Because males and females with IBD have distinct medical manifestations, prevalence patterns, and therapeutic responses, separate volunteered specimens must be separated depending on illness type and sex. These findings suggest that in terms of gender differential, this is the first research to look at the connections between various microorganisms in the intestinal tract of IBD individuals, including fungi, archaea, bacteria, and viruses at the species (or strain) scale. As regulatory candidates for IBD, two fungus strains, one bacterial species,and two viral species were recognized.Also as candidate sex-related microorganism in non-IBD participants, one fungal strain and one viral species were found. We were unable to identify any Archaea that were linked with inflammatory bowel disease. There were apparent disparities among males and females in their connection with intestinal microbiomes in Crohn's disease. Still, there were more similarities between males and females' intestinal microbiome relationships in ulcerative colitis.
Based on our result for the frequency of microbes, in both diseased and control groups, the fungal biodiversity in fecal specimens was smaller than the bacterial biodiversity; this is in agreement with a previous study . Microsporidia, Ascomycota, and Basidiomycota were among the fungi studied in this research (see table S8supplementary 2). Four EnterocytozoonbieneusiH348,Wickerhamomycesciferrii, Malassezia globosaCBS 7966, and Saccharomyces cerevisiaeS288C fungal strains were found in substantial prevalence in certain diseased or control groups, as shown in Figures 1, 2, and 3, suggesting their significance in the gut microbiota. There has also been evidence of Ascomycota and Basidiomycotadivisionsduring studies of normal subjects . The relative prevalence of S. cerevisiae, M. restricta, and C. Albicans from Saccharomyces, Malassezia, and Candida was higher than any other fungal strains . Inflammatory bowel disease is linked to the fungus Malassezia globosafromMalassezia, which may generate indole ligands (a cytoplasmic transcriptional regulator in epithelial cells) for AhR receptors. Malassezia is the most frequent fungus found on the human epidermis, accounting for more than ten different species.Furthermore, several species have been found in other regions of the body, particularly the gut, indicating that the fungus is widespread in this organ . IBD patients had a greater mean frequency of the fungus Malassezia globosaCBS 7966than control groups in this research. Nevertheless, it was more prevalent among males with UC than males with CD and females experiencing UC (see Table S22 in Supplementary4). Table 6 shows that this strain may be a regulatory candidate for UC illness based on the results.Study found that the most frequent intestine Microsporidian in Iranian patients receiving IBD therapy was the Enterocytozoonbieneusi, which belongs to the genus Microsporidia. In research,E.bieneusi was found nearly exclusively in individuals with normal immune systems and immunodeficiency . With more than 47% prevalence, it was the most frequent intestinal Mycobiom in our research (Figures 1, 2, and 3).Furthermore, this strain was shown to be age-dependent in the females' group (see Table S14 in Supplementary 3) (Females under 30 in the control group had a substantially greater prevalence than females over or equal to 30 in the same group).Wickerhamomycesciferriiis a fungus that produces sphingolipid derivatives and shares its sphingoid bases with the human model in stereochemistry . Due to its controlling the gastrointestinal tract's inflammatory responses, higher sphingolipid levels in various gastrointestinal tract regions are linked to IBD .Nevertheless, it was found as an age-related fungus in our research on females groups (see Table S14 in Supplementary 3). This implies that in the control group, the frequency of this fungus was substantially greater in females under the age of 30 than in females beyond the age of 30 in the same group. IBD metabolism in males may be studied using the existence of this fungus in the intestinal tract. There is no doubt that this fungus is a regulatory candidate in CD illness, as shown in Table 5. It has been reported that the sugar content of Saccharomyces cerevisiae's wall differs in different strains may promote anti-inflammatory or pro-inflammatory characteristics in the host; moreover, its frequency is higher in CD compared to the control group . As a result, the Saccharomyces cerevisiaeS288C strain found in participant stools was shown to be sex-dependent in our research (see Table S15 in Supplementary 3). This implies that the females control group had a substantially greater incidence than males in the same group.
It can be concluded from Figures 1, 2, and 3 that because of their high prevalence at least in one of disease or control groups, the bacterial species, like Faecalibacteriumprausnitzii, Bacteroides vulgatus, Bacteroides stercoris, Bacteroides uniformis, andPrevotellacopri are essential in the gut microbiota.Individuals with IBD have had a high frequency of Faecalibacteriumprausnitziiand Bacteroides vulgatusspecies in agreement with our result. However, According to TableS14 in Supplementary 3, both of these microbes were shown to be age-related in females. This implies that in the control group, the percentage of frequency among females under the age of 30 was substantially higher)lower( than the percentage among females above the age of 30 in the same group, respectively.According to our findings, the frequency of Roseburia intestinalis, which was previously seen alternately in some IBD patients , was lower among IBD females (UC and CD) than among their control group. At the same time, it was greater among males with ulcerative colitis than the control group (see Table S9 in Supplementary2).There was a large amount of Bacteroidesstercorisspecies in the fecal matter of CD patients , and research  found a connection between Bacteroides stercoris species and UC. In our research, its mean percentage was greater throughout females with CD than in UC. In contrast, the percentage was greater in males suffering IBD (UC and CD) than in their controls (see Table S9in Supplementary2). Bacteroides stercorisisa regulatory candidate for UC, as shown in Table 6.Bacteroidesuniformis was less common in patients with UC in research  compared to the control group. In contrast, its frequency in females was found to be age-dependent in our investigation (see Table S14 in Supplementary 3). This implies that in their control group, females below the age of 30 had a reduced percentage of incidence versus females over the age of 30.
It was similarly lower in UC-affected males than CD-affected ones.According to recent research , Prevotellacopri is more prevalent in recently confirmed RA patients; however, in our result, it was shown to be less prevalent in males experiencing CD than those with UC and control(see Table S9 in Supplementary2).
Figures 1, 2, and 3 demonstrate that the Siphoviridae, Unclassified bacterial viruses, Myoviridae, and unclassified Microviridaeviral families were found to be the most prevalent, whereas the Siphoviridae and Myoviridae, Podoviridae, and Inroviridae viral families based on research  were found to be the most common in normal participants. Among subjects with CD and the control group, the most common viral families found were those belonging to the Siphoviridae, Myoviridae, and Podoviridae ones, according to ref. .
It can be shown in Figures1, 2, and 3 that a greater frequency was found for five virus types, including uncultured crAssphage, Parabacteroides phage YZ-2015b, Bacteroides phage B124-14, Streptococcus virus phiAbc2, and BeAn 58058 virus.UnculturedcrAssphage as CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and play an essential role in the viral nucleus of a person . Researchers found that it was more common among UC participants than in the control group , whereas in our research, Uncultured crAssphage was less common in IBD(CD &UC)malesthan in those with control group (see Table S16 in Supplementary3). Table 3 and Figure4suggest it as a regulatory candidate in IBD male patients.Parabacteroides phage YZ-2015b was shown to be more prevalent among individuals with colon cancer , but in our research, it was more(less) common in females(males) with CD than in those with control group (see Table S16Supplementary3). Table 5and Figur 4 show that it can be recommended as a regulatory candidate for CD females (males).Bacteroides phage B124-14's host is Bacteroides fragilis, a bacterium seen in high concentrations in patients with recurrent Crohn’s disease . This bacteriophage has been linked to CD illness, as shown in Table 5. Streptococcus virus phiAbc2 infects Streptococcus thermophilus, a bacteria often found in dairy products . Using a mouse model of IBD, this bacteria decreased inflammation while maintaining the intestinal mucosal barrier . Nevertheless, the proportion of its occurrence among females was shown to be age-dependent in our research (see Table S14 in Supplementary 3). This implies that in thefemales' control group, the frequency was substantially greater in females below the age of 30 than in those above the age of 30.Chlorocebus aethiops and Oryzomys sp. are the bacterial hosts of the phage BeAn 58058 virus, according to reference . It was more frequent among females with IBD than their controls in our research, and it was greater in males with UC than in those with CD and controls (see Table S10 in Supplementary2).
Exploration of age-dependent microbes
According to the wide age range of the participants in our study (Supplementary1)and Introducing some age-related microbes in IBD patients treated in the hospital , Seeking a more
in-depth look at the interactions between gut microbiomes, we first discovered and subsequently eliminated these microbes mentioned above from our targeted various groups (see Table S14 in Supplementary 3).
Exploration of sex-linked microbes
One of the motivations for including sex-linked microorganisms in animal studies may be the varied reactions of males and females to the same illness therapy and the responses of sexual hormones with microbial metabolites . Consequently, in our research on sex separation, we found sex-linked microorganisms (see Table S15 Supplementary 3). The Saccharomyces cerevisiaeS288C strain and the Enterobacteria phage cdtI bacteriophage are some examples.
Evidence of correlation and Differential correlation throughout the human intestinal microbiota between case-control (sex-separated) and case-case groups
A differential correlation was found between the Escherichia phage TL-2011c and Bacteroides_sp_1_1_6 throughout the control and the CD for males, as shown in Table 3. According to the virus-host DB database , Bacteroides_sp_1_1_6 is a host for Escherichia phage TL-2011c. On the other hand, according to Table 1 of the study , the species Bacteroides sp. 1 1 6 is equivalent to B. thetaiotaomicron proposed for the therapy of Crohn's disease .A differential correlation was found between the uncultured crAssphage and unclassified Megamonasthroughout the control and the UC for males, as shown in Table 3. While the relationship between uncultured crAssphage and UC has been documented in research , and confirmation of UC in individuals with a higher prevalence of certain species, such as Megamonas, compared to the controls has been documented .To compare females with CD to those without, we used data from Table 3 to see whether there was any differential correlation in the types of Salmonella phage SSU5 and Clostridium ramosum bacteria. Fermentation products and SCFA found in Clostridium ramosum are toxic to Salmonella strains . It may also cure IBD patients by reducing the number of Tregs in the body . Salmonella enterica subsp. enterica serovar Typhimurium str has been found as a host for Salmonella phage SSU5 in the virus-host DB database . This bacterium may worsen illnesses and cause the development of goblet and Paneth cells inside the intestinal tract. Clostridium ramosum, on the other hand, has been linked to increased uptake of fatty acids and intestinal epithelial cells multiplication .Shigella phage 0.75 Stx and Proteus mirabilis had a differential correlation in the CDM-UCM groups, as shown in Table 3. On the other hand, Proteus mirabilis is critical in the development of Crohn's disease . Viral host database records indicate that the Shigella phage 75/02 Stx is thought to be hosted by the Enterobacteralesorder member Shigella sonnei, while Proteus mirabilis species is also a member of this order .MalasseziaglobosaCBS 7966 and Megamonasrupellensis had a differential correlation in males and females with UC, as shown in Table 6. Additionally, it has been evidenced that Malassezia globose was formerly connected with a pathogenic bacterial species favoring host , and its negative correlation with many bacteria has been shown in UC patients (not seen on CD) .On the other side, one of the criteria used to diagnose UC is the prevalence of the genus Megamonas .A differential correlation was found between theWickerhamomycesciferrii and the Escherichia virus P2 throughout the CD for males and females, as shown in Table 5.Even though this fungus encodes a protein that is homologous with an endogenous virus-like element (EVE) from the Partiviridae family (NCBI: txid1520125). EVE is a viral intervention cycle that has subsequently been discovered as a fungus' antiviral defense mechanism . Further study is needed to determine whether this is the cause for the association between fungus and viruses.Table 3 shows that a differential correlation was found between males experiencing UC and the control group between the Bacteroides phage B40-8 and the Bacteroides stercoris. While Bacteroides fragilis is listed as a host for this phage in the virus-host DB database , that belongs to the same family as Bacteroides stercorisand has been linked to UC illness . Nevertheless, Bacteroides fragilis was more prevalent among males with CD and females suffering UC than their controls in our research (see Table S9in Supplementary2).Men suffering CD and UC had differential correlations between Lactobacillus phage Lc-Nu and Unclassifiable Anaeropsipes, as seen in Table 3. The association of dysbiosis in IBD patients (especially UC) with Unclassified Anaerostipes was determined . Furthermore, certain strains of Anaerostipes throughout the gastrointestinal tracts of patients experiencing Crohn's disease and ulcerative colitis were fewer than controls(in patients with CD less than UC) . Although A probiotic bacteria present in normal females' genitals and urinary system, Lacticaseibacillusrhamnosus  is the host of Lactobacillus phage Lc-Nu to the virus-host DB database .As shown in Table 3, differential correlations between Enterobacteria phage WPhiandFusobacteriumnucleatumwere reported in males experiencing CD and UC in this research. Escherichia is a host for Enterobacteria phage WPhi, according to the ViralZonedatabase. Escherichia phages have also been linked to an intestinal inflammatory response in the epithelium of UC individuals .Patients with acute leukemia's feces and plasma have been shown to have significant levels of Enterobacteria phage WPhi . Fusobacterium nucleatum (a bacteria known to damage the intestinal barrier) produces a rise in its abundance in patient stools and when administered to a mouse model with colitis aggravation of colitis . In both males and females experiencing CD, we found a differential correlation between Enterobacteria phage phi80 and the Lachnospiraceae bacteria 6 1 63FAA, as shown in Table 3. In addition, we identified Enterobacteria phage phi80 infected Escherichiacolibelonging to the Proteobacteria. There was no rise in the prevalence of Proteobacteria in the investigation , but the Lachnospiraceae family experienced an increase in abundance. In the stools of Crohn's disease participants, Escherichia coli (AIEC) was a pathobiont.
Overall Statistical view
A significant number of differential correlations among intestinal microorganisms was shown in Table 4 to be associated with CD illness, particularly in males and females with CD, males with CD, and their controls (or those with UC). This may be partly because males and females are affected differently by Crohn's disease. It also shows a larger shift in the microbial interactions among males with CD than in controls or those with UC. In addition, UC condition was linked to the minimum number of differential correlations between intestinal microorganisms, particularly among females with UC and their controls, males and females experiencing UC, and males with UC and their controls. This indicates that UC is less complicated among males and females than Crohn's disease and that UC illness in males and females is more comparable.A maximum differential correlation among intestinal microorganisms and controls was observed in males sufferingCD, as seen in Table 4. Males with CD had substantially greater frequencies of the microorganisms involved in differential correlation (approximately three-quarters of them) than theircontrol. CD in males may be the most exaggerated form of IBD. According to Table 4, females experiencing UC had the fewest differential correlation among intestinal microorganisms and the controls. As a result, UC in females is the most basic form of IBD (see Table S22Supplementary4).Figures4, 5, and 6 show that, unlike males, females' intestinal microbial interactions had no shared edge among the UC, CD, and controls. Thus, UC and CD disease processes are independent in females. Table 4 shows that one fungal strain, 33 bacteriophages, and seven bacterial species have changed microbial interactions among males and females experiencing CD. Moreover, The microbial connections among males and females suffering UC, including one fungal strain and three species of bacteria, have shifted in our research (e.g., Akkermansiamuciniphila,that a link between the reduction in its frequency and inflammation of the large intestine, has been reported .
Evidence of Bacteriophageimportance
Table 4 shows that bacteriophages had the greatest effect on alterations in the gut microbial interaction network in both patients and controls in our research. Bacteriophages were shown to be effective as only bacterial regulators in research . Study , on the other side, linked bacteriophages to inflammatory aggravation among individuals with inflammatory bowel disease and, for instance, enhanced host immunity throughout participants with Crohn's disease and ulcerative colitis is associated with increasing Caudovirales order. The order Caudovirales was shown to be critical in the makeup of the gut microbiome in people with inflammatory bowel disease (IBD) . A member of this order, the Yersinia phage L-413C, according to Table 5, has been identified as a potential regulation candidate for CD illness and is known to target Yersinia pestis, an enterobacterial plague pathogen . Streptococcus phage Sfi19, Streptococcus phage SpSL1, Enterobacteria phage If1, and Escherichia phage pro147, which are covered by the UC and CD regulation sections, need to be studied more thoroughly (Tables 3, 5, and 6). As shown in research , Partitiviruses cooperate synergistically or competitively in horizontal transmission to its host fungal at the junction of its hyphae, which may indicate a correlation between two different viruses with the common host. More study is required to fully understand the impact of bacteriophages on inflammatory bowel disease since most prior investigations have focused on bacteria.
Alterations in microbial communications among UC and CD conditions
In Bacteroides eggerthii(seen in Figure 5), a decrease in the percentage is known as a stimulant of intestinal colitis .Streptococcus thermophilus is the host of Streptococcus phage TP-J34 and has been found to have beneficial consequences on IBD .