The Studies to correlation between gut microbiota diversity and functional erectile dysfunction

Qiang Geng First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Shaofeng Chen (  wisefeng@126.com ) Tianjin University of Traditional Chinese Medicine Yuan Sun Tianjin University of Traditional Chinese Medicine Yu Zhao First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Zhong Li First Teaching Hospital of Tianjin University of Traditional Chinese Medicine Fu Wang Xiyuan Hospital Guojin Yu Xiyuan Hospital Xiuchuan Yan NexbrioMdeical Laboratory Jun Guo Xiyuan Hospital


Introduction
There are a large number of microorganisms living in the human intestinal tract. The normal gut microbiota is the natural barrier of the human body and plays an important part in maintaining human health (1).The normal gut microbiota in the human body is mainly composed of rmicutes, Bacteroidetes, proteobacteria and actinomycetes. According to the impact on human health, it can be divided into three categories: symbiotic bacteria, opportunistic bacteria and pathogenic bacteria, which are dynamically balanced to maintain the homeostasis of the intestinal environment (2). Intestinal bene cial bacteria (i.e., probiotics) include bi dobacteria and lactobacillus, etc., harmful bacteria include Escherichia coli and enterococcus (3).Changes in the internal and external environment of the body could affect the structure of gut microbiota, resulting in gut microbiota imbalance (4), it could lead to in ammatory bowel disease (5), irritable bowel syndrome(6), non-alcoholic liver disease (7), viral hepatitis(8), metabolic syndrome (9) and other diseases aggravation and rapid progression. Sexual function is a complex phenomenon affected by sex hormones, psychology, nerves and hemodynamics, such as hormone disorder, obesity, stress, anxiety, hypertension, diabetes, etc.Therefore, it is speculated that there is a correlation between erectile dysfunction and gut microbiota distribution. Take into account this inference, this study tested the gut microbiota diversity of patients with erectile dysfunction and the normal control group. healthy volunteers aged 20-40 with an average age of (29.17±2.66) years. There was no statistically signi cant difference in age between the two groups(P 0.05).

Inclusion Criteria
Married men aged 20-40 with regular sexual partners; Participants know and agree to participate in the study; Normal genital and secondary sexual characteristics; No acute or chronic gastrointestinal diseases; No history of severe hypertension, myocardial infarction or other serious cardiovascular disease, diabetes, blood, kidney or liver disease; No history of infectious diseases such as tuberculosis, viral hepatitis and AIDS; Did not participate in other clinical trials in the last 3 months, did not use antibiotics, steroid drugs, did not eat probiotics and probiotics and other microbiological preparations. In the ED group, the diagnostic criteria of mild to moderate ED were satis ed. The IIEF-5 score was between 8 and 21, and the diagnosis was consistent with two attending physician or above. In the HD group, male physicians with the title of associate chief physician or above were interviewed in IIEF-5 and scored 22-25. The study was approved by the Ethics Committee of the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, and was strictly followed during the study implementation. The participants gave informed consent and signed the informed consent.

Exclusion Criteria
Having obvious primary diseases and/or complications of the heart, liver, kidney and nervous system; With other sexual dysfunction, such as decreased libido, orgasm suppression or no orgasm, ejaculation suppression or no ejaculation; Female partners have signi cant sexual dysfunction, such as decreased libido, painful sexual intercourse, etc. drug abuse within 2 years; Acute and chronic in ammation of the reproductive system; Penis deformities, genital deformities that obviously impair erectile function; Patients with serious mental disorders.

Collection samples
According to the instructions, the two groups were instructed to take about 50g of fresh stool specimens before eating food in the morning, taking them into a special specimen bottle respectively, and quickly put them in an ice box for preservation at -80℃. All samples are kept in dry ice and sent to Con dante Future for gene sequencing and data analysis.
1.5 Gene sequencing and data analysis 1.DNA extraction: According to different sample types, the corresponding kit (with kit) is used to extract the sample DNA and remove other non-DNA substances from the sample; 2.DNA quality control: Using 0.8-1% agarose gel to detect the integrity, degradation and degradation degree of DNA; Q-bit instruments and matching DNA-speci c dyes were used to detect the DNA concentration.The A260/A280 and A260/A230 of DNA were detected by namedrop to determine the purity of DNA.3. Library construction: V3 and V4 regions was ampli ed using the universal primers of 16S rRNA gene; The PCR ampli cation products were puri ed and the redundant primers and various components in the PCR reaction system were removed. The puri ed product was supplemented by PCR, and the terminals of the puri ed product were added to the connector for sequencing and the index label sequence for distinguishing each sample. The second round of PCR ampli cation products was puri ed and the redundant primers and various components in the PCR reaction system were removed. 4. Library quality inspection and pretreatment before sequencing: Q-BIT was used to identify the concentration of each library, and all libraries were mixed according to the equal molality;Fluorescence quantitative PCR was used to detect the molality of the mixed library.The total library of sodium hydroxide treatment was single-stranded DNA.5. Clustering: In alumina's Miseq sequencer, the single-stranded DNA generated was hybridized with the single-stranded sequence of Flowcell, and then clusters of single-stranded DNA were formed on Flowcell by bridging PCR ampli cation. Finally, the DNA in the cluster was converted into single-stranded DNA and then Paired end(PE) was sequence. 6. Sequencing: On Miseq, the base sequence of each cluster on FlowCell was observed by simultaneous synthesis and sequencing. 7. Disinformation analysis: Raw Data were rstly quality-controlled, ltered, spliced and removed to obtain Clean Data, then the effective Data were analyzed for Operational Taxonomic Units and species classi cation. According to the OTUs clustering results, species annotation was made on the representative sequence of each OTU to obtain the analogous species information and specie-based abundance distribution. At the same time, OTUs was tested for relative abundance, Alpha diversity calculation, Alpha diversity, etc., so as to obtain species richness and uniformity information in samples, as well as differences between different samples or groups. In order to further explore the differences in community structure between the group samples, ttest, LEfSe and other statistical analysis methods were utilized to test the signi cance of the differences in species composition and community structure between the ED group and the HD group.The data(P 0.05)was seen as the criterion of statistical signi cance.

Analysis of bacterial ora composition of samples
According to OTU annotation results, histogram of relative abundance of species was made for each sample at different classi cation levels, which can visually display the bacterial ora of each sample at different classi cation levels. Figure 1-2 shows the stacking histogram of bacteria with relative abundance greater than 1% in each sample at the classi cation level of genera. The abscess is the sample name, and the ordinate is the relative abundance ratio of corresponding bacteria. "F_ *" means that it cannot be annotated to genus in biological classi cation, but can be annotated to family or other classi cation level.
There was no difference between the genera of the ED-HD group, indicating that there was no statistically signi cant difference between the high abundance bacteria (top10) and the core bacteria (90%) of the ED-HD group. However, the total intergroup ora( 1%) between groups, Alloprevotella was statistically signi cant(Alloprevotella was identi ed only in the HD group.)

Alpha diversity analysis
Alpha Diversity refers to the microbial community Diversity in a speci c region or ecological environment, which is a comprehensive index re ecting the richness and uniformity. The Alpha Diversity analysis of a single sample can re ect the richness and diversity of the microbial community in the sample. Figure 3 shows that the ED group has a relatively low Shannon diversity coe cient.(HD group =5.741,ED group 4.982), indicating that ED group had lower bacterial diversity (P=0.00074X 0.01).

PCoA analysis based on Bate diversity
Principal component Analysis (PCoA, Principal Co-ordinates Analysis) is used to study the similarity or heterogeneity of sample community composition. The closer the distance the sample, showed that the higher the similarity community structure, otherwise the structure indicates that the larger the difference of the community. Figure 4 is the PCoA analysis conducted by calculating the Unweighted Unifrac distance using the relationship between OTUs systems. The x-coordinate represents one principal component, the y-coordinate represents another principal component, and the percentage represents the contribution value of the principal component to the difference of samples. Each point in the gure represents a sample, and samples of the same group are represented by the same color. The results showed that the ED group (blue) and the HD group (red) were clustered respectively, and the two groups were obviously separated, indicating that the structure of the ED-HD group was different.

Comparison of relative abundance between groups of all strains
Through statistical analysis, the genera with signi cant difference in the abundance change between groups can be speci cally identi ed, and the enrichment of the genera with signi cant difference between groups can be obtained. Meanwhile, the size of the difference between groups and the difference between groups can be compared to determine whether the community structure difference between groups is statistically signi cant.

t -test
T-test was used to detect signi cant differences between groups at the level of genera (P 0.05). Results (Table 1 and FIG. 5-6) show six species were signi cantly different between groups, Streptococcus and Subdoligranulum were increased in the ED group than HD group, while in platts bacteria genera, Blautia slaughter's species, Lachnospiraceae NK4A136 group and Roseburia were decreased than the HD group. Table 1 The species were significantly different between ED and HD groups Note * P 0.05 ** P 0.01

LEfSe
In order to nd the speci c bacterial genera of ED, LEfSe analysis was used to compare the different bacterial genera between the two groups. The results (Figure 7) showed that the absolute value of LDA Score of 24 bacterial genera was above 2. It is known that LachnospiraceaeNK4A136 group and Prevotella_9 have the greatest in uence on the difference between groups.

Comments:
1) LDA is a supervised dimension reduction method, while PCA is an unsupervised dimension reduction method 2) LDA dimension reduction can be reduced to the dimension of category number K-1 at most, while PCA does not.
3) In addition to dimension reduction, LDA can also be used for classi cation. 4) LDA selects the projection direction with the best classi cation performance, while PCA selects the direction with the greatest variance for the projection of sample points.
The main advantages of LDA algorithm are as follows: 1) Prior knowledge of categories can be used in dimension reduction, while unsupervised learning such as PCA cannot use prior knowledge of categories.
2) LDA is better than PCA when the sample classi cation information depends on mean value instead of variance.
The main disadvantages of LDA algorithm are as follows: 1) LDA is not suitable for dimensionality reduction of non-Gaussian distribution samples, and PCA also has this problem.
2) LDA dimension reduction can be reduced to the dimension of category number K-1 at most. If the dimension reduction is greater than K-1, LDA cannot be used.There are some evolutionary versions of the LDA that can circumvent the problem.
3) When the sample classi cation information depends on variance instead of mean value, LDA has a poor effect of dimension reduction. 4) LDA may over t the data.

Discussion
Normal erectile function is a complex phenomenon affected by sex hormones, psychology, nerves and hemodynamics, such as hormonal disorders, obesity, stress, anxiety, hypertension, diabetes, etc. Markle con rmed that the intestinal microbiota of sterile mice was regulated by microbiota,and the intestinal microbiota of normal male mice was implanted into female nude mice, which resulted in the sustained increase of testosterone levels in females and the regulation of their autoimmunity. It has also been shown that when female mice were treated with normal intestinal microbiota obtained from donor male mice in the context of impaired reproductive capacity. Testosterone levels were signi cantly increased (10).Microbiot-based therapies have also been used for diseases of the immune system, such as type 1 diabetes, which is linked to abnormal levels of sex hormones (11).In a well-designed study, Yurkovetskiy showed that germ-free 5-week-old mice had higher plasma testosterone concentrations than nonpathogen free (SPF) mice. Whereas SPF mice had elevated testosterone levels after 12 weeks. In addition, mice transplanted with lamentous bacteria or proteobacteria similar to Shigella escherichia coli showed higher serum testosterone concentrations at 12 weeks of age than mice without bacteria. However, the probiotic mixture did do not increase testosterone levels (12).In common with the above, the microbiome can be positively and negatively regulate sex hormone levels based on host physiology, bacterial strains and other factors. These ndings strongly indicate that the gut microbiota can improve sexual function by regulating sex hormone levels. Reiter probiotics had positive effects on testicular size and serum testosterone levels in aging mice (13). Thus it can be observed that using probiotics to regulate human microbiota is another method to improve sexual ability. Normal erectile function depends on normal functional penile blood vessels and cavernous blood vessels. It was found that the growth of gut microbiota in ED patients with diabetes was inhibited, the number of probiotics was signi cantly reduced, but the number of opportunistic pathogens was increased (14).Cho found that serum TMAO was associated with intestinal germicides (15).Spalding found that TMAO could promote vascular endothelial in ammation and enhance smooth muscle cell proliferation, eventually leading to vascular wall brosis and vascular function destruction (16).Sun found that TMAO could lead to vascular endothelial dysfunction through ROS mediated oxidative stress, as well as increased adhesion of monocytes in human umbilical cord vessels and endothelial repair dysfunction (17). Therefore, this study reviewed the association between gut microbiota distribution and erectile dysfunction.
Numerous studies have demonstrated that the pathogenesis of ED is related to the in ammatory response. The streptococci causes in ammation mostly, such as Group A Streptococcus (GAS), which is A gram-positive pathogen and can cause A range of diseases, including deadly invasive infections. And Tomomi study showed that Streptococcus haemal and Streptococcus are associated with systemic diseases such as infective endocarditis and abscess, and increased expression levels of the in ammation-related factor IL-1 mRNA when mice were attacked by Streptococcus hemoglobin (18).The Roxella is comprised of gram-positive anaerobes and is one of the common bacteria that produce shortchain fatty acids (SCFAs), especially butyric acid, which affects colon motility and has immunomaintenance and anti-in ammatory effects (19). Bertillon's found that Pruplatella can induce TLR2 signaling pathways and low levels of P65-mediated in ammation. The results of this study showed that streptococci were up-regulated and Rosteria were down-regulated, and both Streptococcus and Rosteria were associated with in ammatory response, and ED was involved in in ammation, therefore, streptococcus and Rosteria were associated with ED.
Obesity increases the likelihood of erectile dysfunction (20). Studies have further shown a link between human gut ora and obesity. Modi cation of the characterization of Roxella may affect various metabolic pathways, resulting in various diseases such as irritable bowel syndrome, obesity, type 2 diabetes, neurological diseases and allergies (19).Miao Ping con rmed that the rats with metabolic syndrome had obvious gut microbiota imbalance, and the content of opportunistic pathogens such as streptococcus increased (21).The results of this study showed that the number of Streptococcus in ED patients was up-regulated, while the number of Rosteria was down-regulated.
In this study, patients were selected to detect the fecal bacterial diversity of the with functional ED and HD groups. Results of Alpha diversity analysis showed that the ED group had lower bacterial diversity. It was further con rmed that there were signi cant differences between the two groups in the level of colony and genus.Level analysis, and also found that the number of Streptococcus (Streptococcus) and Subdoligranulum (rare small cocci) genera in the ED group increased, while the number of Prevotella_9 (Prevola_9), Blautia (Brautia), Lachnospiraceae NK4A136 group (NK4A136 group of Trichospiraceae) and Roseburia (Rhodoria) decreased.
In summary, erectile dysfunction is associated with colonic microbial diversity in men. Gut microbiota can regulate male erectile function by regulating hormone levels, in ammatory mediators and various factors.
Gut microbiota is a promising and promising option for the treatment of erectile dysfunction in men in the future.

Declarations Author Contributions
Shaofeng Chen, Geng Qiang, and Jun Guo conceived and designed the study, analyzed the data and wrote the manuscript. Fu Wang, and Guojin Yu designed, performed and analyzed the experiments shown in Figure 1-3   The distribution of abundance bacteria and core bacteria in ED and HD groups. Note There was no difference between the genera of the ED-HD group. Alloprevotella was statistically signi cant.

Figure 2
The distribution of abundance bacteria and core bacteria in ED groups. Note Alloprevotella was not identi ed only in the ED group.   The Box gure of signi cant differences species between ED-HD groups The Box gure of signi cant differences species between ED-HD groups(except Prevotella) Figure 7 LDA value distribution histogram