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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A 5-year-old male Beagle dog produced ejaculates with a high percentage of spermatozoa with abnormal morphology, especially sperm tail defects. Although libido and semen volume were normal, ejaculates showed asthenospermia, oligozoospermia, and teratozoospermia. The spermatozoa exhibited morphologic defects affecting the flagellum, mainly coiled tails with or without macrocephalia (33.5 ± 2.1%), bent tails (18.3 ± 3.4%), and proximal cytoplasmic droplets (6.7 ± 2.8%). The peripheral plasma testosterone level was 2.76 ± 0.21 ng/mL. The resistive index and the pulsatility index from marginal and intratesticular vessels measured by Doppler ultrasound showed higher values in the right testicle than in the left testicle. Histologic evaluation revealed focal reduction in the number of germ cells and sperm in the seminiferous tubules in the right testicle. This is the first report that describes simultaneously the presence of sperm tail defects in the ejaculate and changes in the blood flow of testicular vessels in the dog.

The use of high-throughput sequencing techniques has allowed the identification of numerous mutations in genes responsible for severe astheno-teratozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). However, more than half of the analysed cases remain unresolved suggesting that many yet uncharacterised gene defects account for this phenotype. Based on whole-exome sequencing data from a large cohort of 167 MMAF-affected subjects, we identified two unrelated affected individuals carrying a homozygous deleterious mutation in CFAP70, a gene not previously linked to the MMAF phenotype. One patient had a homozygous splice variant c.1723-1G>T, altering a consensus splice acceptor site of CFAP70 exon 16, and one had a likely deleterious missense variant in exon 3 (p.Phe60Ile). The CFAP70 gene encodes a regulator protein of the outer dynein arms (ODA) strongly expressed in the human testis. In the sperm cells from the patient carrying the splice variant, immunofluorescence (IF) experiments confirmed the absence of the protein in the sperm flagellum. Moreover, IF analysis showed the absence of markers for the ODAs and the central pair complex of the axoneme. Interestingly, whereas CFAP70 staining was present in sperm cells from patients with mutations in the three other MMAF-related genes ARMC2, FSIP2 and CFAP43, we observed an absence of staining in sperm cells from patients mutated in the WDR66 gene, suggesting a possible interaction between two different axonemal components. In conclusion, this work provides the first evidence that loss of CFAP70 function causes MMAF and that ODA-related proteins may be crucial for the assembly and/or stability of the flagellum axoneme in addition to its motility.

OBJECTIVE: The purpose of this study is to analyze the sperm morphology of a Chinese man affected with multiple morphological abnormalities of the sperm flagella (MMAF) and observe the intracytoplasmic sperm injection (ICSI) outcome.
METHODS: A Chinese man was diagnosed with multiple morphological abnormalities of the sperm flagella by semen analysis and electron microscopy. Testicular spermatozoa were injected intracytoplasmically, and the following ICSI results were observed.
RESULTS: All the spermatozoa from his ejaculate were immotile and morphologically abnormal in the flagellum. In transmission electron microscopy assays, most spermatozoa showed disorganized fibrous sheath, accompanied by distortion of various cytoskeletal components, and missing of the central pair microtubules. Testicular sperm was injected to the oocytes in two ICSI cycles, with fertilization rates of 45.5 and 40.0%. Finally, a healthy female infant was delivered at the second ICSI cycle.
CONCLUSIONS: Fertilization and pregnancy could be achieved by intracytoplasmic sperm injection, regardless of severe flagellar defects. ICSI is effective for MMAF-affected man, and testicular sperm is an alternative when no motile sperm is available.

OBJECTIVE: To compare subgroups of the human sperm hypoosmotic swelling test subgroups in both recurrent fertilization negative infertile cases with normal semen analysis and fertilization positive controls.
METHODS: This was a prospective case-controlled study performed with normospermic 33 previously fertile male (secondary infertility) and 41 infertile men who had undergone two or three unsuccessful in vitro fertilization attempts. HOS test was investigated in 4 subgroups including HOS 1, HOS 2, HOS 3, and HOS 4 according to the degree of sperm tail swelling and compared between the two groups.
RESULTS: Four subgroups were compared and statistical significance was demonstrated in HOS 1, HOS 3 and HOS 4 tests (p < 0.001) in fertile and infertile men. Highest HOS 1 and lowest HOS 4 grades were determined in Group A. However, no statistical significance was determined between two groups in HOS 2 test which was minimal swelling in sperm tails.
CONCLUSIONS: HOS 1, HOS 3 and HOS 4 subgroups of HOS test are reliable and useful methods providing important information regarding the sperm function. A high HOS test 1 grade plus a low HOS test 4 grade should suggest a fertility problem, despite a normal semen analysis. HOS test subgroups provide additional information in normospermic cases with unexplained infertility.

OBJECTIVE: To characterize a novel flagellar defect involving 98% of sperm tails.
METHODS: Case report.
METHODS: Interdepartmental Centre for Research and Therapy of Male Infertility, Siena, Italy.
METHODS: A 45-year-old infertile man with severe asthenozoospermia.
METHODS: Family history, physical examination, hormonal analysis, microbial assays, semen analysis, transmission and scanning electron microscopy, tubulin distribution investigated by immunocytochemistry, fluorescence in situ hybridization (FISH) for chromosomes 9, 16, 18, X, and Y.
METHODS: Flagellar abnormalities detected by microscopical methods.
RESULTS: An apparent heterogeneity was observed: extremely elongated tails prone to ruptures; coiled tails at different levels with a strongly rolled axoneme or with a curl in the final flagellar segment; and V-shaped, isolated, bent tails. Transmission electron microscopy revealed the presence of normal heads, disorganized flagellar structures, and dynein deficiency. The FISH analysis was normal.
CONCLUSIONS: We report a new sperm defect, characterized by abnormal elongation of the tail, which was prone to ruptures at different levels, concomitant with coiled tails, which were impossible to measure in length. This defect remained constant in different examined ejaculates and applied to the entire sperm population of a sterile man, the son of first-degree cousins, indicating a potential genetic origin.

The annulus is a septin-based ring structure located at the junction of the midpiece (MP) and the principal piece (PP) of spermatozoa flagellum. In the mouse, deletion of Septin 4, a structural component of the sperm annulus, prevents annulus formation and leads to MP-PP disjunction, flagellar bending, asthenozoospermia and male sterility. Testis anion transporter 1 (Tat1) is a germ cell-specific member of the SLC26 anion transporter family and is co-expressed with Septin 4 at the sperm annulus. Interestingly, Tat1 null sperm bear an atrophic annulus, causing a phenotype similar to that of Sept4 null sperm. We searched for Tat1 misexpression and/or mislocalization in spermatozoa from asthenozoospermic subjects (n = 75) and controls by performing an immunofluorescence detection assay on sperm smear preparations. We found one patient showing moderate asthenozoospermia, with 97% of sperm lacking Tat1, Septin 4 and Septin 7 proteins at the annulus. We confirmed the absence of the annulus structure by transmission electron microscopy and observed that spermatozoa from the patient displayed MP-PP disjunction and abnormal mitochondrial organization. We show that the structural defects in sperm are not caused by abnormal transcription or point mutations of the TAT1 and SEPT4 genes; however, although both proteins are expressed, they are not properly localized at sperm annulus. The case we studied, so far unreported in human, confirms the involvement of Tat1 and Septin proteins in the constitution of the annulus, but also raises questions about the function of this structure in human sperm motility.

OBJECTIVE: To determine the meiotic segregation in large-headed, multiple-tailed spermatozoa.
METHODS: Analysis of sperm nuclei by fluorescence in situ hybridization (FISH).
METHODS: University hospital.
METHODS: A 34-year-old man with 100% morphologically abnormal spermatozoa.
METHODS: Dual-color FISH for chromosomes 13 and 21 and triple-color FISH for chromosomes X, Y, and 18 were performed.
METHODS: Aneuploidy rates.
RESULTS: More than 99% of the spermatozoa had abnormal content for chromosomes X, Y, 13, 18, and 21. Diploidy, triploidy, and tetraploidy rates were found to be 18.42%, 6.14%, and 33.99% in triple-color FISH and to be 16.09%, 16.28%, and 38.95% in dual-color FISH.
CONCLUSIONS: Our results and those from other investigators show that large-headed, multiple-tailed spermatozoa are associated with a high rate of polyploidy and aneuploidy. Intracytoplasmic sperm injection should not be recommended to those patients, not only because of its low success rate but also because of its high genetic risk.

An atypical case of infertility associated with severe sperm abnormalities is presented. A 29-year-old man with 4 years of primary infertility had no history of significant illness, and no hereditary pathology or male infertility existed in his family. Physical examination of the patient showed no pathological findings. The analyses of four semen samples showed: sperm count, 67-83 10(6)/ml; 0% motility. The morphological analysis showed absence of flagellum, 14-16%; short-tail spermatozoa, 45-64%; coiled tails, 12-17%; and an abnormal proportion of spermatids and spermatocytes. Normal spermatozoa were found in 11-16%. Endocrine profile was found within the normal range. Testicular biopsy revealed impaired spermatogenesis. Scanning electron microscopy revealed sperm heads with intact nuclei and acrosomal regions. To our surprise, some of the stunted tails were uniflagellate. To our knowledge, this is a very uncommon case of sperm tail defect.

OBJECTIVE: To report a case of 100% teratozoospermia in a patient whose family history presented a high degree of consanguinity.
METHODS: Case report.
METHODS: University-based andrology clinic.
METHODS: A 27-year-old man undergoing infertility evaluation.
METHODS: None.
METHODS: Serum hormone analysis, standard (World Health Organization) semen quality evaluation, sperm ultrastructure, lymphocytic karyotype, and polymerase chain reaction screening for Y chromosome microdeletions in peripheral blood.
RESULTS: One hundred percent teratozoospermia characterized by shape alterations of the heads and tail agenesis on standard morphology evaluation, which was confirmed using electron microscopy.
CONCLUSIONS: A case of familial infertility in a consanguineous patient characterized by morphological abnormalities of spermatozoa such as tail agenesis, chromatin subcondensation, and residual cytoplasmic droplets.