Asthenoteratozoospermia refers to the decrease or lack of motile sperm and combines with abnormal sperm morphology, which becomes one of the most common factors leading to male infertility (1, 2). Multiple morphological abnormalities of the sperm flagella (MMAF), a specific kind of asthenoteratozoospermia, is featured by aberrant flagellar phenotypes with absent, short, bent, coiled, and/or irregular flagella, which can be easily identified by light microscopy (3). Mammalian sperm flagella consist of a central structure called the axoneme and peri-axonemal structures (the mitochondrial sheath, the fibrous sheath, and the outer dense fibers) (4). The axoneme comprises a central pair of microtubules surrounded by nine peripheral doublet microtubules, which terms a (9 +2) structure (5). Severe ultrastructural abnormalities are observed by electronic microscopy in the flagellar of patients with MMAF, such as a lack of the central pair, with missing or disorganized peripheral doublet microtubules and missing dynein arms (6), which results in male infertility due to impaired or absent sperm motility.

Since first defined in 2014 (3), the genetic investigation of MMAF has been continuously explored. To date, nearly 40 genes have been identified that account for 30%–60% of individuals with MMAF (6). The mutations in DNAH1, CFAP43, and CFAP44 were reported as recurrent causes of MMAF. DNAH1 encodes axonemal inner arm dynein, which is indispensable for flagellar beating (6). CFAP43 and CFAP44 relate to the cilia- and flagella-associated proteins (CFAPs) with WD repeat domains (WDRs) that locate in the inner dynein arm complex tether/tether head, involved in protein interactions (7). Male Cfap43^−/− and Cfap44^−/− mice are sterile and present peri-axonemal and axonemal defects in sperm flagella (7). Furthermore, DNAH2, DNAH6, DNAH17, CFAP65, CFAP70, CEP135, TTC29, and SPEF2, which mainly encode for sperm components, were also mentioned in a minority of patients with MMAF (6).

Considering that flagellar defects appear to influence intracytoplasmic sperm injection (ICSI) outcomes and fetal development (8, 9), studies focusing on the ICSI outcomes and prognosis for patients with MMAF have been reported. Several studies indicated a positive ICSI outcome in the MMAF cohort (4, 10, 11), and others have opposite opinions (9, 12). However, the sample size of these studies was too small to draw convincing conclusions. Furthermore, the neonatal outcomes and the information regarding the mutations of patients with MMAF were incomplete. Therefore, it is necessary to present a general overview of the comprehensive ART outcomes of patients with MMAF and thus provide accurate guidance for clinicians. In this context, causative genetic variants in individuals with MMAF were identified by whole exome sequencing (WES) to confirm the genetic pathogenesis of MMAF. Furthermore, an extensive evaluation of ART outcomes, including in vitro fertilization (IVF)/ICSI outcomes, IVF/ICSI success chance, donor semen attempt, neonatal outcomes, and time costs on achieving pregnancy, was conducted in this enlarged MMAF cohort. To create a highly comparable control group, the propensity score matching (PSM) method, which can minimize the discrepancies between the different groups of patients, was performed. The propensity score aims to remove the effects of confounding in multiple clinical and genetic analyses. The current study is designed to provide precise genetic counseling and offer individual ART instruction for patients with MMAF.

In this study, we analyzed clinical phenotypes, genetic variants, and overall ART outcomes of patients in the cohort of MMAF. The results suggested that nearly half of the patients with MMAF were identified in the pathogenic genetic variants and patients with MMAF presented compromised ART outcomes, including lower embryo developmental potential (decreased fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyte rate) and impaired clinical outcomes (reduced CLPR), compared with the cohort of oligoasthenozoospermia.

MMAF is a specific kind of asthenoteratozoospermia, which is highly suspected to be associated with genetic mutations (19). WES and Sanger sequencing are common methods to detect abnormal mutations. According to previous studies, WES has uncovered high frequencies of mutations in DNAH1, CFAP44, and CFAP43, which covered approximately one-third of all MMAF cases (5). AK7, CFAP69, CEP135, AKAP3, and AKAP4 were also related to MMAF with strong genetic evidence (5). In the current study, WES was performed in 23 patients with MMAF and presented the mutation in DNAH1, DNAH11, CFAP43, FSIP2, and SPEF2. A high frequency of mutation in DNAH1 was in line with the previous conclusion (20). Different gene mutations observed in patients with MMAF might be linked to the common components among the sperm centrosome, the mitotic spindle, and flagella. The SEM showed aberrant flagellar phenotypes with absent, short, bent, coiled, and/or irregular flagella in the MMAF group. According to the TEM, the abnormal ultrastructure of sperm flagella was presented with an absent central complex and/or a disorganized axonemal structure. The identification of genetic mutations combined with ultrastructural evaluation is not only pivotal for revealing mechanisms underlying spermiogenesis in individuals with MMAF but also important for improving treatment choices and providing adequate genetic counseling for patients.

Sperm immobility is the most common feature of individuals with MMAF; thus, affected couples are only able to conceive via ICSI. To date, the relationship between MMAF and ART outcomes has been investigated. Our results with an enlarged sample and strictly statistical comparison showed a significant decrease in fertilization rate, 2PN cleavage rate, blastocyst formation rate, and available blastocyte rate, and presented lower CLPR in the cohort of MMAF. Therefore, the poor ART outcomes exhibited in our study suggest that MMAF may affect the procedure of fertilization and decrease the quality of embryos. A similar association has already been reported between individuals with MMAF carrying CFAP65, DNAH17, and CEP135 mutations and compromised ICSI outcomes (21–23). Meanwhile, Fauque et al. (9) observed that infertile patients with ultrastructural abnormalities of the sperm flagellum could disturb early embryonic development. In contrast, some studies presented comparable results in laboratory and clinical outcomes in the cohort of the MMAF and control groups (4, 10, 11, 24). The reason for the inconsistent results may be partly due to the limited sample size and the selection criterion of the control group. Most of the previous studies were case reports or small-scale studies that may be affected by individual differences. In addition, although no significant differences were found in several previous studies, the ART outcomes still declined in the MMAF group. On the other hand, male infertility patients caused by oligoasthenozoospermia were strictly chosen as the control group, as patients with some factors such as testicular sperm extraction (TESE) and total teratozoospermia may affect ICSI outcomes. Moreover, PSM was used to set the control group to eliminate the bias as much as possible, thus resulting in more robust and reliable data.

The reasons for unsatisfactory ART outcomes in the MMAF cohort are worth pondering. Published studies have shown that most of the genetic mutations of MMAF were related to the flagellum (6). The sperm flagellum sustains sperm motility, which is indispensable for sperm progression. Therefore, most patients with MMAF presented immotile sperm, which made it difficult for embryologists to distinguish the viable and dead sperm and then affected the fertilization progress. Currently, chemical substances, such as pentoxifylline and theophylline, which can partly induce sperm motility, were optional effective methods for embryologists to identify and select viable sperm (25, 26). In addition, some causative mutated genes in patients with MMAF, such as DNAH6 (27), CFAP65 (12), SPEF2 (28), and CFAP69 (6), were reported in connection with abnormal sperm head and acrosome, which is the main Ca²⁺ storage sites in the sperm (29). Ca²⁺ signal is pivotal to sperm function, including hyperactivation, chemotaxis, and acrosome reaction (29, 30). The impairment of acrosome may lead to male subfertility and fertilization failure via Ca²⁺ signal deficiency. Furthermore, MMAF may affect the structure of the sperm, such as chromatin or the integrity of the sperm centrosome, which are essential for regulating syngamy and first mitosis following fertilization and the developmental potential of the embryo (12, 31). Taken together, the compromised ART outcomes in individuals with MMAF were reasonable.

One of the strengths of the current study is the comprehensive assessment of the ART outcomes and a long-term follow-up until the live birth for patients with MMAF, including CLPR, CLBR, the application of donor semen, and time to pregnancy. CLPR and CLBR could provide a more precise estimate of treatment efficacy for couples and summarize the chance of a pregnancy/live birth over an entire ART period, which have been suggested as more suitable ways of evaluating ART outcomes (32, 33). As presented in the study, the CLPRs were significantly lower in the MMAF group and the CLBRs were also below the oligoasthenospermia group, whether in the aspect of embryo transfer times or complete cycles, indicating compromised ART outcomes in the cohort of MMAF. Although patients with MMAF can achieve successful pregnancy via ICSI, more ovarian stimulation cycles are needed to obtain pregnancy according to our results, which means more time and cost will be spent on the ART process. Furthermore, eight cycles with donor semen in patients with MMAF were included in the current study to individually evaluate the ART pregnancy success in these special groups. Because most couples used donor semen from the second complete cycle, the CLPR and CLBR increased substantially from the second complete cycle in the MMAF+DS group, which were similar to the control group and higher than the MMAF group. Remarkably, donor sperm could offer patients with MMAF better ART outcomes. Based on the current results, strong evidence was offered to the clinicians in the counseling and therapy guidance of MMAF. Individualized treatment can be developed depending on the patient’s intentions and demands, including the expectation of successful pregnancy/live birth; concerns about heritage, time, and cost planning on the ART; and the economic condition.

Furthermore, this is the first study to focus on the neonatal outcomes of the MMAF cohort. The present study reported the delivery of healthy babies after transfer with embryos originating from sperm with MMAF. Seventeen babies were born including two pairs of twins in the MMAF group. One case with major malformation was reported in the twin group. In general, the neonatal outcomes of the MMAF group were similar to the control group, which indicated that embryos derived from the MMAF group have the capacity to develop normally and may lead to babies with no major malformations. Studies with larger sample sizes and long-term follow-ups are still needed to determine the effect of MMAF on newborns. Moreover, given that MMAF has a strong relationship with genetic mutations, the potential for transmission of MMAF to offspring is a leading concern. Therefore, patients with MMAF need to be well informed of the possibility of heritable mutations and the risk of infertility.

However, there were still several limitations. Firstly, owing to the low incidence of MMAF, this was a retrospective single-center study with a small sample size. Then, WES was not performed in all patients with MMAF. Further exploration of the potential genetic mutations or underlying mechanisms in patients without identified mutations via WES is needed. The relationship between different gene mutations and the compromised ART outcomes in the MMAF cohort was also worth exploring. Finally, long-term follow-up of the neonates needs to be addressed in the future.

In addition, for patients with MMAF, the prognosis of ICSI treatment may differ among the results of genetic diagnosis; thus, a systematic genetic screening can help physicians not only diagnose with certainty but also provide precise treatment instructions and pregnancy prediction based on the types of genetic mutations. Furthermore, expanding the mutational and phenotypic spectrum of MMAF genes can lay a foundation for the diagnostic screening kits of MMAF.

In general, genetic mutations account for part of the pathogenesis in MMAF. MMAF may exert deterioration effects on the gamete and embryonic development potential with a compromised ART outcome compared with a male-infertile control group. Results from the analysis of genetics and ART outcomes may provide clinicians with more elaborate and individual evidence in MMAF counseling. Furthermore, large-scale studies and more in-depth mechanism research are still needed to guide future clinical trials.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by the Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

RL: Writing – review & editing, Writing – original draft. MW: Writing – review & editing. JZ: Writing – review & editing. RM: Writing – review & editing. CW: Writing – review & editing. LG: Writing – review & editing. YC: Writing – review & editing. LJ: Writing – review & editing. LZ: Writing – review & editing.