Chronic inflammation of the colon (colitis) is a known risk factor for inflammatory-driven colorectal cancers (id-CRCs), and intestinal microbiota has been implicated in the etiology of id-CRCs. Manipulation of the microbiome is a clinically viable therapeutic approach to limiting id-CRCs. To understand the microbiome changes that occur over time in id-CRCs, we used a mouse model of id-CRCs with the treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS) and measured the microbiome over time. We included cohorts where the microbiome was restored using cage bedding swapping and where the microbiome was depleted using antibiotics to compare to untreated animals. We identified consistent increases in Akkermansia in mice receiving horizontal microbiome transfer (HMT) via cage bedding swapping, while the control cohort had consistent longitudinal increases in Anaeroplasma and Alistipes. Additionally, fecal lipocalin-2 (Lcn-2), a marker of intestinal inflammation, was elevated in unrestored animals compared to restored and antibiotic-treated counterparts following HMT. These observations suggest a potential role for Akkermansia, Anaeroplasma, and Alistipes in regulating colonic inflammation in id-CRCs.

Host diet and gut microbiota interact to contribute to perioperative complications, including anastomotic leak (AL). Using a murine surgical model of colonic anastomosis, we investigated how diet and fecal microbial transplantation (FMT) impacted the intestinal microbiota and if a predictive signature for AL could be determined. We hypothesized that a Western diet (WD) would impact gut microbial composition and that the resulting dysbiosis would correlate with increased rates of AL, while FMT from healthy, lean diet (LD) donors would reduce the risk of AL. Furthermore, we predicted that surgical outcomes would allow for the development of a microbial preclinical translational tool to identify AL. Here, we show that AL is associated with a dysbiotic microbial community characterized by increased levels of Bacteroides and Akkermansia. We identified several key taxa that were associated with leak formation, and developed an index based on the ratio of bacteria associated with the absence and presence of leak. We also highlight a modifiable connection between diet, microbiota, and anastomotic healing, potentially paving the way for perioperative modulation by microbiota-targeted therapeutics to reduce AL.

Background Neutropenic fever (NF) occurs in >70% of hematopoietic cell transplant (HCT) recipients, without a documented cause in most cases. Antibiotics used to prevent and treat NF disrupt the gut microbiota; these disruptions predict a higher posttransplantation mortality rate. We hypothesized that specific features in the gut microbial community may mediate the risk of NF. Methods We searched a large gut microbiota database in allogeneic HCT recipients (12 546 stool samples; 1278 patients) to find pairs with NF (cases) versus without NF (controls) on the same day relative to transplantation and with a stool sample on the previous day. A total of 179 such pairs were matched as to the underlying disease and graft source. Several other important clinical variables were similar between the groups. Results The gut microbiota of cases on the day before NF occurrence had a lower abundance of Blautia than their matched controls on the same day after transplantation, suggesting a protective role for Blautia. Microbiota network analysis did not find any differences in community structure between the groups, suggesting a single-taxon effect. To identify putative mechanisms, we searched a gut microbiome and serum metabolome database of patients with acute leukemia receiving chemotherapy and identified 139 serum samples collected within 24 hours after a stool sample from the same patient. Greater Blautia abundances predicted higher levels of next-day citrulline, a biomarker of total enterocyte mass. Conclusions These findings support a model in which Blautia protects against NF by improving intestinal health. Therapeutic restoration of Blautia may help prevent NF, thus reducing antibiotic exposures and transplantation-related deaths. In this multicenter multiomics study, we found that intestinal Blautia may protect against neutropenic fever in allogeneic transplant recipients by augmenting the intestinal epithelium. Blautia restoration may reduce the incidence of neutropenic fever, subsequent antibiotic exposures, and dysbiosis-related complications.

Previous studies have shown that the gut microbiota of patients with acute myeloid leukemia (AML) is disrupted during induction chemotherapy; however, the durability of microbiota changes is unknown. This is an important knowledge gap, because reduced microbiota diversity at the time of stem cell transplantation weeks to months after the initial chemotherapy has been associated with higher mortality after transplantation. By sequencing the gut microbiota in 410 longitudinal stool samples from 52 patients with AML, we found that, during inpatient chemotherapy, the gut microbiota is stressed beyond its ability to recover its original state. Despite major reductions in antibiotic pressure and other disturbances to the microbiota after hospital discharge, the trajectory of microbiota recovery yields new communities that are highly dissimilar to baseline. This lasting shift in the gut microbiota is relevant for subsequent phases of curative therapy and is a potential target for novel microbiota protective/restorative interventions. This trial was registered at www.clinicaltrials.gov as #NCT03316456.

Network-based approaches offer a powerful framework to evaluate microbial community organization and function as it relates to a variety of environmental processes. Emerging studies are exploring network theory as a method for data integration that is likely to be critical for the integration of 'omics' data using systems biology approaches. Intricacies of network theory and methodological and computational complexities in network construction, however, impede the use of these tools for translational science. We provide a perspective on the methods of network construction, interpretation and emerging uses for these techniques in understanding host-microbiota interactions.

Mycophenolate mofetil (MMF) is an important immunosuppressant used after allogeneic hematopoietic cell transplantation (HCT). MMF has a narrow therapeutic index, and blood concentrations of mycophenolic acid (MPA), the active component of MMF, are highly variable. Low MPA concentrations are associated with the risk of graft-versus-host disease (GVHD), whereas high concentrations are associated with toxicity. Reasons for variability are not well known and may include the presence of beta-glucuronidase-producing bacteria in the gastrointestinal tract, which enhance MPA enterohepatic recirculation (EHR) by transforming MPA metabolites formed in the liver back to MPA. This study was conducted to determine whether individuals with high MPA EHR have a greater abundance of beta-glucuronidase-producing bacteria in their stool and higher MPA concentrations compared with those with low EHR. We conducted a pharmacomicrobiomics study in 20 adult HCT recipients receiving a myeloablative or reduced-intensity preparative regimen. Participants received MMF 1 g i.v. every 8 hours with tacrolimus. Intensive pharmacokinetic sampling of MMF was conducted before hospital discharge; total MPA, MPA glucuronide (MPAG), and acyl-glucuronide metabolite (acylMPAG) were measured. EHR was defined as the ratio of MPA area under the concentration-versus-time curve (AUC)(4-8) to MPA AUC(0-8). Differences in stool microbiome diversity and composition, determined by shotgun metagenomic sequencing, were compared above and below the median EHR (22%; range, 5% to 44%). The median EHR was 12% in the low EHR group and 29% in the high EHR group. MPA troughs, MPA AUC(4-8), and acyl-glucuronide metabolite (acylMPAG) AUC(4-8)/AUC(0-8) ratio were greater in the high EHR group compared with the low EHR group (1.53 mu g/mL versus.28 mu g/mL [P = .0001], 7.33 hour.mu g/mL versus 1.79 hour.mu g/mL [P = .0003], and .33 hour.mu g/mL versus .24 hour.mu g/mL [P = .0007], respectively). MPA AUC(0-8) was greater in the high EHR group than in the low EHR group, and the difference trended toward significance (22.8 hour.mu g/mL versus 15.3 hour.mu g/mL; P = .06). Bacteroides vulgatus, Bacteroides stercoris, and Bacteroides thetaiotaomicron were 1.2- to 2.4-fold more abundant (P = .039, .024, and .046, respectively) in the high EHR group. MPA EHR was positively correlated with B. vulgatus (rho = .58; P <= .01) and B. thetaiotaomicron (rho = .46; P < .05) and negatively correlated with Blautia hydrogenotrophica (rho = -.53; P < .05). Therapeutic MPA troughs were achieved in 80% of patients in the high EHR group but in no patients in the low EHR group. There was a trend toward differences in MPA AUC(0-8) and MPA concentration at steady-state (mu g/mL) between the high EHR group versus the low EHR group (P = .06). MPA EHR was variable. Patients with high MPA EHR had greater abundance of Bacteroides species in stool and higher MPA exposure compared with patients with low MPA EHR. Therefore, Bacteroides may be protective against poor outcomes, such as graft-versus-host disease, in some patients but may increase the risk of MPA adverse effects in others. These data need to be confirmed and studied after oral MMF therapy. (C) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Induction chemotherapy for patients with acute myeloid leukemia (AML) is a unique clinical scenario. These patients spend several weeks in the hospital, receiving multiple antibiotics, experiencing gastrointestinal mucosal damage, and suffering severe impairments in their immune system and nutrition. These factors cause major disruptions to the gut microbiota to a level rarely seen in other clinical conditions. Thus, the study of the gut microbiota in these patients can reveal novel aspects of microbiota-host relationships. When combined with the circulating metabolome, such studies could shed light on gut microbiota contribution to circulating metabolites. Collectively, gut microbiota and circulating metabolome are known to regulate host physiology. We have previously deposited amplicon sequences from 566 fecal samples from 68 AML patients. Here, we provide sample-level details and a link, using de-identified patient IDs, to additional data including serum metabolomics (260 samples from 36 patients) and clinical metadata. The detailed information provided enables comprehensive multi-omics analysis. We validate the technical quality of these data through 3 examples and demonstrate a method for integrated analysis.

COVID-19 precautions decrease social connectedness. It has been proposed that these measures alter the gut microbiota, with potential clinical consequences. We tested this hypothesis in patients with acute myeloid leukemia (AML) receiving inpatient chemotherapy, a population with extensive exposure to the nosocomial setting and at high risk for infections. Hospitalized patients with AML contributed stool samples to a biorepository protocol that was initiated before COVID-19 and continued without change through the pandemic. Patient-, disease-, and treatment-related characteristics remained the same in the two eras and the only change in clinical care was the implementation of COVID-19 precautions in March 2020. The incidence of all-cause nosocomial infections during the pandemic was lower than in the pre-COVID-19 era. Multivariable analysis revealed an imprint of COVID-19 precautions in the gut microbiota as a viable mechanistic explanation. In conclusion, COVID-19 precautions alter the gut microbiota, thereby mediating pathogen susceptibility and nosocomial infections.

Objective: Clostridioides difficile (formerly Clostridium difficile) is among the most reported hospital and community-acquired gastrointestinal infections. Spores produced by this bacterium cause the initial disease and recurrent C. difficile infections (rCDI) and survive for long periods in the environment. Killing of C. difficile spores in carpeting and upholstery is very challenging without destruction or alteration of the fiber matrix material. In this study, we evaluated a fiber-safe standardized method for inhibiting germination of C. difficile spores recovered from in carpeting and upholstery material and thereby reduce the risk of disease incidence in households and nursing care facilities. Methods and Results : In this study, we found that the most effective procedure for the recovery of C. difficile spores from short-fiber textile surfaces used an ammonium phosphate-gelatin buffer, stainless steel beads to extract the spores from the carpet samples, and the addition of a heat activation step prior to plating. Virasept®, a known sporicide containing hydrogen peroxide and peroxyacetic acid, was evaluated for marked reduction of viable C. difficile spore number in carpet and fabric models. After a 30 min contact time, application of the sporicide at ~ 75 ml m2 of carpet (or 51.2 ml m2 upholstery fabric) resulted in a 3-log reduction in germination of five different C. difficile spore types (a ~99.9% decrease), representing five different Nap biotypes. Despite effective inhibition of C. difficile spore germination, Virasept treatment did not visibly damage or discolor carpet or fabric fibers. Conclusions: Results of this study show the potential effectiveness of a robust regimen for the practical treatment of carpeting and upholstery fabric in nursing care facilities, and residential homes to prevent community- and environmentally-acquired reinfection and recurrence of CDI in susceptible individuals.

Human microbiota-associated (HMA) mouse models offer a valuable approach to study the role of intestinal microbiota in the development of obesity. In this study, we used an HMA model to evaluate whether engraftment of human obese or lean microbiota, from each of three donors, could recapitulate host phenotypes under conventional and specific-pathogen-free housing. Microbiota engraftment was correlated with donor relative abundances of the class Bacteroidia (Spearman’s ρ = 0.73, P ≤ 0.001), and one obese donor resulted in significant weight gain (P ≤ 0.003) and compromised insulin sensitivity under conventional housing. SPF housing partially blunted phenotypic response. Results of this study indicate that our HMA model partially recapitulates obese phenotypes under conventional housing and highlights a need to consider donor-specific effects as well as housing conditions when studying the role of the microbiota in obesity.

AbstractDespite antibiotic prophylaxis, most patients with acute leukemia receiving mucotoxic chemotherapy develop neutropenic fever (NF), many cases of which remain without a documented etiology. Antibiotics disrupt the gut microbiota, with adverse clinical consequences, such as Clostridioides difficile infection. A better understanding of NF pathogenesis could inform the development of novel therapeutics without deleterious effects on the microbiota. We hypothesized that metabolites absorbed from the gut to the bloodstream modulate pyrogenic and inflammatory pathways. Longitudinal profiling of the gut microbiota in 2 cohorts of patients with acute leukemia showed that Akkermansia expansion in the gut was associated with an increased risk for NF. As a prototype mucolytic genus, Akkermansia may influence the absorption of luminal metabolites; thus, its association with NF supported our metabolomics hypothesis. Longitudinal profiling of the serum metabolome identified a signature associated with gut Akkermansia and 1 with NF. Importantly, these 2 signatures overlapped in metabolites in the γ-glutamyl cycle, suggesting oxidative stress as a mediator involved in Akkermansia-related NF. In addition, the level of gut microbial–derived indole compounds increased after Akkermansia expansion and decreased before NF, suggesting their role in mediating the anti-inflammatory effects of Akkermansia, as seen predominantly in healthy individuals. These results suggest that Akkermansia regulates microbiota-host metabolic cross talk by modulating the mucosal interface. The clinical context, including factors influencing microbiota composition, determines the type of metabolites absorbed through the gut barrier and their net effect on the host. Our findings identify novel aspects of NF pathogenesis that could be targets for precision therapeutics. This trial was registered at www.clinicaltrials.gov as #NCT03316456.

BACKGROUND & AIMS: A better understanding of prognostic factors within the heterogeneous spectrum of pediatric Crohn's disease (CD) should improve patient management and reduce complications. We aimed to identify evidence-based predictors of outcomes with the goal of optimizing individual patient management. METHODS: Asurvey of 202 experts in pediatric CD identified and prioritized adverse outcomes to be avoided. A systematic review of the literature with meta-analysis, when possible, was performed to identify clinical studies that investigated predictors of these outcomes. Multiple national and international face-to-face meetings were held to draft consensus statements based on the published evidence. RESULTS: Consensus was reached on 27 statements regarding prognostic factors for surgery, complications, chronically active pediatric CD, and hospitalization. Prognostic factors for surgery included CD diagnosis during adolescence, growth impairment, NOD2/CARD15 polymorphisms, disease behavior, and positive anti-Saccharomyces cerevisiae antibody status. Isolated colonic disease was associated with fewer surgeries. Older age at presentation, small bowel disease, serology (anti-Saccharomyces cerevisiae antibody, antiflagellin, and OmpC), NOD2/CARD15 polymorphisms, perianal disease, and ethnicity were risk factors for penetrating (B3) and/or stenotic disease (B2). Male sex, young age at onset, small bowel disease, more active disease, and diagnostic delay may be associated with growth impairment. Malnutrition and higher disease activity were associated with reduced bone density. CONCLUSIONS: These evidence-based consensus statements offer insight into predictors of poor outcomes in pediatric CD and are valuable when developing treatment algorithms and planning future studies. Targeted longitudinal studies are needed to further characterize prognostic factors in pediatric CD and to evaluate the impact of treatment algorithms tailored to individual patient risk.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

The magnitude of microbiota perturbations after exposure to antibiotics varies among individuals. It has been suggested that the composition of pre-treatment microbiota underpins personalized responses to antibiotics. However, this hypothesis has not been directly tested in humans. In this high-throughput amplicon study, we analyzed 16S ribosomal RNA gene sequences of 260 stool samples collected twice weekly from 39 patients with acute leukemia during their ~ 4 weeks of hospitalization for chemotherapy while they received multiple antibiotics. Despite heavy and sustained antibiotic pressure, microbial communities in samples from the same patient remained more similar to one another than to those from other patients. Principal component mixed effect regression using microbiota and granular antibiotic exposure data showed that microbiota departures from baseline depend on the composition of the pre-treatment microbiota. Penalized generalized estimating equations identified 6 taxa within pre-treatment microbiota that predicted the extent of antibiotic-induced perturbations. Our results indicate that specific species in pre-treatment microbiota determine personalized microbiota responses to antibiotics in humans. Thus, precision interventions targeting pre-treatment microbiota may prevent antibiotic-induced dysbiosis and its adverse clinical consequences.

Background: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. Objective: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosislike disease, early-onset seizures, and renal and lung disease. Methods: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double stranded nucleic acids. Results: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. Conclusion: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease. (J Allergy Clin Immunol 2021;148:381-93.)

ABSTRACT IMPACT: By restoring the gut microbiota in patients with acute myeloid leukemia exposed to antibiotics, we will reduce infections during and after curative-intent chemotherapy. OBJECTIVES/GOALS: Infection is a leading cause of death in acute myeloid leukemia (AML). Antibiotics disrupt the gut microbiota, promoting secondary infections. Through a double-blind randomized placebo-controlled phase 2 trial, we will determine whether microbiota restoration using fecal microbiota transplantation (FMT) prevents infections in AML patients. METHODS/STUDY POPULATION: 72 intensively treated AML patients at our institution are randomized in a 2:1 ratio to FMT (arm A) or placebo (arm B). After completing each course of antibacterial antibiotics, patients receive one study treatment. Up to 3 study treatments are administered over 3 months. FMT is delivered as a third-party oral product containing microbiota (˜5x10^11 bacteria) in 4-6 capsules. Stool samples are collected before and after each study treatment. The primary endpoint is 4-month overall infection rate. 16S rRNA gene sequencing of stool samples is used to determine specific taxa that are under- or over-represented in samples preceding infections and compare the two arms for key microbiome features including diversity and composition. Bloodstream infection within 7 days after FMT counts towards stopping rule. RESULTS/ANTICIPATED RESULTS: Five patients have been enrolled: 4 have received 1 dose and 1 received 2 doses. The only adverse event (possibly related to study treatment) has been grade 1 abdominal pain in 1 patient. Notably, no bloodstream infection has occurred. All planned samples have been collected and are sequenced in batches. We expect arm A patients to experience fewer infections and fewer intestinal blooms of pathobionts, and both arms to experience intestinal blooms before specific infections. An interim efficacy analysis will be performed at half total enrollment. DISCUSSION/SIGNIFICANCE OF FINDINGS: Current supportive care during intensive chemotherapy is fundamentally anti-microbial and results in dysbiosis, with detrimental consequences. We will establish the evidence for FMT as a restorative strategy in AML patients. This is the first randomized placebo-controlled trial of repeated FMT, with potential implications to other cancers.

Introduction: Despite antibiotic prophylaxis, most patients with acute myeloid leukemia (AML) develop neutropenic fever (NF) during intensive chemotherapy, suggesting a non-infectious etiology in many cases. In addition, escalated antibiotics used to treat NF increase the risk of Clostridioides difficile infection, promote pathogen colonization, prolong hospitalization, and increase healthcare costs. More effective prevention of NF, preferably using non-antibiotic approaches, is needed. We hypothesized that a longitudinal analysis of the circulating metabolome may reveal novel aspects of NF pathogenesis and identify potential targets for new preventative interventions. Methods: We analyzed 128 longitudinal serum samples from 17 intensively treated adult patients with AML between hospital admission and day 28 of chemotherapy. Samples were collected between 6-8 AM every Mon and Thu. Samples were subjected to ultrahigh performance liquid chromatography-tandem mass spectrometry. Results: All patients developed NF. A total of 1,031 metabolites were identified. Principal components analysis of the circulating metabolome could not resolve individual patients (Fig. 1a). In contrast, pre- vs. post-NF samples were partially clustered (Fig. 1b), suggesting a metabolomic shift associated with NF. After correcting for false discovery, 26 and 27 metabolites were higher in pre- and post-NF samples, respectively (q<0.05, |log fold-change| >1; Fig. 1c). The most significant metabolite that was different between post- and pre-NF samples was citrulline, with a mean concentration ratio of 0.65 between the two groups (q<10-5, Fig. 1c). Citrulline is a known biomarker for total enterocyte mass and its lower levels in post-NF samples indicate intestinal tissue damage as a potential etiology for NF. Another notable metabolite was 3-indoxyl sulfate (3-IS), a tryptophan metabolite and biomarker of gut microbiota diversity and clostridia abundance. 3-IS levels also decreased in post-NF (post vs. pre ratio: 0.45, q=0.02; Fig. 1c), suggesting a protective role for commensal microbiota against NF. Indoles act via the aryl hydrocarbon receptor to repair the intestinal epithelial barrier. Sparse partial least squares discriminant analysis (sPLS-DA) further improved group separation (Fig. 1d). Significantly altered metabolites in the first analysis along with the top 50 metabolites in sPLS-DA were fed into a random forest which generated the final list of 47 metabolites with largest contributions to group separation, including 3-IS and several citrulline metabolites (top 10 metabolites in Table 1). The most frequent metabolites on this list were those in amino acid (n = 17) and lipid (n = 14, including a secondary bile acid) pathways. Conclusions: This first-time analysis of the circulating metabolome in AML patients with NF suggests NF as a metabolic derangement rather than an infectious event in many patients. Augmenting the intestinal epithelium and maintaining a commensal clostridia-rich gut microbiome may help prevent NF. In addition, our list of altered metabolites introduces an unexplored niche for the development of novel, non-antibiotic-based approaches to prevent NF.

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males ( DNAAF2/ KTU , DNAAF4/ DYX1C1 , DNAAF6/ PIH1D3 , DNAAF7/ZMYND10 , CFAP300/C11orf70 and LRRC6 ). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.