Review CGE-00778-2023
Review: CCDC65 , encoding a component of the axonemal Nexin-Dynein Regulatory Complex, is required for sperm flagellum structure in humans.

The manuscript is relevant to the area; however it may be enhanced, particularly some sentences need to be clarified and the findings are overdone.

Abstract
“In order to characterize novel genetic causes of male infertility, we analyzed whole exome sequencing data from a cohort of 167 individuals displaying multiple morphological abnormalities of the sperm flagella (MMAF)”. All those patients had asthenozoospermia? In 167 patients only two had potential pathogenic variants?? Which are the main differences among those two patients and the rest of the cohort? This should be further discussed.
Introduction
“To date, over 50 genes have been reported with pathogenic mutations inducing PCD, most of these mutations affecting the assembly, anchoring, or structure of the axonemal dynein arms5, which constitute multiprotein complexes regulating the beating of motile cilia and flagella” This sentence is quite confusing and not totally accurate. It can induce the idea that only dynein arms are important for ciliary beating. Please reformulate.
Materials and methods
Western and immunofluorescence, authors should normalize data and perform the relative quantification of the protein expression.

Results/Discussion
Authors refer to two patients but then only report results about one. Authors should clarify this.
“Considering that the CCDC65 mutations identified here affect all three protein isoforms (Figure 1) and that GAS8 mutations are associated with PCD and asthenozoospermia19, it is likely that the previously reported CCDC65 mutated patients also present sperm structural and motility defects; this, however, remains to be formally demonstrated” this relation between ccdc65 and gas8 should be clarified. Otherwise, readers do not understand these conclusions.

Authors should be more careful in the conclusions made. With results from only one individual we can not state “ Importantly our work establishes that mutations in the PCD associated gene CCDC65 also cause asthenozoospermia…”. Authors can say that their work suggest something ..

The authors improved the clarity of the text and made some scientific improvements. However, in my opinion, minor improvements are still needed.

Abstract
"We analyzed whole exome sequencing data from a cohort of 167 individuals displaying multiple morphological abnormalities of the sperm flagella (MMAF) and identified two unrelated patients carrying distinct homozygous truncating variants in CCDC65, a gene for which mutations were previously associated with Primary Ciliary Dyskinesia (PCD)"
In my opinion, this should not be in the abstract. This is from a previous work, thus it could induce confusion in the readers. I consider that authors should make a brief introduction, particularly regarding the relation between ATZ and PCD, and then refer that the authors have analyzed two patients with ATZ with a mutation in a gene typical from PCD. Otherwise, it can raise the question among readers regarding the remaining individuals. The information regarding the cohort is corretly referred in materials and methods, but should not be in the abstract.

Results section
Line 151 "In control individuals, we observed signal along the flagella."
According to figure 3A (CCDC65 staining), the staining is mostly in middle piece of flagella, I see only a faint staining in the axoneme section. Also the merge image with tubulin, is not compatible with a distribution of CCDC65 over the flagella, if so, we should see a yellow staining. The same for GAS8 staining.
Regarding SPAG6 and DNALI1 staining it is quite strange to observe an intense staining in the head.
Authors should verify the images