Are ICSI outcomes impaired in cases of severe asthenozoospermia with multiple morphological abnormalities of the flagellum (MMAF phenotype)?
Despite occasional technical difficulties, ICSI outcomes for couples with MMAF do not differ from those of other couples requiring ICSI, irrespective of the genetic defect.
Severe asthenozoospermia, especially when associated with the MMAF phenotype, results in male infertility. Recent findings have confirmed that a genetic aetiology is frequently responsible for this phenotype. In such situations, pregnancies can be achieved using ICSI. However, few studies to date have provided detailed analyses regarding the flagellar ultrastructural defects underlying this phenotype, its genetic aetiologies, and the results of ICSI in such cases of male infertility.
We performed a retrospective study of 25 infertile men exhibiting severe asthenozoospermia associated with the MMAF phenotype identified through standard semen analysis. They were recruited at an academic centre for assisted reproduction in Paris (France) between 2009 and 2017. Transmission electron microscopy (TEM) and whole exome sequencing (WES) were performed in order to determine the sperm ultrastructural phenotype and the causal mutations, respectively. Finally 20 couples with MMAF were treated by assisted reproductive technologies based on ICSI.
Patients with MMAF were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella compared with those in sperm from fertile control men. A quantitative analysis of the several ultrastructural defects was performed for the MMAF patients and for fertile men. The ICSI results obtained for 20 couples with MMAF were compared to those of 378 men with oligoasthenoteratozoospermia but no MMAF as an ICSI control group.
TEM analysis and categorisation of the flagellar anomalies found in these patients provided important information regarding the structural defects underlying asthenozoospermia and sperm tail abnormalities. In particular, the absence of the central pair of axonemal microtubules was the predominant anomaly observed more frequently than in control sperm (P < 0.01). Exome sequencing, performed for 24 of the 25 patients, identified homozygous or compound heterozygous pathogenic mutations in CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29 and MAATS1 in 13 patients (54.2%) (11 affecting MMAF genes and 2 affecting primary ciliary dyskinesia (PCD)-associated genes). A total of 40 ICSI cycles were undertaken for 20 MMAF couples, including 13 cycles (for 5 couples) where a hypo-osmotic swelling (HOS) test was required due to absolute asthenozoospermia. The fertilisation rate was not statistically different between the MMAF (65.7%) and the non-MMAF (66.0%) couples and it did not differ according to the genotype or the flagellar phenotype of the subjects or use of the HOS test. The clinical pregnancy rate per embryo transfer did not differ significantly between the MMAF (23.3%) and the non-MMAF (37.1%) groups. To date, 7 of the 20 MMAF couples have achieved a live birth from the ICSI attempts, with 11 babies born without any birth defects.
The ICSI procedure outcomes were assessed retrospectively on a small number of affected subjects and should be confirmed on a larger cohort. Moreover, TEM analysis could not be performed for all patients due to low sperm concentrations, and WES results are not yet available for all of the included men.
An early and extensive phenotypic and genetic investigation should be considered for all men requiring ICSI for severe asthenozoospermia. Although our study did not reveal any adverse ICSI outcomes associated with MMAF, we cannot rule out that some rare genetic causes could result in low fertilisation or pregnancy rates.
No external funding was used for this study and there are no competing interests.
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We illustrate shape mode analysis as a simple, yet powerful technique to concisely describe complex biological shapes and their dynamics. We characterize undulatory bending waves of beating flagella and reconstruct a limit cycle of flagellar oscillations, paying particular attention to the periodicity of angular data. As a second example, we analyze non-convex boundary outlines of gliding flatworms, which allows us to expose stereotypic body postures that can be related to two different locomotion mechanisms. Further, shape mode analysis based on principal component analysis allows to discriminate different flatworm species, despite large motion-associated shape variability. Thus, complex shape dynamics is characterized by a small number of shape scores that change in time. We present this method using descriptive examples, explaining abstract mathematics in a graphic way.

Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift-associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift-associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly.

According to Bergey\'s Manual of Systematic Bacteriology, the Rickettsiales are \'…bacteria with typical gram-negative cell walls and no flagella\'. The recently sequenced genome of \'Candidatus Midichloria mitochondrii\', a divergent lineage within the order Rickettsiales capable of invading mitochondria in ixodid ticks, revealed the presence of 26 putative flagellar genes. Open questions in relation to this observation are whether these genes are expressed and whether they possess the domains expected for the flagellar function. Here we show that: (a) the putative flagellar proteins of \'Ca. M. mitochondrii\' actually possess the conserved domains and structural features required for their function in a model bacterium; (b) the seven flagellar genes of \'Ca. M. mitochondrii\' that have been tested are expressed at the RNA level; and (c) the putative flagellar cap gene of this bacterium (FliD) is expressed at the protein level, and can be stained within the bacterium and at its surface. Beside the specific questions that we have addressed that relate to the first evidence, to our knowledge, for a flagellar apparatus in a member of the order Rickettsiales, we present here novel tools (recombinant protein and antibodies) that will facilitate the study of \'Ca. M. mitochondrii\'.

Peripheral blood smear examination is the most specific as well as the most common test performed for the diagnosis of malaria. Schizonts, ring forms (trophozoites) and gametocytes are the stages of malarial parasite that are commonly seen in the peripheral blood smear of a patient. Here, we report an extremely rare case of a 40-year-old male patient who presented with Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear with review of the literature. Exflagellation of microgametes in malarial parasites is only seen in the definitive host, mosquito, and is very unusual to see during the developmental phases in the intermediate host, human. It is important to recognize these exflagellated microgametes in the peripheral blood smear as they may lead to diagnostic confusion with organisms such as spirochetes and trypanosomes.

OBJECTIVE: To describe the ultrastructure of spermatozoa from a patient with complete asthenozoospermia that resulted in live births following blastocyst culture.
METHODS: Analyses of spermatozoa from a 36 year old patient were performed using light and electron microscopy. The hypo-osmotic swelling test was used to select spermatozoa for intracytoplasmic sperm injection. Embryos were cultured to the blastocyst stage.
RESULTS: 100% of the spermatozoa had dynein arm deficiency with secondary defects varying from 3-17%. Six oocytes were injected; five fertilized normally and one was digynic. All five zygotes formed good quality blastocysts. Three blastocysts were cryopreserved and two blastocysts were transferred. Twin females were born at 37 weeks.
CONCLUSIONS: The hypo-osmotic swelling test can be used to select viable immotile ejaculated spermatozoa from a patient with dynein arm deficiency and can produce excellent fertilization rates and blastocyst development resulting in live births.

Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered \'irreducibly complex\' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems (\'molecular bricolage\'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

BACKGROUND: Human infections with non-O1, non-O139 V. cholerae have been described from Laos. Elsewhere, non cholera-toxin producing, non-O1, non-O139 V. cholerae have been described from blood cultures and ascitic fluid, although they are exceedingly rare isolates.
METHODS: We describe a farmer who died with Vibrio cholerae O21 bacteremia and peritonitis in Vientiane, Laos, after eating partially cooked apple snails (Pomacea canaliculata) and mussels (Ligumia species). The cultured V. cholerae were non-motile. PCR detected ompW and toxR gene regions but not the ctxA, ompU, omp K and TCP gene regions. Although the organisms lacked flagellae on scanning electron microscopy, they possessed the Vibrio flagellin flaA gene.
CONCLUSIONS: Severe bacteremic non-O1, non-O139 V. cholerae is reported from Laos. The organisms were unusual in being non-motile. They possessed the Vibrio flagellin flaA gene. Further research to determine the reasons for the non-motility and virulence is required.

A recent paper, which will surely figure centrally in the debate between evolutionists and Intelligent Design creationists, proposes a (perhaps too simple) scheme for the evolution of bacterial flagella.

OBJECTIVE: The aim of the study was to characterize a spirochaete isolated from the lesions of a cow with digital dermatitis (DD).
RESULTS: The characterization was on the basis of its light and electron microscopic appearance, enzymic profile and DNA sequence analysis of its flagellin and 16S rRNA genes. The spirochaete was 6-8-microm long and 0.2-0.3 microm in diameter, and possessed seven to eight periplasmic flagella, with three to five helical turns. The enzymic profile of the bacterium resembles, but is not identical to that of Treponema brennaborense. Its flagellin gene sequence was identical to that of Treponema phagedenis but distinct from that of an ovine spirochaete. Analysis of a 1477-bp region of the 16S rRNA genes indicated that this is a Treponema species and that it is indistinguishable from some isolates made from cases of bovine DD in the United States. Finally, electron microscopy revealed the presence of myovirus-like bacteriophage particles in all cultures of the treponeme examined.
CONCLUSIONS: The spirochaete isolate was identified as a Treponema species closely related to some isolates from the United States (by 16S rDNA) and to T. phagedenis (by flagellin gene sequence) and is associated with bacteriophage particles.
CONCLUSIONS: The fact that the isolates with the same or very similar 16S rDNA sequences have been obtained from cases of bovine DD in cattle in different countries at different times, lends further support to the hypothesis that treponemes play a role in the pathogenesis of this disease.