to the ER, and indicated that these genes may play a role in implantation by affecting proliferation and angiogenesis [ 21 ] . MVD reflects the number of microvessels per unit volume and is determined by measuring specific antigens, such as the endothelial cell adhesion factor CD34, to count blood vessels and determine the MVD as a quantitative measure of angiogenesis [ 22 ] . The MVD in a tissue reflects the abundance of the blood supply to the site. CD138 is a recombinant protein, a plasma cell-specific indicator, and a transmembrane proteoglycan expressed in stratified epithelium versus simple epithelium, and elevated levels of CD138 reflect inflammatory changes in the endometrium that are detrimental to embryo implantation [ 23 ] . In the present study, endometrial thickness was reduced and the glandular area was decreased in HU-treated mice, suggesting endometrial insufficiency, possibly associated with reduced estrogen and progesterone levels due to low ovarian reserve function, and further reducing endometrial ER expression and inhibition of endometrial vascular growth, resulting in an inadequate endometrial blood supply and an inflammatory state. The administration of ZC granules effectively increased the number of oocytes, improved oocyte quality, increased AMH levels, reversed the declining ovarian reserve function, increased E 2 and progesterone levels, and restored the reproductive and hormone-secreting potential of the ovaries. The elevated hormone levels led to elevated expression of ERs in the endometrium, an enhanced blood supply, suppression of the HU-induced inflammatory state of the endometrium, and restoration of endometrial function, thus eventually restoring the reproductive ability of the mice. HU-induced DOR has been reported, but its effects on the gut microbiota and serum metabolome remain unclear. Drug intake is an important factor affecting the gut microbiota and metabolism, and HU, as a potent chemotherapeutic agent, may have important effects on these factors. We investigated this hypothesis by 16s sequencing of fecal samples from mice 21 days after drug administration and by analysis of the serum metabolome. Analysis of β-diversity based on Bray–Curtis distances showed that HU significantly affected the gut microorganisms in mice, as revealed by the significant separation of the HU and CON group samples in PCA. The results for the serum metabolome showed similar significant differences to the gut microbiome, indicating that HU significantly affected both the gut microbiome and the serum metabolome. Correlation analysis also revealed a significant correlation between the gut microbiome and the follicle count in mice, suggesting that changes in the gut microbiome and metabolome might contribute to the decrease in ovarian reserve function. ZC granules improved the HU-induced effects on the gut microbiota and metabolites, restoring their β-diversity close to the control group; the composition of the gut microflora and the corresponding metabolite levels following treatment with HU and ZC granules were not significantly different from the normal state, and were significantly different from those in HU-treated mice without ZC granules. The significant effects of HU and ZC granules suggest that the decrease in ovarian reserve function caused by HU exposure, and the amelioration of this condition by ZC granules, can be explained by changes in intestinal microbes and metabolites affecting ovarian pathology. We further analyzed the intestinal flora and metabolic differences in mice treated with HU. A species stacking plot showed that the proportion of Lactobacillaceae was most-significantly reduced in the HU group compared with the CON group. Lactobacillaceae are important probiotics in the intestinal tract and are relevant for intestinal ecological stability and health. Lactobacillus spp. have been reported to produce lactic acid, bacteriocins, and hydrogen peroxide in the endometrial microbial environment, to inhibit pathogens and establish a favorable environment for embryo implantation; however, the effect of Lactobacillus spp. in the intestinal flora on reproduction has not been studied [ 24 – 25 ] .We identified 897 metabolites in metabolome samples and analyzed the differences between the HU and CON groups by univariate analysis. We found that prostaglandin A2 (PGA2), nicotinamide riboside, niacinamide, serotonin, kynurenic acid xanthurenic acid, melatonin, hydroxykynurenamine, deoxycorticosterone, tetrahydrocortisone, corticosterone, progesterone, ornithine, 5'-methylthioadenosine, spermidine, and 15 other metabolites were significantly differentially expressed between the CON and HU groups. Enrichment analysis of these differential metabolites screened by t- tests identified several differential metabolic pathways, including arachidonic acid metabolism, tryptophan metabolism, spermidine and spermine biosynthesis, steroidogenesis, and nicotinate and nicotinamide metabolism, as associated with decreased ovarian reserve function. PGA2 plays a key role in arachidonic acid metabolism, via its G protein-coupled cell surface receptor, to affect oocyte maturation, ovulation, and volume expansion. PGA2 levels were significantly lower in the HU group compared with the CON group in the current study. Low levels of PGA2 inhibit the maturation of oocytes in the ovary, preventing secondary follicles from developing into mature follicles, thus leading to a decrease in the number of recruitable follicles in the ovarian cortical area and a decrease in oocyte quality, and thus to decreased ovarian reserve function [ 26 ] . Tryptophan metabolism may affect oocyte development and follicle quality through immunity. Tryptophan can be converted to melatonin, which can in turn delay the aging of oocytes in post-ovulatory mice via the SIRT1 MnSOD-dependent pathway, and can improve the inhibitory effect of bisphenol A on oocyte meiosis and fertilization and improve oocyte quality. HU downregulated the tryptophan metabolic pathway and reduced pathway activity, thus exerting a similar effect to downregulation of the arachidonic acid metabolism pathway [ 27 ] . Putrescine in the spermidine and spermine metabolic pathway is a precursor of spermine synthesis and has been reported to play an important role in granulosa cell luteinization. High levels of putrescine are also produced during ovulation in the ovary. HU decreased putrescine levels in this metabolic pathway and increased levels of spermine, leading to abnormal ovulation. In addition, the few mature follicles that are generated cannot be expelled, thus blocking ovulation and exacerbating the consequences of inadequate ovarian reserve function [ 28 ] . Steroid hormones are involved in many biological and physiological functions. Cholesterol is a precursor of steroid hormone synthesis. The levels of steroid hormones affect follicular growth and development. Wang et al. carried out bioinformatics analysis and showed that steroid-related genes were enriched in patients with reduced ovarian reserve function, suggesting that the steroid pathway may be related to this reduction in ovarian reserve. In the current study, we found that the steroid pathway was significantly affected in HU-treated mice, thus confirming that downregulation of this pathway reflected a decrease in ovarian reserve function. The downregulation of progesterone levels in the steroid pathway also indicated the downregulation of fertility, thus corroborating the decrease in ovarian reserve function [ 29 ] . Niacin and nicotinamide are two forms of water-soluble vitamin B3, also known as vitamin PP. As essential components of coenzymes, they are involved in anabolism and catabolism and play important roles in carbohydrate, lipid, and protein metabolism, and in the regulation of oxidative stress. Previous studies also indicated that niacin and nicotinamide metabolic pathways may be associated with decreased ovarian reserve function [ 24 ] . The above five metabolic pathways were either experimentally determined or bioinformatically predicted to be related to ovarian reserve function. The present results further corroborated the roles of these pathways as markers of ovarian reserve function by comparing their expression between DOR and normal mice. The coincidental deviations of the gut microbiome and metabolome during treatment with HU and ZC granules suggest that changes in the metabolome might be correlated with changes in gut microorganisms. We therefore performed a correlation analysis to identify significantly changed gut microorganisms and metabolites, and found that all metabolites, except serotonin, were significantly correlated with gut microbes, strongly suggesting that the HU-induced decrease in ovarian reserve function was mediated via changes in metabolic pathways caused by altered gut microbes. The correlation between Lactobacillus spp. and PGA2 was of particular interest, due to their significant HU-induced changes in the intestinal microbial community and metabolite pathways, respectively, and their strong (p < 0.01) positive correlation, as reported previously [ 30 , 31 ] . We therefore propose that HU gavage decreased the abundance of Lactobacillus spp. in the mouse intestine, which in turn resulted in downregulation of PGA2 levels. Low levels of PGA2 may then decrease arachidonic acid metabolism, resulting in inhibition of oocyte maturation in the ovary and a lack of progression to secondary follicles, ultimately resulting in a decrease in ovarian reserve function. In contrast, ZC granules can restore the abundance of Lactobacillus spp. and thus upregulate the activity of arachidonic acid metabolism, thereby restoring ovarian function. Conclusions This study provides novel evidence for the effects of HU on the gut microbiome and metabolome, demonstrating a correlation between changes in the gut microbiome and changes in metabolic pathways. In addition, we demonstrated that the HU-induced decrease in ovarian reserve function was likely to be due to changes in metabolic pathways caused by the gut microbiome. The decrease in ovarian reserve caused by HU could be successfully reversed by ZC granules. The results of this study will help to clarify the pathogenesis of reduced ovarian reserve function and to target clinical treatment. Methods Chemicals and reagents. The following chemicals and reagents were obtained from the noted sources: HU tablets (Qilu Pharmaceutical Co., Ltd., Shandong, China), pregnant mare serum gonadotrophin (PMSG; Solebo Technology Co., Ltd., Beijing, China; no.: 20190602), human chorionic gonadotropin (HCG; Lizhu Group Lizhu Pharmaceutical Factory, Zhuhai, China; no.: 11301010030C), mouse E 2 enzyme-linked immunosorbent assay (ELISA) kit (LMAI Bio, Shanghai, China; no.: LME2020021009), mouse AMH ELISA kit (LMAI Bio, Shanghai, China; no.: LME2020022010), mouse progesterone ELISA kit (LMAI Bio, Shanghai, China; no.: LME202002091), M2 culture medium(Nanjing Aibei Biotechnology Co.,Ltd,Nanjing,China;no.:1912A), anti-CD34 antibody (Boster, Wuhan, China; no.: BA3414), anti-ER-α antibody (Abcam, UK; no.: ab92516), anti-PR-α antibody (Abcam, UK; no.: ab101688), anti-CD138 antibody (Abcam, UK; no.: ab128936). Preparation of ZC granules. As shown in Table 3 , ZC granules comprises Rehmannia glutinosa (Gaertn.) Libosch ex Fisch.et Mey., Dioscorea opposita Thunb., Cornus officinalis Sieb. et Zucc., Lycium chinense Mill., Cuscuta chinensis Lam., Cervi cornu degelatinatum. The plant name has been checked with MPNS ( http://mpns.kew.org ). The herbs were mixed in a ratio of 3: 3: 3: 3: 3: 1 and treated with two rounds of extraction using boiling water. The extracts were then combined and filtered and concentrated under reduced pressure to produce a paste with a density of 1.30 g/cm 3 . The dregs were dried and finely powdered, and then mixed evenly with the paste. The mixture was sprayed into granules and dried. One dose yielded 8.4 g dry powder. The granules were resuspended in physiological saline at the final concentrations of 0.11g/ml,0.23g/ml and stored at 4℃ until use. Table 3 The compositions of ZC granules. Chinese name Accepted scientific name Family Plant part Batch number Amount(g) Shudihuang Rehmannia glutinosa (Gaertn.) Libosch ex Fisch.et Mey. Orobanchaceae Root 2005005S 15g Shanyao Dioscorea opposita Thunb Dioscoreaceae Root 2006003C 15g Shanzhuyu Cornus officinalis Sieb. et Zucc. Cornaceae Fruits 2006003C 15g Gouqizi Lycium chinense Mill. Solanaceae Fruits 2004006S 15g Tusizi Cuscuta chinensis Lam. Convolvulaceae Seed 2007001S 15g Lujiaoshuang Cervi cornu degelatinatum Cervidae Cornu cervi 2003001S 5g The dose selection of Zi Chong granules is based on previous work (Wang, 2018). According to the Chinese Pharmacopoeia, an adult weighting 60 kg should receive a dose of 8.2g of ZC granules per day, and the equivalent dose ratio based on body surface area converted between mice and human was 10. The mice in the ZCL and ZCH groups received approximately 10 and 20 times the dosage was used for adult respectively to intervene in repeated and continuous super ovulation induction in mice to observe the effect of Zi Chong granules on oocyte quality and explore the effects of different drug doses. Chemical composition analysis of ZC granules. The chromatographic analysis of ZC granules was carried out according to the 2015 edition of the Chinese Pharmacopoeia, performing on SHIMADZU LC-40BX3 (SHIMADZU, Japan), using Ultimate UHPLC XB-C18 column (4.6x100mm,1.8µm). The crushed material was obtained by grinding the Zi Chong granules, and after repeated mixing, 2 g of the material was taken. It was then added to 20 ml of 50% methanol, vortexed for 10 minutes, subjected to ultrasonic extraction for 30 minutes, centrifuged, and the supernatant was collected. The volume was adjusted to 20 ml, and the mixture was filtered for UHPLC analysis. The mobile phases were acetonitrile (A) and water (B). The gradient elution condition was as follows: 0–20 min, 5%-30%A; 20–30 min, 30%-100% A; 30–35 min 100% A; 35–37 min 5% A; 37–38 min 5% A; the detection wavelength was 254 nm, the flow rate was 0.5 ml/min, the column temperature was 35℃,the injection volume was 5µl. Animals. Seventy-two specific pathogen-free female Kunming mice, age 4–6 weeks, were purchased from Dashuo Experimental Animal Center (Sichuan, China; certificate no.: SCXK [Sichuan] 2020-030). All mice were housed at room temperature (22 ± 2℃), 45–55% humidity, and a 12 h light/day cycle, with ad libitum access to food and water. This study was approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (2021DL-002). All experiments were performed in accordance with relevant named guidelines and regulations, and the author complied with ARRIVE guidelines. Experimental design. The mice were divided randomly into three groups: control group (CON), model group (HU), and HU + ZC group. Mice in the HU and HU + ZC groups were gavaged with HU suspension at a dose of 400 mg/kg/day, and mice in the CON group were gavaged with an equal amount of saline, for a total of 21 days. Mice in the HU + ZC group were then gavaged with 2.5 g/kg ZC granules daily, and mice in the HU and CON groups were gavaged with the same amount of saline, for a total of 15 days. To ensure uniformity among the groups, mice in the HU and HU + ZC groups were injected intraperitoneally, after gavage, with PMSG (5 IU) + HCG (5 IU) to promote ovulation, and vaginal smears were checked in the CON group to determine the estrous cycle. Mice were anaesthetized with pentobarbital sodium salt (3 mg/ml), at 0.01ml/g mouse body weight, adjust with saline to 15ml volume and administered intraperitoneally. For the first procedure, 12 mice were selected randomly from each group. Mice in the HU and HU + ZC groups were sacrificed under anesthesia at 12 h and 72 h post-ovulation, and mice in the CON group were sacrificed before and after ovulation, respectively. Blood was collected and the ovaries and uterus were removed and fixed in 4% paraformaldehyde buffer for histopathological examination. The day before sacrifice, feces were collected from the mice using sterilized equipment and then frozen rapidly in liquid nitrogen for 16S rRNA gene sequencing. For the second procedure, six mice were selected randomly from each group. Post-ovulation mice in the HU and HU + ZC groups and CON mice in estrus stage were arranged in a 2:1 female: male ratio for mating. For the third procedure, six mice were selected randomly from each group. Mice in the HU and HU + ZC groups were anesthetized and sacrificed 16 h after ovulation, and mice in the CON group were anesthetized and sacrificed after detection of a vaginal plug. The bilateral oviducts were removed and placed in 35 mm dishes to obtain oocytes for staging and counting. ELISA. Serum concentrations of AMH, E 2 , and progesterone were detected by ELISA, according to the instructions in the respective kits. Oocyte staging and counting. Bilateral oviducts from mice in each group were put into 35 mm dishes and gradually torn to release all the oocytes. The oocytes were put into a culture dish containing hyaluronidase to separate the granulosa cells, and the oocytes separated from the granulosa cells were then put into a culture dish containing M2 culture medium and staged and counted under the microscope. MII stage oocytes were identified by uniform cytoplasm, small perivitelline gaps, discharged first polar body and smooth surface, and a clear and uniform zona pellucida. Abnormal oocytes were characterized by an irregular shape, refractive areas or large vacuoles in the cytoplasm, and a thickened or raised zona pellucida. Histopathological analysis of ovarian and uterus tissues. The tissues were fixed in 4% paraformaldehyde buffer, serially dehydrated, embedded in paraffin, sliced into 5-µm thick sections, and stained with hematoxylin-eosin (HE) and sealed with resin glue. The tissue samples were then photographed using a microscope imaging system, and primordial and atretic follicles were counted in the images using Image Pro Plus 6.0. Twenty different views were selected, and the endometrial thickness and glandular area were measured in each view and averaged. In addition, uterine tissue sections were stained immunohistochemically to determine the MVD and CD138 and ER expression. Sections were repaired using pH6 citrate antigen repair solution, rinsed with phosphate-buffered saline (PBS), incubated with peroxidase blocker (3% H 2 O 2 ), rinsed again with PBS, and incubated with primary antibodies at 37℃, followed by rinsing with PBS, incubation with solution A from a two-step anti-rabbit/mouse universal immunohistochemical assay kit (ChemMate™ Envision + HRP) at 37℃, and rinsing with PBS. The sections were then incubated with chromogenic DAB working solution, re-stained with hematoxylin, dehydrated through graded alcohols, transparentized with xylene, and sealed with neutral glue. 16s rDNA gene sequencing of feces. Total DNA was extracted from feces samples. Primers were designed according to the conserved region, and a sequencing junction was added at the end of the primers. Polymerase chain reaction amplification was then performed and the products were purified, quantified, and homogenized to form a sequencing library. The built library was subjected to quality control and then sequenced using an Illumina HiSeq 2500. The original image data were obtained and transformed into raw sequences (sequenced reads) by base calling. The observed operational taxonomic units (OTUs) were clustered according to 97% similarity sequences using USEARCH (version 11.0.667 http://www.drive5.com/usearch/ ) software. Principal coordinate analysis (PCA) was performed using the R package to show the β-diversity of the microbiome between samples. Bacterial classifications were compared among the groups using Wilcoxon’s rank sum test. Based on the 16S rDNA gene sequencing data, the relative abundance of microbial functional categories in the samples was predicted using the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) [ 32 ] software. Subsequently, the significance of the PICRUSt2 predicted results was tested using the limma package in R software for differential analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways (p < 0.05 was considered significant). Non-targeted metabolomics assays. A 100µL serum sample was taken and added to three times the volume of pre-cooled acetonitrile solution. The mixture was shaken at room temperature for 10 minutes, then placed the sample in the refrigerator at -20℃ for 30 mins. The samples were centrifuged at 14000g and 4℃ for 15 mins, and the supernatant was transferred to the mass spectrometry sample bottle. High-performance liquid chromatography-tandem mass spectrometry was performed using a Q Exactive mass spectrometer (Q Exactive, Thermo Scientific) equipped with a Hypesil GOLD (C18) column. Raw data were obtained in positive and negative ion modes, and data were extracted using Compound Discoverer v.1 software. Compound identification was searched using mHU + ZCloud, with ChemSpider. Combined with the built-in database search, the OTCML, a high-resolution mass spectrometry database of important ingredients in the mzVault library, was performed. Statistical analysis. Values for each group were expressed as mean and standard deviation and analyzed using SPSS 25.0 software. Comparisons among the three groups were performed by one-way ANOVA, and comparisons between two groups were performed using the least significant difference method. A p < 0.05 was considered significant. Bray–Curtis distance matrices were calculated using the R project Vegan package (version 2.5.3). Multivariate techniques, including PCA and Bray–Curtis distances, were carried out using the R project Vegan package (version 2.5.3) and plotted using R project ggplot2 package (version 2.2.1). Welch’s t -tests, Kruskal–Wallis H tests, and ANOSIM tests were carried out using the R project Vegan package (version 2.5.3). Correlations between the gut microbiome and metabolome were calculated using the R-package Psych and plotted using the pheatmap package. Abbreviations AFC Antral Follicle Counting AMH Anti-Müllerian Hormone HU Hydroxyurea COH Controlled ovarian hyperstimulate CON Control group DOR Diminished Ovarian Reserve ER Estrogen Receptor E 2 Estradiol GF Germ free HE Hematoxylin-Eosin KEGG Kyoto Encyclopedia of Genes and Genomes MVD Microvessel density OTUs Operational Taxonomic Units PCA Principal Coordinate Analysis PGA2 Prostaglandin A2 SPF Specific Pathogen-free ZC Zi Chong granules Declarations Acknowledgments Project supported by Hainan Province Clinical Medical Center. Author contributions W. D.: Investigation; Data curation; Writing-original draft. X. G.: Software; Data curation; Writing-original draft. H. L.: Project administration; Funding acquisition; Writing-review and editing. Z. L.: Writing-review and editing. L. X.: Data curation; Writing-review and editing. Y. L.: Data curation. Q. W.: Writing-review and editing. R. C.: Funding acquisition. S. L.: Methodology; Supervision. Competing interests The authors declare no competing interests. Data availability statements Illumina sequencing reads were uploaded to the SRA under accession number PRJNA1070711.Any other data supporting this study's conclusions are available from the corresponding author upon request. Funding This work was supported by the Ministry of Science and Technology of the People's Republic of China [grant number: 2018YFC1704305]. References Pecker, L. H., Hussain, S., Christianson, M. S., & Lanzkron, S. Hydroxycarbamide exposure and ovarian reserve in women with sickle cell disease in the Multicenter Study of Hydroxycarbamide. Br J Haematol. 191,880–887(2020). Pecker, L. H., Hussain, S., Mahesh, J., Varadhan, R., Christianson, M. S., & Lanzkron, S. Diminished ovarian reserve in young women with sickle cell anemia. Blood. 139, 1111–1115(2022) Sampson, M., et al. Perturbation of the developmental potential of preimplantation mouse embryos by hydroxyurea. Int J Environ Res Public Health .7,2033-44(2010). Expert group of Consensus on Clinical Diagnosis &Management of Diminished Ovarian Reserve, Reproductive Endocrinology &Fertility Preservation Section of Chinese Society on Fertility Preservation under Chinese Preventive Medicine Association. Consensus on clinical diagnosis and management of diminished ovarian reserve. J. Reprod. Med. (2020) Kamimura, I., et al. Gonadal steroid hormone secretion during the juvenile period depends on host-specific microbiota and contributes to the development of odor preference. Dev Psychobiol. 61,670–678(2019). Shen, R. L., Dang, X. Y., Dong, J. L., & Hu, X. Z. Effects of oat β-glucan and barley β-glucan on fecal characteristics, intestinal microflora, and intestinal bacterial metabolites in rats. J Agric Food Chem.60,11301–8(2012). He, S., et al. The Gut Microbiome and Sex Hormone-Related Diseases. Front Microbiol. 28,711137 (2021). Elgart, M., Stern, S., Salton, O., Gnainsky, Y., Heifetz, Y., Soen, Y. 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Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci U S A .90,11162-6(1993). Winuthayanon, W., Hewitt, S. C., Orvis, G. D., Behringer, R. R., Korach, K. S. Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses. Proc Natl Acad Sci U S A.107,19272–7(2010). Hawkins Bressler, L., et al. Poor Endometrial Proliferation After Clomiphene is Associated with Altered Estrogen Action. J Clin Endocrinol Metab.106,2547–2565(2021). Sun, C., et al. Tumor angiogenesis and bone metastasis - Correlation in invasive breast carcinoma. J Immunol Methods.452,46–52(2018). Chen, Y. Q., Fang, R. L., Luo, Y. N., & Luo, C. Q. Analysis of the diagnostic value of CD138 for chronic endometritis, the risk factors for the pathogenesis of chronic endometritis and the effect of chronic endometritis on pregnancy: a cohort study. BMC womens health.16,60(2016). Song, H., Qin, Q., Yuan, C., Li, H., Zhang, F., Fan, L. (2021). Metabolomic Profiling of Poor Ovarian Response Identifies Potential Predictive Biomarkers. Front Endocrinol (Lausanne) .23,774667(2021) Koedooder, R., et al. Identification and evaluation of the microbiome in the female and male reproductive tracts. Hum Reprod Update.25,298–325(2019). Liang, C., et al. UHPLC-MS-MS analysis of oxylipins metabolomics components of follicular fluid in infertile individuals with diminished ovarian reserve. Reprod Biol Endocrinol.19,143(2021). Xiao, J., Song, J., Sa, Y., Yuan, L., Guo, J., Sun, Z. The Mechanisms of Improving IVF Outcomes of Liu-Wei-Di-Huang Pill Acting on DOR Patients. Evid Based Complement Alternat Med.31,5183017(2020). Tao, Y., et al. Can peri-ovulatory putrescine supplementation improve egg quality in older infertile women? J Assist Reprod Genet. 36,395–402(2019). Chen, Y., et al. Arsenic exposure diminishes ovarian follicular reserve and induces abnormal steroidogenesis by DNA methylation. Ecotoxicol Environ Saf.241,113816(2022). Hu, Y., et al. Flavonoids in Amomum tsaoko Crevost et Lemarie Ameliorate Loperamide-Induced Constipation in Mice by Regulating Gut Microbiota and Related Metabolites. Int J Mol Sci.13,7191(2023). Burrello C., et al. Fecal Microbiota Transplantation Controls Murine Chronic Intestinal Inflammation by Modulating Immune Cell Functions and Gut Microbiota Composition. Cells. 28,517(2019). Vomstein, K., et al. Uterine microbiota plasticity during the menstrual cycle: Differences between healthy controls and patients with recurrent miscarriage or implantation failure. J Reprod Immunol. 151,103634(2022). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.png SupplementaryFigure2a.png SupplementaryFigure2b.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3869320","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":270208334,"identity":"333ae153-8a7f-4a7b-b91b-9a2a73a80150","order_by":0,"name":"Wenran Dong","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenran","middleName":"","lastName":"Dong","suffix":""},{"id":270208335,"identity":"e1317ac6-13ad-438c-93b6-5d9c9356390d","order_by":1,"name":"Xinyu Guo","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Guo","suffix":""},{"id":270208336,"identity":"4300bfe6-40fb-4e17-b117-c6c2f7d6db05","order_by":2,"name":"Hua Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYJACyQ8VpGqRljgDJNlI0SLB20aKFvn2ww9vSM67k88v33zsAWOODWEtBmfSjC0Ktz2znNnGlm7AuC2NCC0SPGwSktsOGxgc4zGTYNx2mAiHzQBq4Z0D1/KfsBaGGyAtDXAtB4hwGNAv1hLHnhlItqWlSSRuSybCYcAQu/mh5o4BP/PhYxIft9kR4TAIgLongWgNcC2jYBSMglEwCrABAKaIM+ZKjMOxAAAAAElFTkSuQmCC","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Lu","suffix":""},{"id":270208337,"identity":"e7581146-adc7-4000-a17f-af123d496609","order_by":3,"name":"Zhibin Liu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhibin","middleName":"","lastName":"Liu","suffix":""},{"id":270208338,"identity":"e7aebc8d-6a5a-421d-9448-ea92db0ef5f4","order_by":4,"name":"Lan Xie","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Xie","suffix":""},{"id":270208339,"identity":"b612f3cc-4916-4729-a95a-c72f8cc803ab","order_by":5,"name":"Yi Liu","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Liu","suffix":""},{"id":270208340,"identity":"17fab1e1-6d9c-48ca-bed9-b45e72589cb4","order_by":6,"name":"Qian Wan","email":"","orcid":"","institution":"Chengdu Neo-Life Hope Medical Testing Lab. 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The HPLC chromatograms of ZC granules, including Morroniside (1), Chlorogenic acid (2), Loganin (3), Hyperoside (4), Isoquercitrin (5), Acteoside (6).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/65b27bbc5381d44b8db70bdc.png"},{"id":50524783,"identity":"9d07e04d-7c93-4fe4-8dde-243e54b227bd","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1727045,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ZC granules on ovarian reserve function in HU-treated mice. (\u003cstrong\u003ea\u003c/strong\u003e) Ovaries stained with HE. Black arrows indicate primordial follicles; red arrows indicate atretic follicles. (\u003cstrong\u003eb\u003c/strong\u003e) AMH, (\u003cstrong\u003ec\u003c/strong\u003e) E\u003csub\u003e2\u003c/sub\u003e, and (\u003cstrong\u003ed\u003c/strong\u003e) progesterone levels. Numbers of (\u003cstrong\u003ee\u003c/strong\u003e) MII stage, (\u003cstrong\u003ef\u003c/strong\u003e) abnormal, and (\u003cstrong\u003eg\u003c/strong\u003e) total oocytes. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/7b62b1495d6505f5e4c17c53.png"},{"id":50524790,"identity":"164a8b66-cb94-4408-9a97-9bd642e8aa0d","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3176162,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ZC granules on endometrial receptivity in mice exposed to HU. (\u003cstrong\u003ea\u003c/strong\u003e) Endometrial HE staining. (\u003cstrong\u003eb\u003c/strong\u003e) MVD, ER α, and CD138 immunohistochemical staining. (\u003cstrong\u003ec\u003c/strong\u003e) Endometrial thickness and (\u003cstrong\u003ed\u003c/strong\u003e) endometrial gland area. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/92485f62839aa199e9ffd94e.png"},{"id":50524785,"identity":"ec254020-ce28-4acd-b6d6-cc9dd6304b71","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267940,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of gut microbiota in mice treated with HU, HU+ZC, and CON, respectively, at the (\u003cstrong\u003ea\u003c/strong\u003e) phylum level and (\u003cstrong\u003eb\u003c/strong\u003e) family level. (\u003cstrong\u003ec\u003c/strong\u003e) Abundance (filtered count and log-transformed count) of Lactobacillaceae in mice treated with HU, HU+ZC, and CON, respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/c94d07e66c2be20144e9f8a1.png"},{"id":50524962,"identity":"72ca6158-60fd-4eaf-97bf-2647ecb313a2","added_by":"auto","created_at":"2024-02-01 21:54:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90510,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Box plot of Bray–Curtis distance and (\u003cstrong\u003eb\u003c/strong\u003e) PCA plot of β-diversity of gut microbiome in mice treated with HU, HU+ZC, and CON, respectively.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/31d9e9f77e44b0a9743c8a33.png"},{"id":50524787,"identity":"8f9bd1ea-0b9e-48c5-8854-f94f4fd4263e","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":306354,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Metabolome PCA analysis in mice treated with HU, HU+ZC, and CON, respectively. (\u003cstrong\u003eb\u003c/strong\u003e) Differential metabolite cluster heatmap. (\u003cstrong\u003ec\u003c/strong\u003e) Differential metabolite enrichment analysis between HU and CON groups and (\u003cstrong\u003ed\u003c/strong\u003e) differential metabolite enrichment analysis between HU+ZC and CON groups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/691269dacfac347f608e98ea.png"},{"id":50524789,"identity":"6415bc63-6f5b-461e-8305-c347b5b4e22b","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6791062,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heat map between gut microbes and oocytes, calculated by Pearson correlation.The color gradient from red to blue represents the correlation between the microorganism and metabolite shifting from positive to negative, with darker colors indicating stronger correlations. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/8c1c93077297cf62d1736ba4.jpg"},{"id":50524794,"identity":"828568ff-aad2-422f-97d5-8326df8510e1","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6273483,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of correlation between gut microbes and metabolites, calculated by Pearson correlation.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/57bcfdd56eab547af0dee1b6.jpg"},{"id":55580767,"identity":"75541378-8ef3-42b2-912b-4de241919585","added_by":"auto","created_at":"2024-04-30 07:40:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3887809,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/0195e480-34b8-4722-afdb-0de14a3c03e0.pdf"},{"id":50524792,"identity":"3f288f67-cbf6-403a-82e7-56bcf451d1a8","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2238194,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/738c9949ac856ca2e67953e7.png"},{"id":50524786,"identity":"53dea1bb-490a-4dd9-ae69-26e0c050ea35","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":624441,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2a.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/5d7c0bb309ecb8b5b30c7357.png"},{"id":50524791,"identity":"b940b15d-8f2e-459d-ac6a-e7393a260783","added_by":"auto","created_at":"2024-02-01 21:46:19","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":497034,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2b.png","url":"https://assets-eu.researchsquare.com/files/rs-3869320/v1/1c78ca13fea228982fc5dc85.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zi Chong granules improve hydroxyurea-induced decrease in ovarian reserve function","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydroxyurea (HU) is an oral chemotherapeutic agent that is widely used to treat various diseases, including sickle cell disease, chronic granulocytic leukemia, and other cancers. However, its clinical use can result in side effects, such as bone marrow suppression and anemia, as well as toxic effects on the human reproductive system, including diminished ovarian reserve (DOR) in women. A multicenter study of patients with sickle cell disease treated with HU and followed for 10 years found that its administration was strongly associated with low anti-M\u0026uuml;llerian hormone (AMH) levels, leading to premature decline of ovarian reserve \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Another study found a significant decrease in the antral follicle counts (AFC) in the ovaries of women exposed to HU compared with controls \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. HU also caused reduced ovarian reserve function, as well as causing embryonic cell death and embryonic malformations, in experimental animals \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, although HU has been shown to decrease ovarian reserve function, the mechanism responsible for its toxic effects on the reproductive system remain unclear.\u003c/p\u003e \u003cp\u003eCurrent treatments for DOR-related infertility caused by HU include pretreatment with drugs such as dehydroepiandrosterone and coenzyme Q10, controlled ovarian hyperstimulation, in vitro fertilization and embryo transfer, sex hormone therapy, bone marrow stem cell transplantation and ovarian injections of platelet-rich plasma to improve fertility, but evidence for the efficacies of these approaches is currently lacking and patient conception rates are generally\u0026thinsp;\u0026lt;\u0026thinsp;40% \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.There are thus no recognized drugs that can effectively mitigate HU reproductive toxicity, and strategies for improving HU-induced DOR are urgently required.\u003c/p\u003e \u003cp\u003eRecent evidence suggests that the intestinal flora may be an important environmental factor contributing to abnormal reproductive system function. Fecal sex hormone concentrations in germ-free (GF) mice were lower than in specific pathogen-free (SPF) mice, However, after colonizing GF mice with the microbiota of SPF mice, the fecal hormone levels of the GF mice increased \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Multiple mechanistic studies have identified a possible correlation with β-glucuronidase production by gut microbes \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.In addition, the gut microbiome and its metabolites, such as short-chain fatty acids, have been implicated in inflammation and immunity, and may likewise play a key role in reproductive disorders \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Elgart et al. found that Drosophila gut bacteria could inhibit egg formation, possibly associated with deficiency of intestinal acetate \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eZi Chong (ZC) granules traditional Chinese medicine formula consists of \u003cem\u003eRehmannia glutinosa (Gaertn.) DC., Dioscorea oppositifolia L. and Cornus officinalis Siebold \u0026amp; Zucc.\u003c/em\u003e, have been used in clinical practice for over 20 years and have shown efficacy in patients with DOR, mostly due to ovarian insufficiency caused by a decrease in the number or quality of oocytes, accompanied by reduced levels of sex hormones and AMH and a decrease in the number of sinus follicles (AFC). In the past ten years, our research group has continued to clarify the efficacy of Zi Chong granules in ovarian reserve function through clinical observations. Some studies have included infertile patients with premature ovarian failure, premature ovarian insufficiency, and endometriosis follicular development disorders. The controlled pre-post study was conducted. Compared with pre-treatment, Zi Chong granules promoted the growth and development of antral follicles and endometrial hyperplasia in patients. The improvement of ovarian dysfunction may be related to the improvement of blood supply to the ovaries and uterus \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In addition, for patients with premature ovarian insufficiency, Zi Chong granules can increase AMH, reduce FSH, and increase the number of antral follicles, suggesting a tendency to improve ovarian function \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The above studies all provide scientific basis for Zi Chong granules to improve ovarian function. A previous study found that ZC granules could promote estrogen secretion from granulosa cells and follicle development in mice \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. ZC granules also induced the differentiation of human embryonic stem cells to granulosa cells, and the promotion of granulosa cell differentiation and development by this compound reinforced its positive regulatory effect on follicular development \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In addition, intestinal infusion of ZC granules improved follicular development in young rats and increased estradiol (E\u003csub\u003e2\u003c/sub\u003e) levels in ovariectomized mice, showing estrogen-like effects \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. These results suggest that ZC may improve reproductive function by regulating the intestinal microenvironment. However, it is not clear if ZC granules can mitigate HU-induced reproductive toxicity to restore fertility in DOR mice, and its potential mechanism of action via the intestinal bacteria also remains unclear.\u003c/p\u003e \u003cp\u003eThis study therefore aimed to observe the effects of HU gavage on the reproductive system and investigate the pharmacodynamic effects of ZC granules on reproductive function in mice. We also used 16s rRNA and targeted metabolomics techniques to detect the intestinal bacteria and blood metabolites in mice before and after HU gavage, and the differential intestinal bacteria and metabolic pathways regulated by ZC granules, in order to explore the mechanism of HU reproductive toxicity and the restoration of fertility by ZC granules following DOR.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemical Compositions of Zi Chong granules\u003c/h2\u003e \u003cp\u003ePhytochemical analysis of the ZC granules was conducted using an UHPLC. The chemical compound peaks of ZC granules were identified by standards based on the specific retention time of each compound. Six main compounds were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), including \u003cem\u003eMorroniside, Chlorogenic acid, Loganin, Hyperoside, Isoquercitrin, Acteoside.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eZC reversed ovarian reserve function in mice exposed to HU\u003c/h2\u003e \u003cp\u003eAccording to our preliminary research, given the better treatment effect achieved with high-dose of ZC granules, mice in the high-dose ZC granules were selected for subsequent research (Supplementary Fig.\u0026nbsp;1). We investigated the effect of HU on ovarian reserve function in mice and the ability of ZC granules to reverse the effect. Oocytes in the CON and HU\u0026thinsp;+\u0026thinsp;ZC groups mostly comprised a single layer of flattened follicular cells in the periphery, i.e., primordial follicles, while oocytes in the HU group mostly had indistinct or absent structures and the zona pellucida was crinkled and depressed, suggesting that they were mostly atretic follicles. These observations suggested that HU accelerated abnormal follicular apoptosis in mice, while ZC granules prevented excessive follicular atresia and hindered the depletion of primordial follicles by HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The low AMH levels in the HU group compared with the CON group further indicated that HU reduced ovarian reserve, impaired ovarian function, and reduced oocyte quality. Compared with the HU group, AMH levels were increased in the HU\u0026thinsp;+\u0026thinsp;ZC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating that ZC granules restored ovarian reserve function. We also examined estrogen and progesterone levels to assess ovarian function. Serum E\u003csub\u003e2\u003c/sub\u003e levels were 34.66\u0026thinsp;\u0026plusmn;\u0026thinsp;10.48 pmol/L in the CON group, but were decreased by about 37.19% in the HU group compared with the CON group, and increased about 6.71-fold in the HU\u0026thinsp;+\u0026thinsp;ZC group compared with the HU group. Progesterone levels showed a similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). In addition, staging and counting of expelled oocytes after COH showed significant decreases in total oocytes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and stage MII oocytes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a significant increase in abnormal oocytes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the HU group compared with the CON group, all of which effects were reversed in mice treated with ZC granules (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eZC improved endometrial receptivity in mice exposed to HU\u003c/h2\u003e \u003cp\u003eThe endometrium is regulated by ovarian hormone levels, and low ovarian reserve function can lead to poor endometrial tolerance. We therefore examined the endometrium by HE staining. Compared with the HU group, the endometrium was thicker in the CON group (343.00\u0026thinsp;\u0026plusmn;\u0026thinsp;70.09 \u0026micro;m), with more glands and dilated ducts and visible protein secretion, suggesting that HU resulted in a thinner endometrium. Compared with the HU group, mice in the HU\u0026thinsp;+\u0026thinsp;ZC group had a thicker endometrium with more glands but no significant dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), suggesting that ZC granules could partly restore endometrial thickness. The endometrial thickness and glandular areas were quantified, and the results were consistent with the trends indicated in the images, with a significant difference between the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and d). We further assessed the endometrial blood supply by determining the MVD, manifested as brownish-yellow granules in the cytoplasm of vascular endothelial cells. The granules were regular in morphology and uniformly distributed in the CON group, but were concentrated and decreased in density in the HU group. However, the granules showed normal morphology and increased density in the HU\u0026thinsp;+\u0026thinsp;ZC group compared with the HU group. Endothelial ERα levels detected by immunohistochemistry showed similar trends to MVD. We also carried out CD138 staining to assess the inflammatory status of the endometrium. CD138 was localized in the cytoplasm and cell membrane, with low expression in the endometrial mesenchyme in the CON group suggesting few plasma cells, compared with high expression in the HU group, suggesting more plasma cells, with reduced expression in the HU\u0026thinsp;+\u0026thinsp;ZC group compared with the HU group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eZi Chong granules regulated the dysbiosis of gut microbiome species caused by hydroxyurea\u003c/h2\u003e \u003cp\u003eWe investigated if the contribution of HU to the symptoms of DOR in mice and the improvement in symptoms following ZC treatment were related to the gut microbiota. We performed 16S rRNA sequencing of fecal samples from mice in the HU, HU\u0026thinsp;+\u0026thinsp;ZC, and CON groups to characterize the differences in their gut microbiomes. Species stacking plots for the gut microbiome at the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) showed that \u003cem\u003eBacteroidetes\u003c/em\u003e was the most common phylum in all groups. HU significantly decreased the proportion of thick-walled phyla compared with the CON group, and this change was reversed in the HU\u0026thinsp;+\u0026thinsp;ZC group. In addition, the proportion of \u003cem\u003eProteobacteria\u003c/em\u003e increased in the HU group relative to the CON group. At the family level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), the proportion of \u003cem\u003eLactobacillaceae\u003c/em\u003e was significantly decreased while \u003cem\u003ePrevotellaceae\u003c/em\u003e were significantly increased in the HU group compared with the CON group, with no significant differences in proportions between the HU\u0026thinsp;+\u0026thinsp;ZC and CON groups. The most significant changes were in the abundance of \u003cem\u003eLactobacillaceae\u003c/em\u003e, and both filtered and log-transformed counts showed that ZC granules improved the HU-impaired levels of intestinal microorganisms in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Overall, these results suggest that the addition of HU altered the composition of the gut microbiome in mice and that ZC granules reversed this effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eZi Chong granules restored the structure of gut microbiome in DOR mice\u003c/h2\u003e \u003cp\u003eβ-Diversity analysis of the gut microbiota based on OTU abundance was used to visualize the clustering of the microbial communities in each group. The box line plot of β-diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) showed that the HU\u0026thinsp;+\u0026thinsp;ZC group was more similar to the CON group than the HU group was to the CON group. The PCA plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) showed that axis 1 (PCA1) explained 39.9% of the variability and axis 2 (PCA2) explained 17.2% of the variability. The PCA results showed that the gut microbial species differed significantly between the HU group and the CON group (r\u0026thinsp;=\u0026thinsp;0.73, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with almost complete separation between the samples. The ANOSIM test results (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that the between-group differences were greater than the within-group differences, but the HU\u0026thinsp;+\u0026thinsp;ZC and CON groups had smaller R-values than the remaining two comparisons and showed a tighter aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOSIM results for β-diversity of bacterial communities in mice treated with HU, HU\u0026thinsp;+\u0026thinsp;ZC, and CON, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHU/CON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHU/HU\u0026thinsp;+\u0026thinsp;ZC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHU\u0026thinsp;+\u0026thinsp;ZC/CON\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.566\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eZC granules restored the metabolome in DOR mice\u003c/h2\u003e \u003cp\u003eWe determined the effect of ZC granules at the metabolite level by examining the serum metabolomes in each group of mice by PCA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The metabolome in the HU group was significantly separate from that in the CON group, while the metabolome in the HU\u0026thinsp;+\u0026thinsp;ZC group showed significant aggregation with the CON group(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This indicated that the CON group was more similar to the HU\u0026thinsp;+\u0026thinsp;ZC group at the metabolite level, suggesting that HU significantly altered the serum metabolome and that ZC granules had a significant restorative effect on the serum metabolome. A heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) showed that HU significantly decreased the levels of 40 metabolites and increased the concentrations of about 10 metabolites, while treatment with ZC granules significantly restored the levels of these metabolites, these metabolites can also be viewed in volcano plots (Supplementary Fig.\u0026nbsp;2a, b). KEGG enrichment analysis confirmed that HU gavage significantly altered many metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), and that ZC granules restored these pathways, similar to levels in the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOSIM results for PCA analysis of metabolome in mice treated with HU, HU\u0026thinsp;+\u0026thinsp;ZC, and CON, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHU/CON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHU/HU\u0026thinsp;+\u0026thinsp;ZC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHU\u0026thinsp;+\u0026thinsp;ZC/CON\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between gut microbiomes and follicle counts\u003c/h2\u003e \u003cp\u003eTo determine if HU-induced changes in gut microbes were responsible for DOR in mice, we examined the correlations of the different strains with the total numbers of oocytes, MII oocytes, and abnormal oocytes in the ovaries using Pearson\u0026rsquo;s correlation coefficient, and found that most of the significantly changed gut microbes were significantly correlated with the numbers of MII and abnormal oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation between gut microbiomes and metabolome\u003c/h2\u003e \u003cp\u003eIn order to determine which gut microorganisms were altered by HU and may thus affect metabolite levels, we examined the correlations between different microorganisms and serum metabolites using Pearson\u0026rsquo;s correlation coefficients, to further investigate the specific mechanism by which HU decreased ovarian reserve function. Notably, \u003cem\u003eLactobacillaceae\u003c/em\u003e showed a strong positive correlation with most metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study demonstrated significant differences between HU-treated and untreated mice, with HU reducing ovarian reserve function, lowering sex hormone levels, and inducing endometrial hypotolerance. We further investigated the mechanism responsible for the HU-induced decrease in ovarian reserve function in mice by analyzing the gut microbiome and serum metabolome. ZC granules effectively improved the HU-induced low ovarian reserve function in mice, and this improvement was closely related to alterations in metabolic pathways caused by improvements in intestinal microbiology.\u003c/p\u003e \u003cp\u003eDOR is caused by a decrease in the number and/or quality of oocytes, leading to inadequate ovarian function and resulting in reduced fertility, accompanied by decreased AMH levels and a decreased AFC \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In this study, HE staining showed a decrease in the number of primordial follicles and increase in the number of atretic follicles in the ovary in HU-treated mice, as well as decreases in mature oocytes obtained by COH and an increase in abnormal oocytes, indicating a decrease in the number and quality of oocytes in mice. E\u003csub\u003e2\u003c/sub\u003e is a steroid that is produced from androstenedione in the follicular membrane in follicular cells through the metabolism of ovarian granulosa cells hormones \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, while progesterone is an endogenous steroid secreted by ovarian luteal cells formed by follicles that have expelled oocytes after ovulation in the female ovary. The low serum E\u003csub\u003e2\u003c/sub\u003e and progesterone levels in HU-treated mice suggest that HU induced ovarian functional impairment in DOR mice. Overall, these results suggest that HU caused reproductive toxicity and DOR in mice. AMH is a member of the transforming growth factor-β family. It is expressed in follicular granulosa cells in the ovaries of reproductive-age women and plays an important role in follicular growth and development by controlling the formation of primary follicles, via inhibiting the recruitment of excess follicles by follicle-stimulating hormone \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Changes in AMH precede changes in follicle-stimulating hormone and E\u003csub\u003e2\u003c/sub\u003e, and changes in AFC can reflect the numbers of sinus and antral follicles in the ovary and indicate ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The decrease in serum AMH levels in mice after HU intervention corroborates the decline in ovarian reserve function induced by HU in mice.\u003c/p\u003e \u003cp\u003eThe uterus is a direct target organ for estrogen and progesterone. Estrogen and its receptor bind to the endometrium and subsequently activate various protein factors in the nucleus to initiate mRNA transcription, activate cell mitosis, and regulate endometrial thickness and function. The ER is the main driver of estrogen action, and ER-deficient mice were shown to have a dysplastic and infertile uterus \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Mice with epithelial-specific deletion of ER-α exhibited abnormal expression of estrogen-responsive genes and failure of implantation \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. A prospective clinical study also found that women with low endometrial thickness exhibited abnormal ER expression patterns and differential expression of genes that bind to the ER, and indicated that these genes may play a role in implantation by affecting proliferation and angiogenesis \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. MVD reflects the number of microvessels per unit volume and is determined by measuring specific antigens, such as the endothelial cell adhesion factor CD34, to count blood vessels and determine the MVD as a quantitative measure of angiogenesis \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The MVD in a tissue reflects the abundance of the blood supply to the site. CD138 is a recombinant protein, a plasma cell-specific indicator, and a transmembrane proteoglycan expressed in stratified epithelium versus simple epithelium, and elevated levels of CD138 reflect inflammatory changes in the endometrium that are detrimental to embryo implantation \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In the present study, endometrial thickness was reduced and the glandular area was decreased in HU-treated mice, suggesting endometrial insufficiency, possibly associated with reduced estrogen and progesterone levels due to low ovarian reserve function, and further reducing endometrial ER expression and inhibition of endometrial vascular growth, resulting in an inadequate endometrial blood supply and an inflammatory state.\u003c/p\u003e \u003cp\u003eThe administration of ZC granules effectively increased the number of oocytes, improved oocyte quality, increased AMH levels, reversed the declining ovarian reserve function, increased E\u003csub\u003e2\u003c/sub\u003e and progesterone levels, and restored the reproductive and hormone-secreting potential of the ovaries. The elevated hormone levels led to elevated expression of ERs in the endometrium, an enhanced blood supply, suppression of the HU-induced inflammatory state of the endometrium, and restoration of endometrial function, thus eventually restoring the reproductive ability of the mice.\u003c/p\u003e \u003cp\u003eHU-induced DOR has been reported, but its effects on the gut microbiota and serum metabolome remain unclear. Drug intake is an important factor affecting the gut microbiota and metabolism, and HU, as a potent chemotherapeutic agent, may have important effects on these factors. We investigated this hypothesis by 16s sequencing of fecal samples from mice 21 days after drug administration and by analysis of the serum metabolome. Analysis of β-diversity based on Bray\u0026ndash;Curtis distances showed that HU significantly affected the gut microorganisms in mice, as revealed by the significant separation of the HU and CON group samples in PCA. The results for the serum metabolome showed similar significant differences to the gut microbiome, indicating that HU significantly affected both the gut microbiome and the serum metabolome. Correlation analysis also revealed a significant correlation between the gut microbiome and the follicle count in mice, suggesting that changes in the gut microbiome and metabolome might contribute to the decrease in ovarian reserve function. ZC granules improved the HU-induced effects on the gut microbiota and metabolites, restoring their β-diversity close to the control group; the composition of the gut microflora and the corresponding metabolite levels following treatment with HU and ZC granules were not significantly different from the normal state, and were significantly different from those in HU-treated mice without ZC granules. The significant effects of HU and ZC granules suggest that the decrease in ovarian reserve function caused by HU exposure, and the amelioration of this condition by ZC granules, can be explained by changes in intestinal microbes and metabolites affecting ovarian pathology.\u003c/p\u003e \u003cp\u003eWe further analyzed the intestinal flora and metabolic differences in mice treated with HU. A species stacking plot showed that the proportion of \u003cem\u003eLactobacillaceae\u003c/em\u003e was most-significantly reduced in the HU group compared with the CON group. \u003cem\u003eLactobacillaceae\u003c/em\u003e are important probiotics in the intestinal tract and are relevant for intestinal ecological stability and health. \u003cem\u003eLactobacillus spp.\u003c/em\u003e have been reported to produce lactic acid, bacteriocins, and hydrogen peroxide in the endometrial microbial environment, to inhibit pathogens and establish a favorable environment for embryo implantation; however, the effect of \u003cem\u003eLactobacillus spp.\u003c/em\u003e in the intestinal flora on reproduction has not been studied \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.We identified 897 metabolites in metabolome samples and analyzed the differences between the HU and CON groups by univariate analysis. We found that prostaglandin A2 (PGA2), nicotinamide riboside, niacinamide, serotonin, kynurenic acid xanthurenic acid, melatonin, hydroxykynurenamine, deoxycorticosterone, tetrahydrocortisone, corticosterone, progesterone, ornithine, 5'-methylthioadenosine, spermidine, and 15 other metabolites were significantly differentially expressed between the CON and HU groups. Enrichment analysis of these differential metabolites screened by \u003cem\u003et-\u003c/em\u003etests identified several differential metabolic pathways, including arachidonic acid metabolism, tryptophan metabolism, spermidine and spermine biosynthesis, steroidogenesis, and nicotinate and nicotinamide metabolism, as associated with decreased ovarian reserve function.\u003c/p\u003e \u003cp\u003ePGA2 plays a key role in arachidonic acid metabolism, via its G protein-coupled cell surface receptor, to affect oocyte maturation, ovulation, and volume expansion. PGA2 levels were significantly lower in the HU group compared with the CON group in the current study. Low levels of PGA2 inhibit the maturation of oocytes in the ovary, preventing secondary follicles from developing into mature follicles, thus leading to a decrease in the number of recruitable follicles in the ovarian cortical area and a decrease in oocyte quality, and thus to decreased ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTryptophan metabolism may affect oocyte development and follicle quality through immunity. Tryptophan can be converted to melatonin, which can in turn delay the aging of oocytes in post-ovulatory mice via the SIRT1 MnSOD-dependent pathway, and can improve the inhibitory effect of bisphenol A on oocyte meiosis and fertilization and improve oocyte quality. HU downregulated the tryptophan metabolic pathway and reduced pathway activity, thus exerting a similar effect to downregulation of the arachidonic acid metabolism pathway \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePutrescine in the spermidine and spermine metabolic pathway is a precursor of spermine synthesis and has been reported to play an important role in granulosa cell luteinization. High levels of putrescine are also produced during ovulation in the ovary. HU decreased putrescine levels in this metabolic pathway and increased levels of spermine, leading to abnormal ovulation. In addition, the few mature follicles that are generated cannot be expelled, thus blocking ovulation and exacerbating the consequences of inadequate ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSteroid hormones are involved in many biological and physiological functions. Cholesterol is a precursor of steroid hormone synthesis. The levels of steroid hormones affect follicular growth and development. Wang et al. carried out bioinformatics analysis and showed that steroid-related genes were enriched in patients with reduced ovarian reserve function, suggesting that the steroid pathway may be related to this reduction in ovarian reserve. In the current study, we found that the steroid pathway was significantly affected in HU-treated mice, thus confirming that downregulation of this pathway reflected a decrease in ovarian reserve function. The downregulation of progesterone levels in the steroid pathway also indicated the downregulation of fertility, thus corroborating the decrease in ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNiacin and nicotinamide are two forms of water-soluble vitamin B3, also known as vitamin PP. As essential components of coenzymes, they are involved in anabolism and catabolism and play important roles in carbohydrate, lipid, and protein metabolism, and in the regulation of oxidative stress. Previous studies also indicated that niacin and nicotinamide metabolic pathways may be associated with decreased ovarian reserve function \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe above five metabolic pathways were either experimentally determined or bioinformatically predicted to be related to ovarian reserve function. The present results further corroborated the roles of these pathways as markers of ovarian reserve function by comparing their expression between DOR and normal mice.\u003c/p\u003e \u003cp\u003eThe coincidental deviations of the gut microbiome and metabolome during treatment with HU and ZC granules suggest that changes in the metabolome might be correlated with changes in gut microorganisms. We therefore performed a correlation analysis to identify significantly changed gut microorganisms and metabolites, and found that all metabolites, except serotonin, were significantly correlated with gut microbes, strongly suggesting that the HU-induced decrease in ovarian reserve function was mediated via changes in metabolic pathways caused by altered gut microbes. The correlation between \u003cem\u003eLactobacillus spp.\u003c/em\u003e and PGA2 was of particular interest, due to their significant HU-induced changes in the intestinal microbial community and metabolite pathways, respectively, and their strong (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) positive correlation, as reported previously \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. We therefore propose that HU gavage decreased the abundance of \u003cem\u003eLactobacillus spp.\u003c/em\u003e in the mouse intestine, which in turn resulted in downregulation of PGA2 levels. Low levels of PGA2 may then decrease arachidonic acid metabolism, resulting in inhibition of oocyte maturation in the ovary and a lack of progression to secondary follicles, ultimately resulting in a decrease in ovarian reserve function. In contrast, ZC granules can restore the abundance of \u003cem\u003eLactobacillus spp.\u003c/em\u003e and thus upregulate the activity of arachidonic acid metabolism, thereby restoring ovarian function.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides novel evidence for the effects of HU on the gut microbiome and metabolome, demonstrating a correlation between changes in the gut microbiome and changes in metabolic pathways. In addition, we demonstrated that the HU-induced decrease in ovarian reserve function was likely to be due to changes in metabolic pathways caused by the gut microbiome. The decrease in ovarian reserve caused by HU could be successfully reversed by ZC granules. The results of this study will help to clarify the pathogenesis of reduced ovarian reserve function and to target clinical treatment.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eChemicals and reagents.\u003c/b\u003e The following chemicals and reagents were obtained from the noted sources: HU tablets (Qilu Pharmaceutical Co., Ltd., Shandong, China), pregnant mare serum gonadotrophin (PMSG; Solebo Technology Co., Ltd., Beijing, China; no.: 20190602), human chorionic gonadotropin (HCG; Lizhu Group Lizhu Pharmaceutical Factory, Zhuhai, China; no.: 11301010030C), mouse E\u003csub\u003e2\u003c/sub\u003e enzyme-linked immunosorbent assay (ELISA) kit (LMAI Bio, Shanghai, China; no.: LME2020021009), mouse AMH ELISA kit (LMAI Bio, Shanghai, China; no.: LME2020022010), mouse progesterone ELISA kit (LMAI Bio, Shanghai, China; no.: LME202002091), M2 culture medium(Nanjing Aibei Biotechnology Co.,Ltd,Nanjing,China;no.:1912A), anti-CD34 antibody (Boster, Wuhan, China; no.: BA3414), anti-ER-α antibody (Abcam, UK; no.: ab92516), anti-PR-α antibody (Abcam, UK; no.: ab101688), anti-CD138 antibody (Abcam, UK; no.: ab128936).\u003c/p\u003e\u003cp\u003e \u003cb\u003ePreparation of ZC granules.\u003c/b\u003e As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, ZC granules comprises \u003cem\u003eRehmannia glutinosa (Gaertn.) Libosch ex Fisch.et Mey., Dioscorea opposita Thunb., Cornus officinalis Sieb. et Zucc., Lycium chinense Mill., Cuscuta chinensis Lam., Cervi cornu degelatinatum.\u003c/em\u003e The plant name has been checked with MPNS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mpns.kew.org\u003c/span\u003e\u003cspan address=\"http://mpns.kew.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The herbs were mixed in a ratio of 3: 3: 3: 3: 3: 1 and treated with two rounds of extraction using boiling water. The extracts were then combined and filtered and concentrated under reduced pressure to produce a paste with a density of 1.30 g/cm\u003csup\u003e3\u003c/sup\u003e. The dregs were dried and finely powdered, and then mixed evenly with the paste. The mixture was sprayed into granules and dried. One dose yielded 8.4 g dry powder. The granules were resuspended in physiological saline at the final concentrations of 0.11g/ml,0.23g/ml and stored at 4℃ until use.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe compositions of ZC granules.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChinese name\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccepted scientific name\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlant part\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBatch number\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmount(g)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShudihuang\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRehmannia glutinosa (Gaertn.) Libosch ex Fisch.et Mey.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrobanchaceae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2005005S\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShanyao\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDioscorea opposita Thunb\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDioscoreaceae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2006003C\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShanzhuyu\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCornus officinalis Sieb. et Zucc.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCornaceae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFruits\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2006003C\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGouqizi\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLycium chinense Mill.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSolanaceae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFruits\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2004006S\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTusizi\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCuscuta chinensis Lam.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConvolvulaceae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSeed\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2007001S\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLujiaoshuang\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCervi cornu degelatinatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCervidae\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCornu cervi\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2003001S\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5g\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dose selection of Zi Chong granules is based on previous work (Wang, 2018). According to the Chinese Pharmacopoeia, an adult weighting 60 kg should receive a dose of 8.2g of ZC granules per day, and the equivalent dose ratio based on body surface area converted between mice and human was 10. The mice in the ZCL and ZCH groups received approximately 10 and 20 times the dosage was used for adult respectively to intervene in repeated and continuous super ovulation induction in mice to observe the effect of Zi Chong granules on oocyte quality and explore the effects of different drug doses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical composition analysis of ZC granules.\u003c/b\u003e The chromatographic analysis of ZC granules was carried out according to the 2015 edition of the Chinese Pharmacopoeia, performing on SHIMADZU LC-40BX3 (SHIMADZU, Japan), using Ultimate UHPLC XB-C18 column (4.6x100mm,1.8µm). The crushed material was obtained by grinding the Zi Chong granules, and after repeated mixing, 2 g of the material was taken. It was then added to 20 ml of 50% methanol, vortexed for 10 minutes, subjected to ultrasonic extraction for 30 minutes, centrifuged, and the supernatant was collected. The volume was adjusted to 20 ml, and the mixture was filtered for UHPLC analysis. The mobile phases were acetonitrile (A) and water (B). The gradient elution condition was as follows: 0–20 min, 5%-30%A; 20–30 min, 30%-100% A; 30–35 min 100% A; 35–37 min 5% A; 37–38 min 5% A; the detection wavelength was 254 nm, the flow rate was 0.5 ml/min, the column temperature was 35℃,the injection volume was 5µl.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimals.\u003c/b\u003e Seventy-two specific pathogen-free female Kunming mice, age 4–6 weeks, were purchased from Dashuo Experimental Animal Center (Sichuan, China; certificate no.: SCXK [Sichuan] 2020-030). All mice were housed at room temperature (22 ± 2℃), 45–55% humidity, and a 12 h light/day cycle, with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. This study was approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (2021DL-002). All experiments were performed in accordance with relevant named guidelines and regulations, and the author complied with ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e \u003cb\u003eExperimental design.\u003c/b\u003e The mice were divided randomly into three groups: control group (CON), model group (HU), and HU + ZC group. Mice in the HU and HU + ZC groups were gavaged with HU suspension at a dose of 400 mg/kg/day, and mice in the CON group were gavaged with an equal amount of saline, for a total of 21 days. Mice in the HU + ZC group were then gavaged with 2.5 g/kg ZC granules daily, and mice in the HU and CON groups were gavaged with the same amount of saline, for a total of 15 days. To ensure uniformity among the groups, mice in the HU and HU + ZC groups were injected intraperitoneally, after gavage, with PMSG (5 IU) + HCG (5 IU) to promote ovulation, and vaginal smears were checked in the CON group to determine the estrous cycle. Mice were anaesthetized with pentobarbital sodium salt (3 mg/ml), at 0.01ml/g mouse body weight, adjust with saline to 15ml volume and administered intraperitoneally.\u003c/p\u003e\u003cp\u003eFor the first procedure, 12 mice were selected randomly from each group. Mice in the HU and HU + ZC groups were sacrificed under anesthesia at 12 h and 72 h post-ovulation, and mice in the CON group were sacrificed before and after ovulation, respectively. Blood was collected and the ovaries and uterus were removed and fixed in 4% paraformaldehyde buffer for histopathological examination. The day before sacrifice, feces were collected from the mice using sterilized equipment and then frozen rapidly in liquid nitrogen for 16S rRNA gene sequencing.\u003c/p\u003e\u003cp\u003eFor the second procedure, six mice were selected randomly from each group. Post-ovulation mice in the HU and HU + ZC groups and CON mice in estrus stage were arranged in a 2:1 female: male ratio for mating.\u003c/p\u003e\u003cp\u003eFor the third procedure, six mice were selected randomly from each group. Mice in the HU and HU + ZC groups were anesthetized and sacrificed 16 h after ovulation, and mice in the CON group were anesthetized and sacrificed after detection of a vaginal plug. The bilateral oviducts were removed and placed in 35 mm dishes to obtain oocytes for staging and counting.\u003c/p\u003e\u003cp\u003e \u003cb\u003eELISA.\u003c/b\u003e Serum concentrations of AMH, E\u003csub\u003e2\u003c/sub\u003e, and progesterone were detected by ELISA, according to the instructions in the respective kits.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOocyte staging and counting.\u003c/b\u003e Bilateral oviducts from mice in each group were put into 35 mm dishes and gradually torn to release all the oocytes. The oocytes were put into a culture dish containing hyaluronidase to separate the granulosa cells, and the oocytes separated from the granulosa cells were then put into a culture dish containing M2 culture medium and staged and counted under the microscope. MII stage oocytes were identified by uniform cytoplasm, small perivitelline gaps, discharged first polar body and smooth surface, and a clear and uniform zona pellucida. Abnormal oocytes were characterized by an irregular shape, refractive areas or large vacuoles in the cytoplasm, and a thickened or raised zona pellucida.\u003c/p\u003e\u003cp\u003e \u003cb\u003eHistopathological analysis of ovarian and uterus tissues.\u003c/b\u003e The tissues were fixed in 4% paraformaldehyde buffer, serially dehydrated, embedded in paraffin, sliced into 5-µm thick sections, and stained with hematoxylin-eosin (HE) and sealed with resin glue. The tissue samples were then photographed using a microscope imaging system, and primordial and atretic follicles were counted in the images using Image Pro Plus 6.0. Twenty different views were selected, and the endometrial thickness and glandular area were measured in each view and averaged.\u003c/p\u003e\u003cp\u003eIn addition, uterine tissue sections were stained immunohistochemically to determine the MVD and CD138 and ER expression. Sections were repaired using pH6 citrate antigen repair solution, rinsed with phosphate-buffered saline (PBS), incubated with peroxidase blocker (3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), rinsed again with PBS, and incubated with primary antibodies at 37℃, followed by rinsing with PBS, incubation with solution A from a two-step anti-rabbit/mouse universal immunohistochemical assay kit (ChemMate™ Envision + HRP) at 37℃, and rinsing with PBS. The sections were then incubated with chromogenic DAB working solution, re-stained with hematoxylin, dehydrated through graded alcohols, transparentized with xylene, and sealed with neutral glue.\u003c/p\u003e\u003cp\u003e\u003cb\u003e16s rDNA gene sequencing of feces.\u003c/b\u003e Total DNA was extracted from feces samples. Primers were designed according to the conserved region, and a sequencing junction was added at the end of the primers. Polymerase chain reaction amplification was then performed and the products were purified, quantified, and homogenized to form a sequencing library. The built library was subjected to quality control and then sequenced using an Illumina HiSeq 2500. The original image data were obtained and transformed into raw sequences (sequenced reads) by base calling. The observed operational taxonomic units (OTUs) were clustered according to 97% similarity sequences using USEARCH (version 11.0.667 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.drive5.com/usearch/\u003c/span\u003e\u003cspan address=\"http://www.drive5.com/usearch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) software. Principal coordinate analysis (PCA) was performed using the R package to show the β-diversity of the microbiome between samples. Bacterial classifications were compared among the groups using Wilcoxon’s rank sum test. Based on the 16S rDNA gene sequencing data, the relative abundance of microbial functional categories in the samples was predicted using the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e software. Subsequently, the significance of the PICRUSt2 predicted results was tested using the limma package in R software for differential analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways (p \u0026lt; 0.05 was considered significant).\u003c/p\u003e\u003cp\u003e \u003cb\u003eNon-targeted metabolomics assays.\u003c/b\u003e A 100µL serum sample was taken and added to three times the volume of pre-cooled acetonitrile solution. The mixture was shaken at room temperature for 10 minutes, then placed the sample in the refrigerator at -20℃ for 30 mins. The samples were centrifuged at 14000g and 4℃ for 15 mins, and the supernatant was transferred to the mass spectrometry sample bottle. High-performance liquid chromatography-tandem mass spectrometry was performed using a Q Exactive mass spectrometer (Q Exactive, Thermo Scientific) equipped with a Hypesil GOLD (C18) column. Raw data were obtained in positive and negative ion modes, and data were extracted using Compound Discoverer v.1 software. Compound identification was searched using mHU + ZCloud, with ChemSpider. Combined with the built-in database search, the OTCML, a high-resolution mass spectrometry database of important ingredients in the mzVault library, was performed.\u003c/p\u003e\u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Values for each group were expressed as mean and standard deviation and analyzed using SPSS 25.0 software. Comparisons among the three groups were performed by one-way ANOVA, and comparisons between two groups were performed using the least significant difference method. A p \u0026lt; 0.05 was considered significant.\u003c/p\u003e\u003cp\u003eBray–Curtis distance matrices were calculated using the R project Vegan package (version 2.5.3). Multivariate techniques, including PCA and Bray–Curtis distances, were carried out using the R project Vegan package (version 2.5.3) and plotted using R project ggplot2 package (version 2.2.1). Welch’s \u003cem\u003et\u003c/em\u003e-tests, Kruskal–Wallis H tests, and ANOSIM tests were carried out using the R project Vegan package (version 2.5.3). Correlations between the gut microbiome and metabolome were calculated using the R-package Psych and plotted using the pheatmap package.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAFC \u0026nbsp; \u0026nbsp; Antral Follicle Counting\u003c/p\u003e\n\u003cp\u003eAMH \u0026nbsp; \u0026nbsp;Anti-M\u0026uuml;llerian Hormone\u003c/p\u003e\n\u003cp\u003eHU \u0026nbsp; \u0026nbsp; \u0026nbsp;Hydroxyurea\u003c/p\u003e\n\u003cp\u003eCOH \u0026nbsp; \u0026nbsp;Controlled ovarian hyperstimulate\u003c/p\u003e\n\u003cp\u003eCON \u0026nbsp; \u0026nbsp;Control group\u003c/p\u003e\n\u003cp\u003eDOR \u0026nbsp; \u0026nbsp;Diminished Ovarian Reserve\u003c/p\u003e\n\u003cp\u003eER \u0026nbsp; \u0026nbsp; \u0026nbsp;Estrogen Receptor\u003c/p\u003e\n\u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Estradiol\u003c/p\u003e\n\u003cp\u003eGF \u0026nbsp; \u0026nbsp; \u0026nbsp;Germ free\u003c/p\u003e\n\u003cp\u003eHE \u0026nbsp; \u0026nbsp; \u0026nbsp;Hematoxylin-Eosin\u003c/p\u003e\n\u003cp\u003eKEGG \u0026nbsp; Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eMVD \u0026nbsp; \u0026nbsp;Microvessel density\u003c/p\u003e\n\u003cp\u003eOTUs \u0026nbsp; \u0026nbsp;Operational Taxonomic Units\u003c/p\u003e\n\u003cp\u003ePCA \u0026nbsp; \u0026nbsp; Principal Coordinate Analysis\u003c/p\u003e\n\u003cp\u003ePGA2 \u0026nbsp; \u0026nbsp;Prostaglandin A2\u003c/p\u003e\n\u003cp\u003eSPF \u0026nbsp; \u0026nbsp; Specific Pathogen-free\u003c/p\u003e\n\u003cp\u003eZC \u0026nbsp; \u0026nbsp; \u0026nbsp;Zi Chong granules\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProject supported by Hainan Province Clinical Medical Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eW. D.:\u0026nbsp;\u003c/strong\u003eInvestigation; Data curation; Writing-original draft. \u003cstrong\u003eX. G.:\u0026nbsp;\u003c/strong\u003eSoftware; Data curation; Writing-original draft. \u003cstrong\u003eH. L.:\u0026nbsp;\u003c/strong\u003eProject administration; Funding acquisition; Writing-review and editing. \u003cstrong\u003eZ. L.:\u0026nbsp;\u003c/strong\u003eWriting-review and editing. \u003cstrong\u003eL. X.:\u0026nbsp;\u003c/strong\u003eData curation; Writing-review and editing. \u003cstrong\u003eY. L.:\u0026nbsp;\u003c/strong\u003eData curation. \u003cstrong\u003eQ. W.: \u0026nbsp;\u003c/strong\u003eWriting-review and editing. \u003cstrong\u003eR. C.:\u0026nbsp;\u003c/strong\u003eFunding acquisition. \u003cstrong\u003eS. L.:\u0026nbsp;\u003c/strong\u003eMethodology; Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIllumina sequencing reads were uploaded to the SRA under accession number PRJNA1070711.Any other data supporting this study\u0026apos;s conclusions are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Science and Technology of the People\u0026apos;s Republic of China [grant number: 2018YFC1704305].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePecker, L. H., Hussain, S., Christianson, M. S., \u0026amp; Lanzkron, S. Hydroxycarbamide exposure and ovarian reserve in women with sickle cell disease in the Multicenter Study of Hydroxycarbamide. Br J Haematol. 191,880\u0026ndash;887(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePecker, L. H., Hussain, S., Mahesh, J., Varadhan, R., Christianson, M. S., \u0026amp; Lanzkron, S. Diminished ovarian reserve in young women with sickle cell anemia. Blood. 139, 1111\u0026ndash;1115(2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampson, M., et al. 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J Reprod Immunol. 151,103634(2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3869320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3869320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydroxyurea (HU) is an antitumor drug. However, HU exposure is associated with diminished ovarian reserve (DOR). Zi Chong granules, a Chinese Medicine, can protect against DOR, but little is known regarding its underlying mechanisms of DOR treatment, and thus the target of the present study. Female KM mice were randomly divided into three groups: the control group (Con), the hydroxyurea group (HU), and the Zi Chong group (ZC). The ovaries and uterus of mice were examined histologically by H\u0026amp;E. The levels of anti-Mullerian hormone (AMH), estradiol (E\u003csub\u003e2\u003c/sub\u003e), and progesterone (P) were quantified using ELISA kits. The number and quality of oocytes were assessed, and endometrial receptivity was evaluated by immunohistochemistry. 16S rDNA gene sequencing was used to analyze the composition and abundance of gut microbiome in feces, and non-targeted metabolomics was performed to detect serum metabolite profiles. Correlation analysis was performed to explore the relationships between different gut microbiota and differential metabolites. The results showed that ZC granules increased the number of primordial follicles in the ovaries, reduced excessive follicular atresia, restored low AMH, upregulated estrogen and progesterone secretion, and increased the number of mature oocytes after ovulation promotion. It also increased thickness of uterine endometrium and the number of glands, resulting in increased endometrial microvessel density (MVD), enhanced endometrial blood supply, reduced CD138 expression levels and endometrial inflammation. HU decreased the abundance of Lactobacillus spp. in mouse intestines and decreased arachidonic acid metabolism, tryptophan metabolism, spermidine and spermine biosynthesis, steroidogenesis and nicotinate and nicotinamide metabolism. Correlation analysis revealed that HU exerted its side effects by altering the gut microbiome and bacteria-derived metabolites, while ZC granules could reverse DOR partly depends on regulating gut microbiota and metabolites. Together, ZC granules may be a potential therapy for alleviating HU-induced DOR.\u003c/p\u003e","manuscriptTitle":"Zi Chong granules improve hydroxyurea-induced decrease in ovarian reserve function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-01 21:46:14","doi":"10.21203/rs.3.rs-3869320/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc4a42b3-70d3-49ff-94dd-de803aeaf101","owner":[],"postedDate":"February 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28477585,"name":"Biological sciences/Drug discovery/Pharmacology"},{"id":28477586,"name":"Health sciences/Diseases/Reproductive disorders/Infertility"}],"tags":[],"updatedAt":"2024-07-27T13:23:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-01 21:46:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3869320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3869320","identity":"rs-3869320","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}